Columbia ®nton*ftp intljeCttpofltogark College of 3$i}y&itim& ants gmrgeong lUbrarp CLINICAL PATHOLOGY Digitized by the Internet Archive in 2010 with funding from Open Knowledge Commons http://www.archive.org/details/clinicalpathologOOpant CLINICAL PATHOLOGY BY P. N. PANTON, M.A., M.B., B.C.Cantab. Clinical Pathologist to the London Hospital ; formerly Assistant Director of the Louis Joiner Clinical Laboratory. St. Thomas's Hospital. With 13 Plates (11 Coloured) and 45 Illustrations in the Text. PHILADELPHIA P. BLAKISTON'S SON & CO. 1012, WALNUT STREET 1913 Printed in Great Britain. I 1 <} - I % ? 1 PREFACE. This book represents an attempt to describe in a reasonable compass such laboratory investigations, whether chemical, histological or bacteriological, as have a practical bearing upon the diagnosis and treatment of disease ; to give some account of their meaning, and to assess, so far as possible, their value in practice. Many of the smaller text-books of "Clinical Pathology" deal with the subject only in part ; the complexity of methods in the larger works renders them more suitable to special investigators, and it was felt that a book of intermediate size might be of use to the student and practitioner. The reduction in size has been mainly arrived at by avoidance of a reduplication of methods, in preference to the omission of any essential branch of the subject. Where several methods for the same object are in use, one, or at most two, are described in full : the remainder are omitted altogether. Much of scientific interest has been necessarily sacrificed to the practical application of diagnostic methods, and the book has consequently no pretensions to consideration as anything more than an adjunct to clinical medicine. No list of references is given, and the names of authorities, unless definitely associated with a particular reaction, are for the most part omitted. In describing some of the more special investigations I have made use of, among other works, Sequeira's " Diseases of the Skin," Plimmer's "Practical Physiological Chemistry," Von Jaksch and Garrod's " Clinical Diagnosis," Sahli's " Diagnostic Methods," and Daniels and Alcock's " Tropical Medicine and vi PREFACE. Hygiene." The last work has also been of assistance to the artist in some of his drawings of the higher parasites. I am originally indebted to Mr. L. S. Dudgeon for many of the methods of procedure recorded here and for numerous details of technique, which have been of the greatest value to me in practice. The illustrations, with very few exceptions, have been specially drawn for this book from actual preparations. Mr. Shattock has kindly provided me with some of the specimens of intestinal parasites, and Dr. Turnbull with others. My colleague, Dr. Tidy, has most kindly read and revised the manuscript and has assisted me with his advice upon numerous particulars. Mr. A. C. Hudson has also helped me freely with the final revision. CONTENTS. CHAPTER I. II. SECTION I.— THE BLOOD PAGE 1—99 The Normal Blood — The Primary Blood Diseases 3 The Secondary Blood Changes — The Blood Changes occurring in Children . . . .17 III. The Methods oe Examining the Blood ... 32 IY. The Blood Serum — Agglutinins and Opsonins . 48 V. The Blood Serum {continued) — Complement Fixation Tests— The "Wassermann Beaction ... 65 VI. The Parasitology of the Blood . . . .81 VII. The Chemical and Physical Examination of the Blood 92 XI XII XIII SECTION II.— BACTERIOLOGY 100—188 VIII. Introductory — Table of Classification . . .101 IX. The Cocci — The Gram-positive Bacilli . . .115 X. The Gram-negative Bacilli — Spirilla — Strepto- triche^e — hyphomycetes . . . .' .132 Bacteriological Methods — General and Special. 147 Vaccines — Anti-sera 164 Preparation of Culture Media — Staining Reagents 177 SECTION III.— PUNCTURE FLUIDS . 189-211 XIV. General Procedure — Pleural Fluids — Peri- cardial Fluids 190 XV. Peritoneal Fluids — Cerebro-spinal Fluids - Synovial Fluids — Cysts, etc 202 SECTION IV.— THE UBINE 212—284 XVI. Routine Examination — Variations in Amount — Variations in Appearance 213 XVII. Variations in Acidity, and Acidosis — Variations in Specific Gravity — Urea — Proteids— Carbo- hydrates 228 viii CONTENTS. CHAPTER PAGE XVIII. Urinary Deposits — Urinary Calculi . . . 248 XIX. Special Investigations oe the Urine—Bacterio- logy of the Urino-genital Tract . . . 266 SECTION Y.— THE ALIMENTAEY SYSTEM .... 285-345 XX. The Mouth— The Stomach 286 XXI. The Pancreas — The Liver — The Spleen — The Peritoneum 303 XXII. The FiECES 316 XXIII. The Parasitology of the F.eces .... 329 SECTION VI.— THE EYE AND SKIN 346-360 XXIV. The Eye and Conjunctival Sac — The Skin . . 347 SECTION VII.— THE. RESPIRATORY TRACT .... 361-372 XXV. The Nose- The Sputum 362 SECTION VIII.— HISTOLOGY . . 373-433 XXVI. The Examination of Sections — The Inflammations — The Degenerations 374 XXVII. Neoplasms — Simple Tumours 391 XX yi II. Carcinomata — Sarcomata — Other Tumours — Cysts 403 XXIX. Histological Methods 421 INDEX 435 LIST OF PLATES. PLATE — ^ PAGE I. Normal and Abnormal Blood Cells To face 5 II. Pernicious Anaemia, Acute Inflammation 9 III. 11 IT. Lymphoid Leukaemia .... 14 V. Malarial Parasites 87 VI. 90 VII. Absorption Spectra 92 VIII. 115 IX. 132 X. The Cells of Puncture Fluids 192 XI. G-lucosazone Crystals, etc. . 246 xn. 250 XIII. 258 SECTION I. THE BLOOD. CHAPTEE I. The Normal Blood — The Primary Blood Diseases. CHAPTEE II. The Secondary Blood Changes— The Blood Changes occurring in Children. CHAPTEE III. The Methods of Examining the Blood. CHAPTEE IV. The Blood Serum — Agglutinins and Opsonins. CHAPTEE V. The Blood Serum (c.ontin ued)— Complement Fixation Tests— The "Wasser- mann Eeaction. CHAPTEE VI. The Parasitology of the Blood. CHAPTEE VII. The Chemical and Physical Examination of the Blood. CLINICAL PATHOLOGY. CHAPTER I. the normal blood — the primary blood diseases. The Normal Blood. The study of the histology of the blood in health is properly a part of physiology and is essential to the appreciation of the changes which take place in disease. The student is advised to refresh his reading of the normal blood before attempting the study of the blood in disease. The following is a brief account of the normal blood. The fresh blood (page 34) . — If a drop of blood, immedi- ately after being shed, be examined under the microscope the red cells will be found to have arranged themselves in the form of long curved rouleaux, few if any red corpuscles remain- ing isolated. Occasional leucocytes will be easily recognised lying between the rouleaux, often in groups of 2 or 3. Small granular-looking bundles of blood platelets will be made out, usually in the near neighbourhood of a collection of leucocytes. After a few minutes a delicate network of fibrin, appearing like a fine cobweb, will spread through the film, being densest in the vicinity of the blood platelets. Later the rouleaux will break up and the individual red cells will lose their shape and become crenated. The red corpuscles are round, or almost round, biconcave discs having an average diameter of 7'5 /x. They are oxyphilic, and in stained preparations the centre of the disc is, owing to the shape of the cell, frequently paler than the periphery. The almost white central area seen in some preparations is no evidence of disease. The number of red cells is subject to considerable individual variation, and is usually given as 5 million per c.mm. This number is much below the average found in the normal adult Englishman, whose red cells vary 4 CLINICAL PATHOLOGY. between 6 and 7 million per c.nini., the number being some- what less in the case of a woman. The number 5 million is, however, still commonly held to represent 100 per cent, of the normal for both sexes, mainly for the sake of simplicity in estimating the colour index. The non-nucleated red cells of the circulating blood arise from the nucleated cells of the red marrow. These nucleated cells are of two main varieties, normoblasts and megaloblasts. The former differ from the circulating red cells in possessing a single deeply and evenly staining nucleus, which fills the greater part of the cell. The megaloblast is usually a larger cell, the cytoplasm of which always shows, though in varying degree, an affinity for the basic dyes, the so-called basic or polychromatophilic degeneration. The colour of the cytoplasm, as stained by Leishman's stain, varies from a bronze to a French grey or even a deep blue, and is probably evidence, not of degeneration, but of immaturity. The nucleus is charac- teristically stippled with alternating light and dark areas, and is, in a good preparation, readily distinguished from the normoblast nucleus. The area of cytoplasm unoccupied by nucleus is in a megaloblast relatively large (see Plate I.). The haemoglobin is reckoned in percentages, and the standard of the amount of haemoglobin present in a normal person is taken as 100 per cent. The percentage of haemo- globin varies very little in health ; a fall below 90 per cent, or a rise above 105 per cent, should be regarded as pathological. The colour index. — By this is meant the index of the haemoglobin-carrying properties of the red cells. The index is obtained by dividing the percentage of haemoglobin by the percentage of red cells per c.mm. For example, a specimen of blood containing 4 million red cells per c.mm., or 80 per cent, of the conventional normal number and 40 per cent, of haemoglobin, will have a colour index of f§ or - 5, indicating that each red cell contains half the normal percentage of haemoglobin. The white corpuscles. — The normal number of leucocytes per c.mm. varies between 5 and 7 thousand, the average number being about 6 thousand. The number of leucocytes is sub- ject to periodic fluctuations. The leucocy tosis (i.e., increase in the number of leucocytes) which occurs after meals is due to an increase in the lymphocytes, and is sufficient to make it PLATE I. :Cv::v, % # * m >tt.' r °i tt ■wr..* ♦ Normal and Abnormal Blood Cells. (Leisb man's Stain.) t * KEY TO PLATE I. MEGALOBL/i57 OOP NORMAL ° cQ > i>nd . 20 to 30 j) 5 to 10 5 J 2 to 5 ) ; "J 5 to 20 55 2 to 5 )j 1 to 3 55 consists of n a wVi i r».Vi m a.\j iyeloblasts, 1->a rliffip.nlf, NORMAL BLOOD—PRIMARY BLOOD DISEASES. 13 increased. Commonly the fall in the number of leucocytes is more marked than the shrinkage of the spleen, and a patient may have a leucopenia with a spleen reaching to the umbilicus. Such a condition might be mistaken for splenic anaemia. These changes are favourable and may persist for some months before a relapse occurs. In the terminal stages of the disease a diminution in the number of leucocytes may be accompanied by the appearance of large numbers of non-granular cells in the blood. These non-granular cells are myeloblasts and in their more usual type are of about the size of large lymphocytes, but differ from them in having a relatively large nucleus, containing 3 or 4 pale pear-shaped nucleoli and an intensely basophilic cytoplasm (Plate III.). A less common type of myeloblast is little larger than the small lymphocyte, and in all probability represents a non-granular stage of the small type of neutro- philic myelocyte ; it is distinguished from the small lympho- cyte by the purplish colour of its cytoplasm. Both these forms of myeloblast are to be found in small numbers in all stages of the disease. A marked increase in their relative numbers is of the gravest significance. Acute myeloid leukaemia is an extremely rare condition, which differs from the ordinary form of the disease in the shortness of its course, in the usual absence of marked splenic enlargement, and in the relatively low leucocytosis. There may be considerable involvement of the lymphatic glands. Mast cells, eosinophils, and myelocytes are present, but the predominant cell may be either a myeloblast or a cell identical with the large hyaline of normal blood or a granular variety of it. Lymphoid leukaemia (Lymphatic leukaemia, Lymphaemia), the least common of the primary anaemias, may occur at any age, but usually attacks children between the ages of 8 and 15. In its typical form lymphaemia is an acute disease, accompanied by fever and severe constitutional disturbance, and terminating fatally in a few weeks. There is a moderate general enlarge- ment of the superficial lymphatic glands and the spleen as a rule is readily palpable. The blood changes are as follow : — The blood flows readily on puncture and the excess of leucocytes is very obvious in the fresh drop under the micro- scope. The leucocytes are seen to be non-granular. Fibrin 14 CLINICAL PATHOLOGY formation is slight and blood platelets are very scanty. The leucocytes are much increased, being commonly about 60,000 per c.mm., a number intermediate between that found in acute inflammation and in myeloid leukaemia. The red cells and haemoglobin are as a rule greatly diminished, and often to the extent found in pernicious anaemia. The colour index not infrequently remains rela- tively high, and may be above the normal. The stained film is very characteristic, almost the entire leucocytes consisting of lymphocytes. The lymphocytes are in the majority of cases of the small variety, but differ in some respects from those of normal blood. They are as a rule larger than the normal cell, but retain the relatively small proportion of cytoplasm to nucleus. The cytoplasm has the same staining reaction as that of the normal cell ; the nucleus is as a rule atypical, taking the basic dye less deeply than normal and being frequently notched or indented after the fashion of the nucleus of the large hyaline. Less commonly the lymphocyte is of the large type, and both varieties, together with intermediate forms, may occur in the same film. Some authorities consider these atypical cells to be non-granular myelocytes produced in the bone marrow and not of lymphoid origin. The relative proportions of large and small lymphocytes may fluctuate greatly from day to day. The total lymphocytes usually comprise from 90 to 99 per cent, of all the leucocytes. The red cells as a rule show the changes usual in any severe secondary anaemia, but nucleated red cells are frequently absent. Both normoblasts and megaloblasts, may however, be found, particularly after a severe haemorrhage. The most important of the blood changes in lymphoid leukaemia consist in a great increase in the total number of leucocytes accompanied by an almost complete replacement of the normal cells by atypical lymphocytes, usually of the small variety. The variety of lymphocyte present is no guide to the acuteness of the disease. The blood changes are practically diagnostic ; it must be remembered, however, that a very high degree of lymphocy- tosis is known to occur in quite small children, apart from this disease. The blood of children below the age of 5 has normally a relatively high number (40 to 50 per cent.) of small lymphocytes, and these cells are the ones most liable to PLATE IV. Acute Lymphoid Leukasmia. (Lymphocytes of Small Type.) (Leishman's Stain.) Chronic Lymphoid Leukasmia. (Lymphocytes of Large Type ; from a case of 3 years' duration.) (Leishman's Stain.) PLATE IV. NOKMAL BLOOD— PRIMARY BLOOD DISEASES. 15 increase in several varieties of diseased conditions, and parti- cularly during an attack of whooping cough. Chronic lymphoid leukaemia is an extremely rare con- dition, resembling in its clinical aspects nryelaeinia rather than lymphgemia. The spleen may be greatly enlarged and the glands little if at all affected. The blood condition resembles that of the acute disease, and the lymphocytes may be of the small or of the large variety. Chloroma is in all probability a variety of acute lymphoid leukaemia, and the blood picture may be identical. Less com- monly the blood changes are myeloid in character. In addition to the clinical features of lymphgemia the patient presents a peculiar greenish coloration of the skin, together with evidence of subperiosteal swellings, most numerous on the bones of the skull. Bilateral proptosis is usual. The sub- periosteal infiltrations are of a bright green colour, which fades on exposure to the air. The green pigment is not present in all cases, and is not confined to this condition. It may be found in the lymph glands in some cases of myelaemia. Chloroma is regarded by some authorities as a form of new growth of the nature of a sarcoma, and the blood condition is considered to be due to a leakage of the malignant cells from the tumours into the circulation. In the usual forms of sarcoma no such leakage of tumour cells appears to occur. The disease seems to affect especially children of the Hebrew race. Summaey. An acquaintance with the normal blood is essential before undertaking the diagnosis of disease, and students are advised to examine the blood of patients not suspected of blood changes in order to appreciate the variations in the normal. The blood of normal individuals differs just as the normal breath sounds differ, and an acquaintance with these variations renders the recognition of diseased conditions certain. The derivation of all leucocytes from cells of one type in the adult is possible but not proven. The simplest classification of the normal leucocytes is into the non-granular mononuclear cells of the lymphoid system and the polynuclear cells of the myeloid system. The primary anaemias are diseases associated with striking 16 CLINICAL PATHOLOGY. changes in the blood of which the causes are unknown. With the exception of chlorosis these are rare diseases. The diagnosis in all of them can be made with tolerable certainty by means of an examination of the blood, provided that the fluctuating nature of the blood changes are recognised and that every advantage is taken of the clinical information derived from the patient. CHAPTER II. THE SECONDARY BLOOD CHANGES — THE BLOOD CHANGES OCCURRING IN CHILDREN. A description of the changes which may be found by means of an ordinary blood examination in every known disease would be obviously impossible, and indeed is unnecessary, since similar pathological conditions underlie different diseases and give rise to similar changes in the blood. The secondary blood changes and their diagnostic signifi- cances are conveniently described under the following five headings, which denote the predominant change associated with various conditions : — (1) An increase in the number of white cells, or leucocytosis. (2) An increase in the number of eosinophil cells, or eosinophilia. (3) A decrease in the number of white cells, or leucopenia. (4) An increase in the number of red cells, or polycythemia. (5) A decrease in the number of red cells, or oligocythemia. (1) Conditions associated with a leucocytosis. Acute inflammation is the most important underlying cause of an increase in the white cells in the secondary blood diseases. A typical example of the acute inflammatory process set up by one of the pyogenic organisms is met with in lobar pneumonia. The blood changes present in this con- dition are as follow : — The blood clots readily, and an increase in the blood platelets, together with an excessive fibrin formation, is seen in the fresh blood. A comparatively mild secondary ansemia is usually present. The leucocytes are markedly increased, often up to 20 or 30,000 per c.mm. The number of the leucocytes does not fall with the temperature, but gradually diminishes as p. 2 18 CLINICAL PATHOLOGY. resolution occurs in the lung. The stained film (Plate II., yields the following differential count : — Polynuclear neutrophils . . 80 to 95 per cent. Eosinophils .... absent. Large hyalines . . . . 5 to 10 per cent. Small and large lymphocytes . 2 to 10 per cent. The leucocytosis is seen to be due mainly to an absolute and relative increase in the polynuclear neutrophils and to a lesser degree to an absolute increase in the phagocytic hyaline cells. The complete, or almost complete, absence of the eosinophils is a very constant feature, and the reappearance of these cells is an indication that the inflammatory process is undergoing resolution. Very occasionally in acute inflammation there is no increase in the leucocytes, and they may even be diminished ; the relative proportion of the leucocytes, however, is altered in the manner described above. In a fatal case of extensive sup- puration in the bile passages the total number of leucocytes was only 3,000 per c.mni., but the polynuclear neutro- phils formed 90 per cent, of the white cells. Such a blood picture occurs in a patient whose protective mechanism is failing to react to the infection, and is of the gravest prognosis. The blood changes of lobar pneumonia are similar to those in all acute inflammatory processes set up by any of the pyogenic organisms, such as the streptococci, staphylococci, colon bacillus and the like, and are of assistance in arriving at a clinical diagnosis. In a case of doubtful appendicitis an inflammatory blood count means that there is acute inflamma- tion in the body, not necessarily in the appendix. It does not mean that actual suppuration has taken place and that immediate surgical interference is necessary on that account. The processes of acute inflammation and suppuration differ only in degree, and so do the changes in the blood. When pus is pent up in the body the leucocytes tend to increase ; w r hen inflammation is resolving the white cells diminish and the eosinophils reappear. A rising leucocyte count in a case of appendicitis is evidence of abscess formation ; a falling count with a diminution in the relative number of polynuclear neutrophils is evidence of resolution. The clinical value of THE SECONDARY BLOOD CHANGES. 19 the blood examination therefore depends not only upon an enumeration of the total number of leucocytes present, but also upon an estimation of the relative percentage of the cells found in the stained blood, and in addition a series of examina- tions may be necessary. Fevers. — Among the pathological processes giving similar changes in the blood are certain fevers of unknown etiology, such as scarlet-fever, small-pox, chicken-pox and rheumatic fever. In carcinoma, particularly in carcinoma of the intestine, an inflammatory blood count is usual, and is probably due to the infective processes set up by the growth. In tuberculosis, in its usual and comparatively localised forms, the blood changes are very different, but in generalised tuberculosis, especially with wide spread glandular involvement, a well-marked inflammatory count is common. If a patient with general glandular enlargement is found to have a marked leucocytosis of the polynuclear variety the condition is extremely likely to be tuberculous. The presence of an inflammatory leucocytosis is always strongly suggestive of one of the above diseases, and helps to distinguish them as a group from any of the affections commonly associated with a leucopenia, such as influenza or typhoid fever. A leucocytosis occurring in typhoid fever indicates that some complication is present — for example, an infection of the bile passages. A continuous, high and rising leucocytic count suggests that an inflammatory process has gone on to pus formation. A low total count with a high relative number of polynuclear neutrophils is of grave prognosis. While an inflammatory leucocytosis is found with all acute septic lesions, an apparent exception must be referred to in tropical abscess of the liver. In this condition, particularly when latent, leucocytosis is frequently absent ; but it must be remembered that the " abscess " is in reality a chronic necrotic condition and does not contain pus. If infection by pyogenic organisms occurs in the necrotic liver a leucocytosis supervenes. (2) Conditions associated with an eosinophilia. Animal parasites are among the most important agents 2—2 20 CLINICAL PATHOLOGY. capable of producing an increase in the eosinophil cells. Any of the common intestinal worms may cause an eosinophilia, and in particular the ankylostoma, which produces in addition a severe anaemia of the secondary type. Trichinosis, hydatid disease, filariasis, and bilharzia disease also produce an eosinophilia. The increase in the eosinophil cells is usually accompanied by a leucocytosis, and the relative percentage of the eosinophils may vary from 5 to 60 per cent., or even more, so that the total increase in these cells may be very considerable. In the case of hydatid disease the eosinophilia may persist even when the cyst has become fibrous and partly calcined. With cysts which have become secondarily infected and have undergone suppuration the eosinophilia disappears. An eosinophilia is not invariable in these parasitic infections, and may be absent in uncomplicated cases. As an aid to diagnosis in doubtful cases, the presence of a well-marked eosinophilia is of value; a negative blood examination is of less significance. Skin lesions. — Eosinophilia is a condition associated with many varieties of affections of the skin. It is present in urticaria, in psoriasis, and in dermatitis herpetiformis. In the latter condition the majority of the cells present in the bullae may be eosinophils. In the specific fevers associated with skin eruptions, and already mentioned as being accompanied by inflammatory changes in the blood, a considerable eosinophilia is present in the early stages. In small-pox the number of eosinophils may be very high ; in chicken-pox and in scarlet-fever the condition is not so marked. Both in small-pox and in chicken- pox eosinophils are also present in the vesicles, but disappear from the blood and from the skin lesions when suppuration takes place. Blood diseases. — As already stated, the eosinophils are increased in myeloid leukaemia and to a less extent in per- nicious anaemia. Following excision of the spleen in animals Ehrlich found the eosinophils increased after a considerable period; a similar result has been noted to occur in human beings, but as a rule the relative increase in these cells is very slight. Spasmodic asthma is accompanied by an increase in the eosinophils of the blood and of the sputum. THE SECONDARY BLOOD CHANGES. 21 Spring catarrh, a rare affection of the eyes, is associated with an eosinophilia in the blood and with large numbers of eosinophil cells in the conjunctival discharge. Gonorrhoea is accompanied by a slight eosinophilia in adults. In children I have seen an eosinophilia of 40 per cent, in a case of gonorrhceal affection of the eye. There was no evidence of intestinal parasites in this case. Occasional eosinophils are seen as a rule in films made from the pus of a gonorrhceal discharge. (3) Conditions associated with a leucopenia. Chronic inflammation. — The blood changes associated with the chronic infective granulomata, tuberculosis, and syphilis are the reverse of those set up by the pyogenic organisms. The total number of leucocytes is diminished usually to between 3 and 4 thousand per c.mm., and the relative number of the lymphocytes is increased. The type of lymphocyte usually increased is the small lymphocyte, a cell identical with, or at any rate very similar to, the lymphoid cell found in the giant cell system of tuberculosis and in the serous effusions associated with tuberculous and syphilitic diseases. In addition to the leucocytic changes there may be a consider- able reduction in the number of red cells and in the percentage of haemoglobin. The colour index is low. A typical blood examination would give the following result : — Red cells Haemoglobin Colour index 3,500,000 per c.mm. 45 per cent. 0-6 White cells . Differential count — 3,000 per c.mm. Polynuclear neutrophils Eosinophils Large hyalines Small lymphocytes Large lymphocytes 40 per cent. 3 4 40 13 100 In acute general tuberculosis, and particularly in wide- spread tuberculous lymphadenitis, the blood changes are 22 CLINICAL PATHOLOGY. frequently those of acute inflammation, as has been mentioned under that heading. Fevers. — Certain fevers are associated with similar blood changes, the more important being typhoid fever, influenza, malaria, and measles. The predominating lymphocyte in typhoid and in malaria may be of the large type. In a febrile case of doubtful nature a complete white cell examination may be of considerable assistance in diagnosing between the acute inflammatory and the non-suppurative affections, as, for example, between a pneumococcal infection and influenza. Pseudo-blood diseases. — By these are meant certain diseases of unknown etiology which affect the hsemopoietic system and which do not give rise to any characteristic changes in the blood, other than the secondary anaemia associated with a leucopenia and a relative lymphocytosis common to so many conditions. These diseases include Hodgkin's disease, or lymphadenoma, splenic anaemia, and Banti's disease. In none of these affections can a diagnosis be made from the results of a blood examination alone. In Hodgkin's disease the general glandular enlargement, together with the increase in the size of the spleen, may lead to a diagnosis of lymphoid leukaemia, and this can be negatived at once by a blood examination. The two conditions, how- ever, have less clinical similarity than might be expected. In lympho- sarcoma and in glandular tuberculosis there is no constant blood change capable of distinguishing the glandular enlargements from those of Hodgkin's disease. A large number of nucleated red cells with the appearance of myeloid leucocytes would be in favour of sarcomatous or carcinomatous glands with other deposits in the bone marrow. The presence of a high polynuclear leucocytosis would be in favour of tuberculous disease. A moderate eosinophilia is not uncommon in Hodgkin's disease. The only certain method of diagnosis, however, consists in the removal of a gland for histological examination, a proceeding which in the case of one of the discrete and superficial glands may readily be carried out under local anaesthesia. Splenic anaemia is, on clinical grounds alone, difficult to distinguish from myeloid leukaemia, but the latter disease can be recognised at once by a blood examination. In splenic THE SECONDARY BLOOD CHANGES. 23 anaemia the red cell and haemoglobin loss may not be great, but the diminution in the number of leucocytes is more marked than in any other condition. The white cells may fall below 1,000 per c.mni. and are commonly between 2,000 and 3,000 per c.mm. Such a blood condition associated with great enlargement of the spleen is scarcely seen in any other disease, with the possible exceptions of kala-azar and malaria. Banti's disease differs from splenic anemia in the additional association of cirrhosis of the liver, together with haematemesis, jaundice, and ascites in the later stages. Banti's disease is usually met with in young adults, splenic anaemia in older people ; but the two diseases are not clearly differentiated, and the two terms may apply to two stages of the same disease or may embrace a variety of different affections. The blood changes are identical. In addition to the diseases already enumerated, this type of leucocytic change is also met with in numerous conditions which affect more noticeably the red cells and haemoglobin. A leucopenia with a relative lymphocytosis has already been stated to occur in chlorosis and pernicious anaemia among the primary blood diseases ; they are also found in the anaemias of malignant growths, among workers in metallic poisons, and among the subjects of malignant malaria and other tropical diseases. (4) Conditions associated with an increase in the number of red cells (polycythaemia or erythraemia). It must be remembered that the normal number of red cells in a healthy male adult is commonly about 6 million per c.mm. and that any number in the neighbourhood of this figure does not constitute polycythaemia. The red cell estimations quoted in some text-books as examples of polycythaemia are practically those of normal persons. The number of red cells is capable of accommodation to circumstances, and is increased at high altitudes, during starvation, and temporarily after the removal of large quantities of fluid from the body, as after tapping an ascitic abdomen. Polycythaemia is accompanied by an increase in the haemoglobin percentage, and is found in the following morbid conditions : — Cardiac failure, accompanied by cyanosis and general 24 CLINICAL ' PATHOLOGY. venous stasis, leads to an increase in the number of red cells in the peripheral circulation. The number frequently varies between 7 and 8 million per c.rnm. Congenital morbus cordis is almost invariably accom- panied by a considerable polycythemia, the number of red cells being commonly above 8 million. Polycythemia may be present when the cyanosis is by no means marked ; it is temporarily reduced by bleeding. Splenic polycythsemia (Erythremia : Osier's disease : Vaquez's disease) is conveniently considered here, although the marked alterations in the blood and our complete ignorance as to the cause of the disease would justify its classifica- tion among the primary blood diseases. It is a comparatively rare affection. The condition is a chronic one, attacking people usually of middle age, and the most noticeable clinical features in an advanced and typical case are the striking plum-coloured complexion and the great enlargement of the spleen. The blood obtained on puncturing the ear is almost black in colour, and so sticky that it is difficult to obtain sufficient for a count without pressure and impossible to spread tbin films of it. If the blood is taken into a test tube and centri- fuged the red cells are found to almost fill the serum, leaving only a small layer of supernatant fluid. The actual volume of blood is greatly increased. The number of red cells is commonly about 10 million per c.mm., and may reach 12 million. The percentage of hemoglobin may be 130 or over. The leucocytes are increased to about double the normal number, and there may be some relative increase in the poly nuclear neutrophils. (5) Conditions associated with a decrease in the number of red cells (oligocythemia). A decrease in the number of red cells is invariably associated with a decrease in the percentage of hemoglobin, and it is this blood condition which is commonly referred to by the loose clinical expression "anemia." If the unqualified term " anemia " is used at all it must be applied to designate a physical sign and never as the diagnosis of a disease. The clinical recognition of the anemic state is apparently impossible. Nothing can be more fallacious than the common THE SECONDARY BLOOD CHANGES. 25 idea that the colour of a patient's face, or even of his lips and conjunctivae, is any guide to the extent of his anaemia. I have known a sallow-complexioned constipated Hebrew with 6 million red cells per c.mm. and 105 per cent, of haemoglobin diagnosed as pernicious anaemia, and I have frequently fallen into the error of expecting in a patient a considerable reduction in the red cells and have found little or none. The erythrocytic mechanism of an adult in ordinary health is very evenly regulated, and any serious drop in the red cells or in the haemoglobin content is to be regarded as a definite indication of organic disease. The following are among the more important conditions associated with a diminution in the red cells and haemoglobin. Haemorrhage is the simplest and most obvious cause of blood loss, and is naturally followed at once by a proportionate loss in red cells and haemoglobin. The red cells formed with great rapidity to replace those lost are deficient in haemoglobin, consequently the colour index quickly falls. After a single haemorrhage the blood readily returns to normal, in periods varying with the amount of blood lost and the recuperative powers of the individual. The blood lost by a normal person during an operation attended with considerable haemorrhage should be replaced in from 2 to 3 weeks. Repeated small haemorrhages may lead to a considerable degree of anaemia, and if the bleeding has escaped observation the condition of the patient may come to resemble that of pernicious anaemia. The following may be given as an example of the condition in such a case. Fresh blood. — Rouleaux formation slight. Fibrin forma- tion normal or excessive. Poikilocytosis present : — Red cells 2,500,000 per c.mm. Haemoglobin 30 per cent. Colour index .. . ... ... 0*6 ,, White cells normal in number or increased. Stained blood. — Polychromatophilia present, but not extreme. Occasional normoblasts seen, but no megaloblasts. White cells often show some increase in the relative numbers of polynuclear neutrophils. Such a blood condition is found not only after haemorrhage, but in numerous conditions associated with " anaemia," and is of the kind referred to as of the chlorosis or of the secondary 26 CLINICAL PATHOLOGY. anaemia type. The state differs from that of chlorosis in that the red cells are commonly more affected and the colour index of necessity higher, though these differences may not be marked, and are of less consequence since the clinical recogni- tion of the disease chlorosis is rarely difficult. It is of great importance to distinguish this secondary type of anaemia from primary or pernicious anaemia. The main differences in the secondary type are the rarity with which the red cells fall below 2 million, a colour index less than 0*8, and the absence of megaloblasts. Metallic poisons. — Workers in arsenic, antimony, lead and similar metals may develop an anaemia of the secondary type. Basic granular degeneration of the red cells is said to be especially characteristic of lead poisoning. Granular degeneration is present rarely in other diseases accompanied by a secondary anaemia, and is seen in its greatest degree in pernicious anaemia. In cases of lead poisoning admitted to a general hospital I have hardly ever seen marked granular degeneration of the red cells. On the Continent, however, the presence and relative proportion of these cells is taken as evidence of the extent to which the lead- worker is affected. The Cachexias of carcinoma, tuberculosis, and syphilis are associated with this type of anaemia, and when the physical signs of these conditions are latent the discovery of serious loss in red cells and haemoglobin is sufficient indication for further investigations. Gout, diabetes, myxoedema, and Addison's disease are among the numerous chronic affections accompanied by erythrocytic changes. In addition, diseases associated with blood changes more especially affecting the leucocytes, and which have been already mentioned, are usually accompanied by red cell changes in addition. The anaemias due to intestinal parasites may be particularly severe. The Blood Changes occueeing in Childeen. Before considering the changes which may be found in the blood of infants it is necessary to appreciate that in children less than 5 years old the normal blood presents several differences from that of adults and tends to react excessively to abnormal stimuli. In infants the total leucocyte count is high and the THE SECONDARY BLOOD CHANGES. 27 lymphocytes relatively numerous. Nucleated red cells are present at birth, and persist for several months after birth. The blood readily reverts to the foetal type in disease, and remarkable fluctuations in the number and character of the cells such as would indicate the gravest disorders in an adult may have little serious meaning in an infant. The blood- forming mechanism in an infant has had much the same time to steady down as its heat-regulating centre, and both may be temporarily disordered by the cutting of a tooth. The extreme characters of the fluctuations in the blood of infants renders the pathology of the blood very obscure and any classification of the blood diseases of children almost impossible. The matter is further complicated by the comparative frequency with which the infantile spleen and lymph glands enlarge. Lymphoid leukaemia, Myeloid leukaemia, and Pernicious anaemia have all been described as occurring in infants, and it is possible that a very few of the reported cases are genuine. Such diagnoses should be made with the utmost reserve and never on the examination of a blood film alone. If that is to be taken as the criterion of our diagnosis, then I have seen an infant pass calmly through an attack of acute lymphoid leukaemia with nothing more deadly than a whoop and another child shake off in rapid succession the onslaughts of myeloid leukaemia and pernicious anaemia. The blood changes of these diseases are better regarded as types of the changes which may accompany some of the known affections of childhood. The blood changes of infants may be divided into : — (1) The primary blood diseases of infants. (2) The secondary blood diseases of infants. (1) The primary blood diseases of infants. Splenic anaemia of infants (Von Jaksch's anaemia : Anaemia infantum pseudoleukaemica) . — This is a disease affecting quite young children and accompanied by great enlargement of the spleen and moderate enlargement of the liver. The affected child may recover completely, or may die, or may improve and be left with a large spleen and subse- quently come into the category of Banti's disease. It is still a matter of dispute as to whether the disease constitutes a 28 CLINICAL jPATHOLOGY. clinical entity or is a condition secondary to rickets or congenital syphilis. Undoubted cases are, however, met with in which no evidence of either disease can be found and in which the Wasserrnann reaction is negative. Such cases present a fairly typical clinical picture and a remarkable, if somewhat varied, blood condition, so that it is reasonable to describe them for the present as examples of the primary anaemias of children. The main features of the blood condition are as follow: — The red cells and haemoglobin percentage are greatly reduced, the former to a number between 1 and 2 million per c.mm., the latter to from 10 to 30 per cent. The white cells are increased often to a marked degree, and frequently number from 30 to 40,000 per c.mm. The stained film is very remarkable, and may display every known kind of blood cell in considerable numbers. Nucleated red cells are always present, and usually in excessive numbers ; 200 may be seen while counting 500 leucocytes. Normoblasts are greatly in excess of megalo blasts. The relative percentage of the leucocytes is very variable, and the predominant cells may be lymphocytic, but are more frequently myeloid. Myelocytes, especially the small type of neutrophilic myelo- cyte, are numerous. More rarely a great enlargement of the spleen may be associated with extreme red cell changes, a high colour index, and little or no change in the leucocytes. Congenital family cholsBmia (Acholuric family jaundice) is a rare and interesting hereditary condition occurring in several members of the same family, and associated with enlargement of the spleen, the presence of bile in the serum, and the usual absence of bile from the urine. The blood in the course of an ordinary examination shows nothing more than a moderate secondary anaemia and the presence of occasional nucleated red cells. The erythrocytes are in reality remarkable by reason of their abnormal fragility. This fragility is demonstrated by the ease with which the cells are haemolysed in hypotonic salt solutions. Normal red cells retain their haemoglobin when placed in solutions of sodium chloride of 0*4 per cent, or less ; the red cells in this disease are haeruolysed in salt solutions approaching the strength of normal saline. Haemophilia is a comparatively rare hereditary disease THE SECONDARY BLOOD CHANGES. 29 transmitted by unaffected females to males and characterised by extensive haemorrhages following trifling causes. Affected children rarely reach the age of maturity. Haemophilia is commonly classified among the purpuras, with which it has probably nothing in common. The essential known morbid change is the alteration in the coagulation time of the blood. The normal coagulation time of the blood as estimated by Wright's method (page 46) is very constant, and is almost invariably in the near neighbourhood of 3| minutes. In haemophilia the time is greatly prolonged, sometimes to as much as half an hour. The coagulation time diminishes after severe haemorrhage or after calcium salts have been given by the mouth. Hemophilics can be distinguished from other patients who may be the subjects of excessive haemorrhage by this marked retardation in the coagulation of their blood. The ordinary blood examination in haemophilia shows nothing characteristic, and the red cells are not excessively fragile, as in acholuric family jaundice. (2) The secondary blood diseases of infants. Rickets and Congenital syphilis are the two most important primary conditions capable of producing marked alteration in the blood of children. A considerable enlarge- ment of the spleen and liver is frequently associated and a condition produced which may be very similar to that of splenic anaemia infantum. The degree of anaemia may be marked and nucleated red cells numerous. In some cases the leucocytes are greatly increased, in others there may be a leucopenia, with a relative lymphocytosis. The blood changes are very varied and rarely present to the degree seen in splenic anaemia, from which the secondary blood diseases are to be diagnosed, partly by the minor enlargement of the spleen and the less obvious blood alteration, but mainly by a recognition of the originating factor. A relative lymphocytosis is common in many affections of childhood, including tuber- culosis, and more especially whooping cough. In these diseases there may be a marked increase in the total leucocytes and a blood picture produced very similar to that of lymphatic leukaemia. The lymphocytes present are usually identical with the small lymphocytes of normal blood, and have a deeply-staining round nucleus. 30 CLINICAL PATHOLOGY. Infantile scurvy (Barlow's disease) is usually associated with a severe anaemia of the secondary type and as a rule with an inflammatory leucocytosis. Purpura in its various forms is not confined to children, but occurs in them more commonly than in adults. The most extensive purpura is not accomj)anied by any characteristic changes in the blood. As a rule there is a mild secondary anaemia and the leucocytes are unaltered. The coagulation time is normal, and there is no increased fragility of the red cells. The serum usually has the property of agglutinating the red cells of unaffected individuals. If the serum of a purpuric individual is added to the washed red cells of a normal person the red cells are thrown into tight clumps similar to the clumps of typhoid bacilli in a positive Widal reaction. The purpuric red cells are not agglutinated by the serum of a similar case. Hemagglutinins in the serum are of scientific rather than of practical interest, and red cell agglutination of the nature usually found in purpura may be present in a variety of conditions and occasionally in apparently healthy individuals (page 58). Summary. The secondary blood changes of adults may be divided into those which mainly affect the leucocytes on the one hand and the red cells on the other. The leucocytic changes are of two main types, namely, the leucocytosis, usually polynuclear in character, set up by the pyogenic organisms, and the leucopenia, with a relative lymphocytosis which accompanies the non-suppurative infections. To these must be added the small group of disorders associated with an eosinophilia. The erythrocytic changes are similarly two. An increase in red cells and haemoglobin is comparatively rare ; a decrease is met with in the great majority of serious and prolonged diseases, and is always an indication of notable organic disturbance. The fluctuations of the blood-forming mechanism in infants are difficult to classify and to interpret owing to the relatively foetal condition of the infantile blood in health and to the excessive response of the mechanism to morbid stimuli. The main changes of the primary blood diseases of adults THE SECONDARY BLOOD CHANGES. 31 may temporarily appear in infants, the diseases themselves very rarely indeed. The condition known as splenic anasmia infantum appears to be a clinical entity, and may be regarded as such in spite of the fact that very similar blood changes may be set up by other conditions, and particularly by rickets or congenital syphilis. CHAPTEE III. THE METHODS OF EXAMINING THE BLOOD. The ordinary routine examination of the blood includes the following investigations : — (1) The unstained blood. (2) The percentage of haemoglobin. (3) The estimation of the number of red cells to the cubic millimetre. (4) The estimation of the number of leucocytes to the cubic millimetre. (5) The stained blood. The essential apparatus comprises : — A microscope. A haenioglobinometer. Blood pipettes and diluting fluid. A haemocytometer. A special blood stain. Slides and cover glasses. A dry swab, ether, and a surgical needle. The majority of these materials are described under their appropriate headings. The microscope is conveniently referred to here. The microscope. — A thoroughly reliable instrument is necessary, not only for the examination of the blood, but for numerous other pathological investigations in common use. For a student who is not hampered b}^ motives of economy or by the previous possession of an inferior microscope the choice is not difficult, if he bears in mind the following points. The microscope should have a firm base, should be provided with a diaphragm and Abbe condenser, with a mechanical stage, with a triple nose-piece for objectives having the focal distances of f , -jt, and ^ inch, and with at most three eye- pieces No. 1 (X 4 diameters), No. 2 (X 6 diameters), No. 4 ( X 10 diameters). The low-power eye-piece should be used for all ordinary work. The mechanical stage should be built THE METHODS OE EXAMINING THE BLOOD. 33 into and form part of the framework of the microscope ; it need not necessarily be graduated. Thoroughly reliable microscopes are manufactured by several of the leading English firms, and there is no necessity to obtain an imported instrument. The ^ inch objectives of home manufacture however, are often inferior to those made by Zeiss, and the additional cost of the Zeiss lens is worth the outlay ; a Zeiss J inch objective is a further but not essential advan- tage. The best English microscope fitted with a Zeiss T X 2 inch objective costs about £21. If necessary the triple nose-piece can be dis- pensed with and a double or even single nose- piece used. A mechani- cal stage can be fitted to almost any microscope, but is seldom entirely satisfactory, since the numerous patterns in use require constant attention and easily get out of order. The stage is a great convenience, but is not absolutely essential, unless Strong's method of counting the leucocytes is used. To obtain the blood. — Rub the lobe of the ear lightly with ether and allow it to dry. With as rapid a movement as possible make a deep puncture with a surgical needle. The novice is apt to deliberately press the needle into the ear, giving the maximum amount of pain and obtaining the minimal quantity of blood. By a rapid stab sufficient blood can be obtained from a sleeping infant without awakening it. The best needle to employ is the ordinary straight Hagedorn No. 9. It should be sterilised before use by rapid passage p. 3 Fig. 1.— The Microscope. 34 CLINICAL PATHOLOGY. several times through a flame. The specialised instruments of torture provided with nearly all forms of blood apparatus should be avoided. The drop of blood must be allowed to flow out, and should never be squeezed out, since anj r pressure upsets the equilibrium between cells and plasma. Sufficient blood can rarely be got from the finger without considerable pressure ; it can always be obtained from the ear. The unstained blood. — Place a cover-glass with one edge flush with the edge of a slide. Hold the two lightly in apposi- tion with the thumb. Place the apposed edges against a drop of blood and the blood will flow between slide and cover-glass. Examine within 10 minutes or so, using the J inch objec- tive. All the available light should be thrown through the condenser, and the diaphragm should then be closed down until the corpuscles stand out clearly. If the diaphragm is left widely open the cells cannot be seen at all. Observe the extent of rouleaux and fibrin formation, the collections of blood platelets, the shape and size of the red cells, and the relative proportion of white to red cells. Gross changes in the blood such as occur in the leukaemias can be recognised at once by this simple process ; some of the minor changes can be observed in no other way, and it should never be omitted. Spirilla, filaria, and trypanosomes can be readily recognised if present. Malarial parasites are more certainly identified with a j 1 ^ inch objective ; the eye is attracted to them by the active dancing of their pigment granules. The hemoglobin. — Some form of apparatus is necessary for the estimation of haemoglobin, the blotting paper method of Tallqvist is too unreliable for anything more than an approximate reading. Haldane's modification of Gower's instrument is the most convenient hsemoglobinometer for general use. The standard of comparison in this form of hsemoglobinonieter consists of a tube containing a 1 per cent, solution of blood having the average percentage of haemoglobin found in the blood of a healthy adult man, and which has been saturated with carbonic oxide. A measured quantity of the blood to be examined is placed in a similar graduated tube, saturated with carbonic oxide and diluted until it matches the standard tube in colour. The readings obtained by this instrument are with THE METHODS OF EXAMINING THE BLOOD 35 practice very exact, and it is claimed that the error does not exceed 1 per cent. ; it should not exceed 5 per cent., and that Fig. 2. — The Normal Unstained Blood. Fig. 3. — The Unstained Blood in Myeloid Leukaemia. 3—2 36 CLINICAL PATHOLOGY. is all that is necessary for ordinary purposes. The instruc- tions for use are supplied with the instrument and are briefly as follow : — 20 c.mm. of blood are drawn up into the pipette and the end of the pipette is wiped. A small quantity of distilled water having been placed in the graduated tube the blood is blown into this, the pipette rinsed up and down with the water and withdrawn. A piece of rubber tubing is affixed to a gas jet by one end and the other end is passed into the tube to near the level of the mixture of blood and water. The gas is allowed Pig. 4. — Haldane's Hteraoglobinometer. to pass for a few seconds, the rubber tube is drawn out with the gas still escaping, the end of the graduated tube closed with the ringer, and the tube slowly inverted several times. The diluted blood saturated with carbonic oxide is then com- pared with the standard tube and carefully diluted further until the colour in the two tubes is exactly matched. The level of the diluted liquid gives the percentage of the haemo- globin. In matching the colour the tubes should be held against the light from the sky and frequently transposed. Oliver's haemoglobinometer as modified and manu- factured by the Tintometer Co., Ltd., of Salisbury, is a more THE METHODS OF EXAMINING THE BLOOD. 37 expensive and somewhat cumbersome apparatus. It is, how- ever, accurate, simple to use, and does not require a supply of coal gas. The tintometer consists of a wooden box illuminated by a candle, and is provided with a small capillary pipette, a mixing chamber, and a long sliding scale containing a series of tinted discs graduated in percentages. The method of use is as follows : — Fill the capillary pipette by holding the fine end against a drop of blood. No suction is required. Wipe the end of the pipette and slip over it a small piece of rubber tubing attached to an ordinary glass pipette rilled with distilled water and provided with a rubber teat. Blow out the blood into the mixing chamber together with sufficient water to exactly fill the chamber. Stir the mixture with the metal handle of the pipette and cover the chamber with the piece of glass provided. If the chamber is accurately filled, one small air bubble rests between the glass and the drop and does not interfere with the observation. Place the chamber in the box, light the candle and shut the door of the box. Slide the graduated scale into the box, and, shading the observing eye with the hand so as to cut off all external light, move the scale in and out until the disc is found which exactly matches the colour of the diluted blood in the chamber. The number of the disc gives the percentage of haemoglobin in the blood. By means of the graduated rider provided to fit over the scale the percentage can be read to within 5 per cent. (Both the above-described instruments are provided by Messrs. Hawksley, of Oxford Street.) The estimation of the number of red cells.— (1) Strong's method (modified). — The necessary apparatus consists of two graduated pipettes, a mixing bottle, a diluting fluid, and a Thoma-Zeiss hamiocytometer. The pipettes are graduated to hold 995 c.mm. and 5 c.mm. respectively. The 5 c.mm. tube has two marks close together ; from the upper mark 5 c.mm. are delivered, at the lower mark they are con- tained. The mixing bottle holds just over 1,000 c.mm., and is provided with a well-fitting stopper. The diluting fluid has the following composition : — •85 gramme sodium chloride. •85 gramme sodium citrate. Commercial (40 per cent.) formalin 1 c.c. Distilled water to 100 c.c. 38 CLINICAL PATHOLOGY. The method of use is as follows : — 995 c.mm. of diluting fluid are measured into the mixing- bottle : 5 c.mm. of blood are drawn up to the lower mark in the small pipette. The end of the tube is wiped, placed in the bottle, the blood blown out, and the pipette rinsed up and down with the mixture of blood and fluid. The mixing bottle is corked, and can be kept until it is convenient to count the cells. The red cells sink to the bottom, and before counting the bottle must be thoroughly shaken. After shaking, a drop Fig. 5. — Strong's Pipettes and Mixing Bottle. of the mixture is transferred to the centre disc of the haerno- cytometer by means of the stopper or a glass rod. The cover- glass is gently lowered on to the drop and stroked down on the haemocytometer with the handles of two mounted needles until circles of coloured rings (Newton's rings) appear between slide and cover-glass. The rings are best seen by holding the slide up to the light and looking down at it in a slanting direction from a little distance above. The size of the drop when flattened out in this manner must be sufficiently large to nearly fill the central platform of the counting slide, but not so large that any of it flows into the trench surrounding the platform. No air bubbles should be present in the drop. The slide is allowed to stand for a few minutes while the cells settle down, and is then placed on a microscope fitted with a No. 2 eye-piece and a \ inch objective. The light is thrown through the condenser, and the diaphragm is shut THE METHODS OF EXAMINING THE BLOOD. 39 down in the same manner as when examining the fresh blood. The microscope must be vertical, otherwise the red cells settle towards the lower part of the slide. The occasional leucocytes which may be present are readily distinguished from the red cells by their shape, colour, and the usual presence of refractile granules. The slide is moved until the ruled area of the central disc is found. This area is ruled in small squares, every fifth square in either direction having a line drawn through the middle of it. The area is in this manner marked off into 16 large squares, each of which contains 16 small squares bounded on every side with small double- ruled squares. A large square occupies a field of the microscope. In counting the red cells only those in the single-ruled squares are considered, and as a minimum all the red cells in 4 large squares, each consisting of 16 small squares, are counted. In cases in which the red cells are much diminished all the 16 large squares should be counted. A certain number of the red cells will be found to impinge on the lines bounding the squares; such cells should only be in- cluded when lying on the left- hand and bottom lines of the squares. The average number of red cells per large square in health is about 120. In order to calculate the number of red cells to the cubic millimetre it should be remembered that the dimensions of a small square are ^oo of a cubic millimetre and that the blood has been diluted 200 times. The number of red cells to the cubic millimetre will therefore be 4000 X 200 X- the average number of red cells per small square. If 400 red cells are counted in 4 large squares containing 64 small squares the number of red cells to the c.mm. of undiluted blood would be 4,000 X 200 X ^r° or 5,000,000. (2) The Thoma-Zeiss method. — The apparatus consists of a combined pipette and mixing chamber, a diluting fluid, and a hsemocytometer. The diluting fluid employed may be ,}-. «, ' ] ■> TT'TT^ *PT °= T 7™t"t; Y o ° ° ;• :••"„:/!' .y. :• f : V .%• te {•. °i y; .v. "/."'•:"*. °' °°° 'T o o ° o o v- >l°i : ' '«" " kvj: •f;.;.; °° - -- ™ J ° ° c Fig. 6.— Field of Hfemo- cytometer. 40 CLINICAL PATHOLOGY. the same as that described under Strong's method, or a mixture may be used which contains a stain for differen- tiating the leucocytes. The staining mixture commonly employed is that of Toison, and has the following com- position : — Methyl violet .... 0'25 gramme. Neutral glycerine .... 30 c.c. Distilled water .... 80 c.c. Add to this a solution of : — Sodium chloride . . . . 1 gramme. Sodium sulphate .... 8 grammes. Distilled water .... 80 c.c. Filter the mixture. To make the dilution draw up the blood in the pipette to the mark 0'5. Wipe the end of the pipette. Place the pipette in a bottle of the diluting fluid and draw up the fluid to the mark 101. Eotate the tube vigorously until blood and fluid are thoroughly mixed in the bulb of the pipette. The blood in the mixing bulb is now in a dilution of 1 in 200. Blow out the fluid in the capillary part of the pipette, also a few drops of the diluted blood in the bulb, and transfer a drop to the platform of the counting slide. Proceed as described under Strong's method. The disadvantages of this method are that it is difficult to be sure of a thorough mixing of the blood and fluid in the bulb, and that the contents of the pipette tend to leak out and necessitate the immediate counting of the cells. In Strong's method the pipette is more easily manipulated, the mixture is readily transported and can be counted at leisure, and the same mixture can be used without further apparatus for an enumeration of the leucocytes. The estimation of the number of white cells. — (1) Strong's method (modified).— The apparatus required is the same as that for the red cells. The same mixture of 5 c.mm. of blood with 995 c.mm. of diluting fluid in a mixing bottle is employed. After thoroughly shaking the bottle draw up the mixture of blood and diluting fluid to the upper of the two marks on the 5 c.mm. pipette. Wipe the end of the pipette. Place a clean slide on the bench and hold the pipette vertically to the slide with the end of the pipette just resting on the centre of the slide. Blow out the fluid in the form of THE METHODS OF EXAMINING THE BLOOD. 41 a drop, lifting the pipette and ceasing to blow just as the last portion of the fluid falls out. Allow the drop to dry. Filter hasniotoxylin on to the slide for 5 minutes. Wash in tap water for 3 minutes. Wipe off excess of water and allow the drop to dry. Do not blot dry. When dry mount in cedar wood oil with a cover slip. (Instead of hematoxylin, Leishman's stain, or any simple nuclear stain, such as carbol thionin, may be used.) Place in the eye-piece of the microscope a flat round metal disc with a central square aperture f of an inch square, or, failing this, make a square hole in a piece of visiting card cut to the size of the eye- piece. All that is required is to obtain a square field for counting the leucocytes in a round drop. Use the £ inch objective. Observe that the nuclei of the leucocytes are stained blue and the red cells are practically unstained. The edge of the drop is clearly defined. To count the leucocytes find the top segment of the drop and move the drop across the field from one side to the other. When the other side is reached mark a red cell on the bottom line of the square and move the drop down exactly 1 square field. Continue moving the drop backwards and forwards across the field until the entire drop has been covered and all the leucocytes have been counted. The number of white cells counted in a normal case would be about 150. The dilution of the blood is 1 in 200, and 5 c.mm. of this dilution have been counted. The number of leucocytes in 1 c.mm. of the undiluted blood would therefore be 150 multiplied by — — -, or 6,000. In cases of leukaemia with an excessive number of white cells the enumeration of the leucocytes in a drop of blood diluted 200 times is too laborious, and a further dilution of the blood is advisable. The further dilution may be made with a Wright's capillary tube (see page 51) in the following way. Make a mark on the tube and draw up to the mark 1 volume of the 1 in 200 dilution and 9 volumes of the diluting fluid. The blood is now diluted 1 in 2,000 times. Blow out the mixture into a watch glass, mix thoroughly, draw up 5 c.mm. of the mixture to the upper mark of the Strong's pipette and proceed as before. The number of leucocytes counted will have to be multiplied by 400 instead of by 40. (2) The Thoma-Zeiss method. --The apparatus required 42 CLINICAL PATHOLOGY. consists of a special pipette, a diluting fluid and a hsemo- cytometer. The diluting fluid consists of a 0"3 per cent, solution of acetic acid in distilled water with sufficient methyl green added to give the fluid a distinct green colour. The acetic water dissolves the red cells and the methyl green stains the nuclei of the leucocytes. The blood is drawn up to the 0"5 mark on the pipette and the diluting fluid to the 11 mark. The pipette is manipulated as described under the enumera- tion of the red cells, and the mixture is put up on the haBinocytometer slide in the same manner. The dilution of the blood is 1 in 20. All the leucocytes in the entire set of 16 large squares are counted. To calculate the number of leucocytes per c.mm. of undiluted blood multiply the average number of leucocytes per small square (i.e., the total number of leucocytes counted in the 16 large squares divided by 256) by 20 times 4,000. The average number of leucocytes counted in the entire set of squares is only abaut 20, and the possible error is considerable. The sole advantage of this method is that it is rather more rapid than Strong's method. For the enumeration of both red cells and white cells Strong's method has certain advantages over the Thoma-Zeiss technique. The latter method is described here because it is sufficiently reliable and is still widely used. To clean pipettes. — All blood pipettes should be cleaned immediately after use. It is sufficient to first suck water up and down the pipettes, then alcohol and then ether. If particles of blood or dust have lodged in the pipette these should first be removed with a thread of fine silver wire. If the blood has been allowed to clot in the tube immerse the tube in 33 per cent, acetic acid, removing the blood with a fine wire at intervals as it softens ; it may take a few days to remove a firm clot, but the process may be considerably hastened by using glacial instead of 33 per cent, acetic acid. If the fine end of a pipette is chipped or notched the pipette is broken and should be discarded. The stained blood. — The materials required are clean slides or cover- slips and a blood stain. If cover-glasses are used for making the blood films it is essential that they should be clean and absolutely free from grease. The best quality of square cover-glasses should be THE METHODS OF EXAMINING THE BLOOD. 43 obtained and boiled in a wide evaporating dish or a sand bath for two hours in the following solution : — Sulphuric acid . ... . .60 c.c. Potassium bichromate . . .60 grammes. Distilled water .... 1,000 c.c. Fresh solution should be added from time to time as evaporation occurs, and the glasses should be occasionally stirred with a glass rod. The glasses should then be trans- ferred to distilled water and washed in it thoroughly with several changes. They should then be placed in absolute alcohol, and when required for use picked out with clean forceps and ignited in a Bunsen flame. Slides should also be of good quality, and can be cleaned in the same manner as the cover-glasses. It is quite sufficient, however, to first rub them with very fine emery paper (the best emery paper for the purpose bears the trade symbol " Hubert 0000 ") and then to place them in absolute alcohol. When they are required for use wipe them dry with a clean cloth and then warm them in the Bunsen flame to drive off the last trace of moisture. The blood stain employed should be either Leishman's or Jenner's ; both are excellent. Leishman's stain can be bought ready made up, but the majority of such solutions are un- satisfactory. It is advisable to buy the stain and the alcohol separately and to make up in the following manner : — Place 1 gramme of the stain (Grubler) in a clean, well -stoppered bottle ; add to it 100 c.c. of the best methyl alcohol. Shake well and stand in a dark cupboard. Shake every morning for a week and keep for at least one month before using. The stock bottle of stain made in this manner keeps indefinitely in ordinary climates, and when required for use is diluted with an equal volume of methyl alcohol, the strength of stain employed being then 0*5 per cent, in methyl alcohol. Jenner's stain can be bought in solution or made up as follows (Strangeways) : — Two stock solutions are kept, a 0'5 per cent, solution of eosin yellow shade (Grubler) in pure methyl alcohol, and a 05 per cent, solution of medicinal methylene blue (Grubler) in methyl alcohol. For use mix eosin solution . . • 12-5 c.c. methylene blue solution . 10'0 c.c In making up both this stain and Leishman's stain it is 44 CLINICAL PATHOLOGY. essential that all bottles and measuring glasses should be chemically clean. The stock bottles should have well-fitting stoppers and should be kept in a dark place. To make the blood films either slides or cover-glasses can be used ; the latter give excellent results in skilled hands ; slides give equally good results, and are to be recom- mended to those who are not in constant practice, since a bad film on a slide is like the curate's egg ; a bad film on a cover- glass is useless. To make a film on a slide place one end of the slide against the drop of blood, taking care not to touch the skin of the ear. Place the slide flat on a smooth firm surface, such as a polished Fie. 7. — To make a Blood Film on a Slide. table, and hold it in position with the thumb and first finger of the left hand. "With the right hand place the end of a second slide in the drop of blood and hold it there until the blood has run across the breadth of the slides. Draw the second slide slowly across the entire length of the first, maintaining an angle of about 45 degrees between the two slides. There should be no pressure whatever beiween the surfaces of the slides, and to ensure this the second slide should be held in the thumb and first finger of the right hand at about the level of their distal joints, the tips of the fingers being supported by the table. The more slowly the film is made the thinner the resulting film. The even spreading of the film is assisted by previously warming the slide in the flame of a spirit lamp. As soon as the film is spread the slide should be waved vigorously in the air to ensure the immediate drying of the film and thus avoid undue shrinkage of the cells. To make films on cover-glasses hold two glasses, one in each hand, by their corners. Place the centre of one glass THE METHODS OF EXAMINING THE BLOOD. 45 against the drop of blood. Apply the centre of the second glass to the drop on the first. Hold the two glasses together until the blood has spread across the glass. Eapidly separate the two glasses. The thinness of the resulting film depends upon the size of the drop of blood and on the rapidity and evenness with which the glasses are separated. If the glasses stick at all, the separation has been unduly delayed and the films are useless. To stain the film.— (1) With Leishman's stain. Cover the film well all over with the 0*5 per cent, stain and leave for 30 seconds. Add about twice the volume of distilled water to the stain. Mix stain and water with a glass pipette until an iridescent scum forms over the surface and leave for 7 minutes. Pour off the stain and cover the film with distilled water only for 2 minutes. Wash off the water with fresh distilled water, wipe clean the back of the slide, and gently blot the film dry with clean blotting paper. To preserve the film mount when dry in Canada balsam with a thin cover-glass. The film is fixed by the methyl alcohol in the first stage, it is stained during the 7 minutes of the second stage, and the colours are differentiated by the distilled water in the third stage. It is essential that the pipettes and beakers used should be first washed out with distilled water and that the water used should be distilled. The water must be neutral to litmus paper and must give no precipitate with silver nitrate. (2) With Jenner's stain. Cover the film with the stain and place a watch glass or inverted dish over the film to prevent evaporation. Stain for 4 minutes. Wash in distilled water until the film becomes a delicate pink tint. Blot dry. The differential count should always be made with a -J^inch objective and preferably with the help of a mechanical stage. Daylight should be used when available. Choose the thinnest and most even part of the film and count as a minimum number 200 leucocytes, tabulating on a piece 46 CLINICAL PATHOLOGY. of paper each leucocyte under its proper heading. In order to obtain the percentage of the leucocytes present in their relative proportions it is not necessary to count 200 consecutive cells so long as care is taken to avoid counting any cell more than once. Special Methods of Examining the Blood. The coagulation time. — A simple and accurate method of estimating the coagulation time of the blood is that of Wright. The necessary apparatus consists of a series of capillary tubes, elastic bands, a beaker, a jug of hot and a jug of cold water, a watch with a second hand, and a thermometer. The capillary tubes are of the same calibre and are provided with a 5 c.mm. mark. The procedure is as follows : — Clean the patient's thumb with ether. Wrap a piece of elastic tubing round the thumb from its base to nearly the tip. Puncture the tip of the thumb with a sterile surgical needle. Draw up blood to the mark on the pipette. (It is not essential to obtain the exact quantity of blood, slight variations in the amount being of less importance than rapidity in the manipu- lation of the tubes.) Note the exact time by the watch. Stretch a flat elastic band over the ends of the tube to prevent water entering. Stand the tube in the beaker filled with water at 37° C. Stir the water occasionally with the thermometer, and keep the temperature constant by adding hot or cold water. Prepare 3 or 4 more capillary tubes in the same way, numbering each tube and taking the time of each. At the end of 3 minutes take out the first tube and blow out the blood. Give the second tube 3 J minutes, and if the blood is still fluid give the third tube 4 minutes, and so on. The tube from which the blood fails to be expelled by blowing gives the coagulation time. The time is a very constant one for normal people of about 3^ minutes. The fragility of the red cells. — The examination of the action of various strengths of salt solutions on the red cells is rarely called for. It is described here because it is a simple proceeding and forms a part of the examination of the blood in acholuric family jaundice. No special apparatus is required. The necessary materials consist of 2 burettes, 10 large watch glasses, sodium chloride, distilled water, a THE METHODS OF EXAMINING THE BLOOD. 47 Wright's capillary tube provided with a rubber teat (page 51), a number of small tubes sealed at one end. The small tubes are readily made from a piece of glass tubing and should be about 2 inches long. The procedure is as follows. Make up exactly a 1 per cent, solution of sodium chloride in distilled water. Fill one burette with the salt solution, the other with distilled water. Eun into the first watch glass 0'9 c.c. of water and O'l c.c. of salt solution (= 0"1 per cent, salt), into the second 0"8 c.c. of water and 0*2 c.c. of salt (= 0'2 per cent, salt), and so on. A series of solutions from O'l per cent, saline to 1 per cent, is thus obtained. Deliver an equal volume of each solution into a double series of the small tubes. Stand the two series of tubes in order in a Petri dish rilled with " plasticine." Make a mark about 2 inches from the end of the Wright's pipette. Obtain blood from the patient's thumb in the same way as for the estimation of the coagula- tion time. Blow out a volume of blood into each tube in the series. Bepeat the process with the blood of a normal person and the second series of tubes. Invert each tube and stand the Petri dish in the incubator at 37° C. for 30 minutes. If no incubator is available the tubes may be stood in warm water or even left at room temperature. In those tubes in which haemolysis occurs the supernatant fluid is tinged red and there is no deposit of red cells. Where haemolysis is absent the salt solution remains colourless and the red cells form a deposit at the bottom of the tube. Other methods. — Other special methods of examination of the blood, such as the estimation of the alkalinity of the blood, and the presence of fat or bile in the serum, are described under the heading of the chemistry of the blood. Certain special methods commonly described and extremely rarely practised are omitted altogether. Such methods include estimations of the viscosity of the blood, and are of some scientific but of no clinical value. CHAPTEK IV. THE BLOOD SEBUM — AGGLUTININS AND OPSONINS. The nature of agglutinins. — Agglutinins are antibodies appearing in the blood in excess in response to infection. The more important agglutinins are those which act upon bacteria, and these have the property of checking the motility of organisms and causing them to come together into clumps or masses. The phenomenon is a very striking one to watch, but its purpose and mode of action are obscure. The bacterial clumps produced by an agglutinating serum acting on a suspension of organisms are not permanent, and the organisms themselves are only temporarily embarrassed, for after an interval the clumps break up again into separate individuals, the bacteria regain their motility and are capable of multi- plying and producing disease. Agglutinating substances may also be present in the serum which have no action upon bacteria, but are capable of agglutinating red blood corpuscles ; such bodies are known as hemagglutinins. The agglutinins are thermo-stable, that is to say, an aggluti- nating serum heated to 60° C. is still capable of clumping bacteria or red cells. In common with other antibodies, the agglutinins are specific, and act only upon the infecting agent or " antigen " which calls them into existence. The agglutinin for the typhoid bacillus, for example, has the power of clumping that bacillus and no other bacteria. It happens, however, that infection by one organism not infrequently leads to a general increase in the antibodies for other, and particularly for closely allied organisms, the increase in the specific antibody being only relatively greater than the increase in the general antibodies. In any febrile condition, such as tuberculosis or influenza, there may be a considerable increase in the amount of agglutinin for the typhoid bacillus. In paratyphoid infections there is almost always a considerable increase in the agglutinin for the typhoid bacillus, but in typhoid infections the increase in the typhoid BLOOD SEEUM— AGGLUTININS AND OPSONINS 49 agglutinin is appreciably greater than that found in any other circumstances. Further, agglutinins are present in the blood in health, but in minute quantities ; they are enormously increased in response to infections. In making use of agglutinins in the clinical diagnosis of disease it is evidently insufficient to demonstrate their presence ; it is essential to estimate their amount. The amount of agglutinin in any serum is judged by progressively diluting the serum until the greatest dilution capable of agglutinating the bacteria is arrived at. The use of agglutinins in diagnosis. — Some infecting agents, such as the tubercle bacillus, do not lead to any appre- ciable production of agglutinin in the blood, and the agglutina- tion test is therefore inapplicable as a mode of diagnosis. In other diseases, such as cholera, the specific agglutinin is manifest only after recovery from the attack. Other organisms, such as the bacillus pyocyaneus, agglutinate spontaneously in saline suspensions. Other organisms again have either not been as yet discovered or are unable to be cultivated outside the body. The agglutination test has therefore a comparatively limited field of application, and is mainly confined to the diagnosis of typhoid and paratyphoid infections, dysentery, and Malta fever. The agglutinins in typhoid fever. — The estimation of the typhoid agglutinin is known as the Griinbaum- Widal reaction and is of the greatest value in diagnosis. A positive reaction is one of the very few pathognomonic physical signs in medicine, since it indicates with certainty the presence of an infection with the typhoid bacillus. If we except the comparatively rare examples of " typhoid carriers," a positive reaction is almost certain evidence of typhoid fever. A classical case of typhoid fever can be safely diagnosed on clinical grounds alone, but the disease is so commonly atypical that the diag- nosis is occasionally made and is frequently confirmed by means of the agglutination test. A negative reaction does not contraindicate typhoid fever, since it commonly happens that the increase in agglutinin is insufficient for a completely positive reaction, and a partial reaction is not conclusive evidence of typhoid fever. A completely negative reaction, however, is strongly opposed to the diagnosis of typhoid fever. A positive reaction is rarely obtained before the end of the p. 4 50 CLINICAL PATHOLOGY. first or the beginning of the second week of the disease, but an appreciable increase in the agglutinins is usual within the first 4 or 5 days of the onset of symptoms. The reaction usually remains positive throughout the course of the disease, and for some weeks, or even a few months, after the tempera- ture has become normal. A partial reaction may persist for a year or more. The reaction is of no particular value as a basis for prog- nosis ; a mild case of typhoid fever may never give a strongly positive agglutination test, and fatal cases may or may not react strongly. In the very early stages of typhoid fever the diagnosis may be made by the isolation of the bacilli from the blood (page 83) ; in the later stages, if the agglutination test is doubtful, the bacilli rnsij be obtained from the faeces (page 342), or less frequently from the urine (page 282). The technique of the Griinbaum-Widal test. —The materials required for the performance of the reaction are as follows : — Serum tubes, Wright's capillary tubes and rubber teat, hollow-ground slides and vaseline, cover-glasses, normal saline, watch glasses, a bowl or small hand-basin containing carbolic acid (1 in 20), and a 24 hours old culture of typhoid bacilli on an agar slope. The "Wright's tubes and the serum tubes can readily be made from glass tubing with the aid of a blow-pipe ; the glass tubing should have an outside diameter of \ inch. To make a Wright's tube hold the glass tubing with one hand at each end and heat it to a red heat as near to one end as it is convenient to hold it, continually rotating the glass. As soon as the heated portion is freely malleable remove it from the flame and separate the two ends evenly, without force and moderately slowly. Fuse the terminal portion of the capillary part of the tube in the flame. As soon as the glass is cool enough to hold repeat the process and make a series of tubes. Leave both ends of the tube sealed until required for use, then nick each end with a file and break them off. The bulbous portion of each tube should be about \\ inches long and the capillary portion about 9 inches. The bore of the capillary part should be nearly equal throughout, tapering very slightly towards the distal end. The tubes are readily made with a little practice, the points BLOOD SERUM— AGGLUTININS AND OPSONINS. 51 requiring experience being the size of the blow-pipe flame, the degree to which the glass is heated, and the rapidity with which the capillary tube is drawn out. The tendency is either to heat the glass insufficiently and produce a short, thick tube, or to draw the ends apart too rapidly and make the tube excessively long and thin. The serum tubes for collecting the blood are made in a similar manner, but the bulbous portion is left longer (about 2 inches), and the capillary part is made shorter and burnt through at its centre, the distal half being left to provide the capillary portion of the next tube drawn out. To obtain the serum cleanse the patient's thumb with ether and let it dry. If the hand is cold place it previously in hot water and dry thoroughly. Wind a piece of fine rubber tubing round the thumb from the base nearly to the nail. With a surgical needle make a sharp, deep stab at the side of Pig. 8.— Serum Tube ; Wright's Tube ; Eubber Teat. the thumb in the line of the digital artery. Avoid the pulp of the thumb. Having broken off both ends of the serum tube, hold one end lightly in the drop of blood, sloping the tube a very little downwards. Keep rotating the tube. When the blood has all entered the tube lay the tube gently down on a flat surface, loose the rubber tubing, rub the thumb briskly with a dry swab, reapply the tubing, and continue to fill the tube. When the bulbous portion of the tube is about half full, wipe the end containing the blood and seal it off in a flame, then seal off the other end. Stand the tube in an upright position for about half an hour or until the serum has separated, then centrifuge the tube at a moderate speed. (For the purposes of this reaction a comparatively small quantity of serum is required, and the centrifuging may frequently be dispensed with.) The slides for the hanging drops should be provided with a central circular depression, but this is not essential. 4—2 52 CLINICAL PATHOLOGY. To prepare the slides, warm them in the Bunsen flame, take up a little vaseline on a glass rod, warm the rod in the flame to melt the vaseline, and then draw the end of the rod round the circular depression in the slide, leaving a substantial ring of vaseline. No vaseline should be allowed to drop into the central depression. The typhoid culture used is of considerable importance. A reliable strain can be obtained from any known laboratory, or the bacilli may be isolated from the blood of a case of early typhoid fever (page 83), or from the spleen post mortem (page 161). From whatever source the bacilli are derived the culture should be examined in two ways — the full cultural characters of the bacillus should be investigated (page 135) and the bacillus should be tested against a known positive serum. The readiness with which various strains of bacilli are aggluti- nated is not constant, and a bacillus is exceptionally met with which has all the cultural characters of the typhoid bacillus, but which is not agglutinated by the most powerful sera. A reliable strain of bacilli once obtained can often be sub- cuitured over an almost indefinite period without change of character. The subculture used for performing the test should have been incubated from the previous day on an agar slope. Before making the suspension put up a subculture from the agar slope into broth to preserve the strain. To make the suspension add warm saline to nearly halfway up the agar slope and replace the cotton-wool plug. Gently shake the tube until the growth is washed off into the saline and the latter has become distinctly milky. It may be neces- sary to assist the washing off of the growth into the saline by gently rubbing the former with a sterile platinum wire. When a milky suspension has been obtained, remove the cotton-wool plug and drop it in the carbolic bowl. Filter the suspension through a small filter paper moistened with warm saline and held in a pair of forceps over a watch glass. Place the culture tube and filter paper in the carbolic and sterilise the ends of the forceps in the Bunsen flame. The object of filtering the suspension is to remove any clumps of bacilli which may be washed off as such from the surface of the medium. The discrete bacilli pass through the filter paper. The reaction is then performed as follows : — With a blue glass-pencil (or with a pen and ink) make a mark on a Wright's BLOOD SERUM— AGGLUTININS AND OPSONINS. 53 pipette about half an inch from the end. By means of the rubber teat, which must fit tightly to the pipette, draw up a volume of the blood serum to be tested to the mark, admit a column of air, and draw up 9 equal volumes of normal (0*85 per cent.) salt solution, leaving a small column of air between each volume. Blow out serum and saline into a clean watch glass and mix thoroughly ; the serum is now diluted to 1 in 10. Draw up a volume of the diluted serum to the mark and an equal volume of the suspension of bacilli. Mix in a watch glass and transfer a sample drop with the pipette to the centre of a cover-glass, spreading out the drop so as to cover about one-fourth the area of the glass. Pick up the cover-glass by pressing on it one of the vaseline ringed slides. Turn the slide over. Press down the cover-glass with a mounted needle over the whole circumference of the vaseline ring so that no air can gain admittance. No vaseline should touch the hanging drop. Mark on the slide the time and the dilution of the serum (1 in 20). Take another volume of the diluted serum in saline and 4 volumes of the suspension of bacilli. Mix and prepare a hanging drop as before. The dilution of the serum is now 1 in 50. Take a third volume of the diluted serum and 9 volumes of the bacillary suspension and prepare a hanging drop in which the serum is diluted 1 in 100. Make a fourth drop from a sample of the typhoid suspension to which no serum has been added and label the slide " control." Examine each slide with the microscope vertical and a -jt inch objective, altering the aperture of the diaphragm until the refractile bacilli are clearly seen. Observe at intervals the motility of the bacilli and the formation of clumps. Com- pare the drops containing serum with the control ; if the bacilli in the latter lose their motility or come together in clumps the test is valueless ; it is, however, extremely rare to meet with a sample of typhoid bacilli which agglutinate spontaneously in suspension. In a positive reaction between serum and bacilli the latter become motionless and collected into com- pact masses easily visible to the naked eye, few if any bacilli remaining isolated between the clumps. After completing the reaction place the slides (after partially slipping off the cover- glasses), the capillary tubes, typhoid suspension, watch glasses, and everything which can possibly have come in contact with the bacilli into the carbolic basin and leave them there till 54 CLINICAL PATHOLOGY. the next day. Make sure that no bacilli have been spilt on the bench, but if they have soak the bench in carbolic. Wash your hands after completing the reaction and do not smoke while it is being performed.* The interpretation of the reaction is based upon the time taken for the completion of the reaction and upon the dilution of serum capable of producing agglutination of the bacilli. "With serum very loaded "with agglutinins clumping ma}^ take place within a few minutes in the 1 in 100 dilution. In a completely negative case no clumping occurs in the 1 in 20 dilution, and the motility of the bacilli may even be accelerated. Complete clumping in the 1 in 20, partial clumping with incomplete loss of motility in the 1 in 50, and little or no reaction in the 1 in 100 is a " partial " reaction. The essentials of a positive reaction are that clumping and loss of motility should be complete in the 1 in 50 dilution within half an hour. Such a reaction is the strongest possible evidence of typhoid fever. Complete absence of reaction in the 1 in 20 dilution is strongly opposed to the diagnosis of typhoid in a febrile case of any duration. A partial reaction is often of assistance when taken in conjunction with the physical con- dition of the patient, and in cases of doubt should be repeated after a few days' interval. It may be stated here, and cannot be too strongly emphasised, that the deductions drawn from any pathological test, even from the Wassermann or Griinbaum-Widal reactions, should never be made from the test alone. The interpretation of the results requires a knowledge of the clinical condition of the patient coupled with an understanding of how the test is performed and what it means. Other methods of performing the reaction are in fairly common use ; the method described above is perhaps the one most widely employed, and is known as the microscopical method. Other observers prefer the macroscopic test, which is performed by mixing diluted serum and a saline suspension of bacilli in a Wright's tube ; sealing the end in a flame, and * It may be said generally of smoking in a laboratory that the atmo- sphere of such places is sufficiently vitiated without indulging in this habit. If smoking is permitted care should be taken never to lay down a pipe or cigarette on the bench, since there is a distinct risk of transferring organisms from the bench to the mouth. BLOOD SERUM— AGGLUTININS AND OPSONINS. 55 standing in a vertical position either at room temperature or in an incubator at 37° C. In a positive reaction a granular precipitate of clumped bacilli forms in the capillary tube and sinks towards the bottom of the tube; the control tube con- taining bacilli in salt solution remains uniformly turbid. The interpretation of the results depends upon the dilution of the serum, the time during which the precipitate forms, and the temperature at which the reaction takes place. The method is a perfectly reliable one. The living bacilli may be substituted by a suspension of dead organisms such as can be obtained ready for use from various sources. The dead bacilli ready prepared are more convenient for those not constantly working in a laboratory, but should only be used when unavoidable and never without adequate controls, since such preparations have been provided in some instances with the bacilli omitted and others with the organisms already clumped. Certain ingenious little automatic test cases are provided by manufacturers for the performance of this reaction. I have only examined one apparatus of this nature and found it worthless. All such mechanical contrivances are apt to deceive and should be avoided. The agglutinins in diseases other than typhoid fever. — Paratyphoid infections are infections not by the typhoid bacillus, but by organisms closely allied to it and known as paratyphoid bacilli. These bacilli are of more than one variety, the organism most commonly met with in this country being that known as b. paratyphosus B. The para- typhoid bacilli give rise to a clinical condition indistinguishable from typhoid fever by other than bacteriological methods. The paratyphoid infections, however, tend to run a milder course. Infection by one of the paratyphoid bacilli may be sus- pected when a case clinically resembling typhoid fever fails to give a positive serum test. In such cases the serum should be tested against stock cultures of one or more of the para- typhoid bacilli, and in addition attempts should be made to isolate the causative organism from the blood, the faeces or the urine. The methods of testing the agglutinating content of the serum or of isolating the bacillus are identical with those employed in the case of the typhoid bacillus. It usually 56 CLINICAL PATHOLOGY. happens, however, that the serum of a paratyphoid infection agglutinates to a considerable extent typhoid bacilli, and the serum must be diluted beyond the point at which one organism is agglutinated but not the other. The separation of the agglutinin for each bacillus is somewhat beyond the scope of ordinary clinical methods. It may be done as follows : — A thick suspension of typhoid bacilli is added to a portion of the serum and the mixture incubated. After incubation the mixture is centrifuged at a high speed and the clear serum pipetted off from the bacilli. The agglutinating property of the serum is then tested in the usual manner with paratyphoid bacilli. The agglutinins for the typhoid bacilli have been removed by the previous saturation of the serum with these bacilli, and the agglutinins for the paratyphoid bacilli are unaffected. A second sample of serum is saturated with paratyphoid bacilli and then tested on typhoid bacilli. Dysentery of the bacillary variety and such of the intestinal affections as may be caused by one or other of the dysentery bacilli give rise to specific agglutinins in the blood. The agglutination test with the serum of dysenteric patients is conducted in exactly the same manner as is the Griinbaum- Widal reaction. The amount and the activity of the agglutinin present in dysentery commonly fall below that produced in typhoid fever, and it is exceptional to meet with a serum capable in dilution of 1 in 50 of completely agglutinating one of the strains of dysentery bacilli in less than one hour. The dysentery bacilli differ from each other in a manner similar to the paratyphoid bacilli ; the most widespread infecting agents of bacillary dysentery are those first described by Shiga in Japan and Flexner in America. Cases of amoebic dysentery give no reaction in their serum with the dysentery bacilli. Malta fever is a disease produced by a coccus, the micro- coccus melitensis, and the serum of infected persons or animals acquires the property of agglutinating this organism. The serum test is a very valuable one in assisting diagnosis, and is performed in the same manner as the typhoid test. The Malta fever coccus can be obtained from the urine of an infected person or animal, or from the blood. If no case is available a culture can be obtained from a reliable laboratory, but it is advisable in performing this reaction that the culture used should be one comparatively recently isolated from the body. BLOOD SEBUM— AGGLUTININS AND OPSONINS 57 The agglutinins as evidence of infection.— The isolation of an organism from some part of the body is not of itself evidence that the organism is actually producing disease. It may also happen that a bacterium, not generally recognised as capable of producing disease, or a variety of bacteria amongst which the infective organism is in doubt, may be isolated in cultures from a lesion, or from the excreta, or from one of the body fluids. In such cases, and particularly when the organism in question is a member of the colon or typhoid group, evidence of the infectivity of the bacterium may be sought for by examining the agglutinating power of the patient's serum upon it, together with that of the serum from a normal person. The presence of agglutinins in the serum in greater than the normal amount for the organism isolated is definite evidence of actual infection by that organism. Absence of agglutinin for the organism is no evidence against its infectivity, since some bacteria give rise to little or no agglutinin in the blood even when they are undoubtedly producing disease. Infection of the urinary tract with the bacillus coli, for example, commonly produces no appreciable rise in agglutinin for this bacillus. The agglutinins as a test for an organism. — Just as a patient with an unknown disease may be proved to be infected with a known organism by the demonstration of agglutinins in his serum for that organism, so a reversal of the process, the testing of an unknown organism with a known serum, may be adduced as a proof of the nature of the organism. A bacillus, isolated from the blood in the first few days of a febrile attack, may be immediately tested with the serum of a patient known to have typhoid fever, or with the serum of an animal which has been immunised to the typhoid bacillus. A positive diagnosis can thus be made at an earlier date than would be possible if the recognition of the bacillus depended upon its full cultural characters. The reaction is most com- monly used in typhoid fever, and it is convenient to have at hand a strongly agglutinating serum of this nature. When such a sample of blood is obtained it should be centrifuged and the clear serum pipetted off" into a sterile capsule. The capsule should be sealed, heated in water at 56° C. for half an hour, and then kept in a cupboard, or preferably on ice. A serum will keep its agglutinating property for several weeks or even months. 58 CLINICAL PATHOLOGY. Hemagglutinins. — The agglutinins described above are those which act upon bacteria. There may also be present in the serum bodies capable of agglutinating red cells, or heernagglutinins. The hemagglutinins have been already referred to under the description of the blood in purpura. The phenomenon most commonly met with is that the serum of the affected person has the power of agglutinating washed normal red cells, but not his own red cells, while his own red cells are not acted upon by a similar agglutinating serum. The reaction is performed by mixing in a Wright's tube 1 volume of the patient's serum and 1 volume of a 10 per cent, suspension of normal washed red cells in normal saline (page 60), incubating for 15 minutes at 37° C, blowing out the mixture on to a slide, and placing a cover-slip on the expelled drop. The red cells will be seen to have run together into large, tight clumps. Very rarely a serum will be found to have the property of agglutinating its own red cells, a phenomenon which can hardly take place in the body, yet it may be seen to have occurred immediately the blood is shed. On making a puncture into the ear in such a case clear serum exuded, followed by a clump of red cells, and it was found almost impossible to make films and quite impossible to make a count of the red cells. Opsonins. The nature of opsonins. — Opsonins were shown by Wright to be substances present in the blood which have the property of acting upon bacteria in such a manner that the phagocytes are able to ingest them. Opsonins are present in considerable amount in health ; they may be either increased or diminished as the result of infection. If the body reacts favourably to infection, the opsonins are increased : if the infection gains the upper hand, the opsonins are diminished. The fluctuations in the opsonic content of the blood in disease are due to alterations partly in the specific opsonin, that is the opsonin which acts only on the specific infecting agent, and partly in the general opsonins capable of acting on organisms other than the infecting agent. Opsonins are destroyed by heating the serum to 60° C. and also by keeping it ; they thus differ from the agglutinins, and closely resemble the complex ment of normal serum, BLOOD SERUM— AGGLUTININS AND OPSONINS. 59 The estimation of the opsonic content of the serum has been made use of as a method of diagnosis and as a means of controlling the treatment of disease by the administration of vaccines. The ratio of the opsonic content of a patient's serum to that of a normal serum is known as the opsonic index. The technique of the opsonic index test. — The materials required are Wright's capillary pipettes, serum tubes, a suspension of the organism to be tested in normal saline, the patient's serum, normal serum and washed normal leucocytes, normal salt solution and normal citrated salt solution, an incubator, a centrifugal machine, watch glasses, slides, staining reagents, etc. The capillary pipettes and serum tubes are of the same nature as those used for the Grunbaum-Widal test. The bacterial suspension is made, when practicable, from a '24 hours old culture on agar of the organism to be tested. The growth is washed off the agar slope by means of normal salt solution and transferred to a clean centrifuge tube. The tube is centrifuged at a moderate speed, and the super- natant fluid (free from clumps of bacteria) is poured off and diluted with saline until a suspension of suitable turbidity is obtained. The strength of the suspension is a matter of much importance and can be gauged by the degree of turbidity, but only as the result of experience. Suspensions of organisms readily ingested by the phagocytes (such as the staphylococcus aureus) should be made thinner than suspensions of organisms less readily ingested (such as the bacillus coll). The natural tendency is to make the suspensions stronger than is desirable. In the case of the tubercle bacillus it is convenient to use the dried bacilli sterilised by heat. A small portion of the growth is rubbed up in a mortar with saline and then centrifuged and treated in the manner described above. The majority of organisms are best used in the living state, since heat sufficient to destroy them may lead to a diminution in their affinity for the ordinary stains. All suspensions should be freshly made for each series of observations, and should be thoroughly mixed before use. The serum is obtained in the same way as for the agglutinin test. In the case of the normal serum it is advisable to take the blood from several normal persons and mix the sera so 60 CLINICAL PATHOLOGY. obtained in one capsule in order to diminish the risk of taking as normal the serum of an infected person. In the case of the tuberculous opsonic index such a normal "pooled" serum is almost essential. The sera used should have been obtained not more than 24 hours previously. The washed normal leucocytes are obtained by pricking the thumb and allowing the blood to drop into a centrifuge tube partly rilled with citrated salt solution having the composition of *85 grammes sodium citrate and "85 grammes sodium chloride in 100 c.c. of distilled water. After every few drops of blood have been added the tube should be inverted in order to mix adequately the blood and the solution. The tube is then centrifuged and the supernatant fluid pipetted off. Normal salt solution is added to the deposit of red cells and leucocytes and the tube inverted two or three times. The tube is again centrifuged, the saline removed and fresh added, and the process again repeated. A final deposit of washed blood cells, the upper layers of which are particularly rich in leucoc} 7 tes, is thus obtained. The centrifugal machine employed must be one capable of starting slowly, running smoothly, and stopping gradually. With a badly-running machine the leucocytes maybe broken up and rendered useless for phagocytic work. The usual varieties of hand-driven machines are for this reason unsuitable, and some more expensive apparatus is desirable. The best form of centrifuge is one in which the carriers are held in a circular disc or plate, which can run free when the power is cut off. The driving force is preferably a jet of water acting on a turbine arrangement attached to the base of the spindle of the plate, but this is only available when a sufficient pressure of water (40 to 50 lbs. to the square inch if a single jet is used or 20 to 30 lbs. with a double jet) can be obtained. When electricity is used as the driving force, it is best applied by a motor and band working on a collar attached to the spindle, and capable of running free when the motor is stopped. The speed of the motor must be regulated by a series of stops connected with the starting lever. This type of instrument, as supplied by Messrs. Maw, Son and Thompson, is depicted in the illustration. To perform the reaction. — Make a mark on a Wright's pipette about 1 inch from the tip. Draw up to the mark a BLOOD SERUM— AGGLUTININS AND OPSONINS 61 volume of the washed cells, and admit a column of air. Draw up a volume of the normal serum, and admit a column of air. Draw up a volume of the bacterial suspension. Blow out the con- tents of the pipette into a clean watch glass and mix them thoroughly. Draw up the mixture into the pipette, seal the end of the pipette in the flame, and remove the rubber teat. Make a note of the time, and place the pipette in the incubator. Eepeat the process, substituting the patient's for the normal serum. At the end of 20 minutes remove each pipette from the incubator, break off the end, slip on the teat, and blow the contents on to a clean slide. With a second slide make a thick film on the first slide as if making a blood film. Dry the film by rapidly waving in the air. Dip the film when dry in a beaker of tap water and keep it there until the Fig. 9. — Centrifugal Machine. haemoglobin is dissolved out of the red cells. Without allow- ing to dry, stain the film. The stain employed depends upon the bacteria present in the suspension. In the case of bacteria which take the ordinary dyes, filter carbol thionin on to the slide and leave for 3 minutes. Wash in tap water and blot dry. In the case of tubercle bacilli, stain for 5 minutes in hot filtered carbol fuchsin. Decolorise in 12 per cent, nitric acid. Wash in tap water. Counterstain for 2 minutes with dilute methylene blue. Wash in water and blot dry. For counting the films, use the oil immersion lens, and choose the thicker parts of the films, preferably towards the edges of the slides. Count 50 or 100 polynuclear leucocytes and the number of organisms contained in them. To estimate the opsonic index divide the number of organisms counted in the slide made from, the tube which contained the patient's serum by the number of organisms counted in 62 CLINICAL PATHOLOGY. the same number of cells on the control or normal slide. Thus, if 100 phagocytes in the pathological mixture contained 300 organisms and in the normal mixture 200, the opsonic index of the patient's serum is 1*5. The number of organisms per phagocyte depends largely on the strength of the bacterial suspension, and the ideal number ingested is an average of about 3 per cell. If the number of bacteria taken up falls much below the proper standard, or if the cells are over full and many of them contain clumps of organisms, the experiment must be repeated. The value of the opsonic index. — The claims of this reaction to a place in clinical medicine are two in number. It has been said that the dosage of vaccines should be regulated by frequent estimations of the opsonic index, on the grounds that a dose given with a falling index may depress the index still further and do harm, and that the nature of the response on the part of the index is an indication of the suitability in size of the dose. It has been further claimed that variations of the opsonic index either above or below the normal to any organism is evidence of infection by that organism. The opsonic index has thus been used both in the treatment and in the diagnosis of disease. Considerable controversy has ranged around the value of this test, and the opinion of the majority at the present would seem to be that, whatever guide the opsonic index may have been in the past as a control of vaccine treatment, it is no longer necessary to rely upon it, and that as a method of diagnosis, the variations in the index being on the whole less than the variations arising from the errors of technique, little dependence is to be placed upon it. The errors in the index due solely to the technique are no doubt considerable, and the whole process is a very artificial one, which estimates not the immunity of the body to a parti- cular infection, but a single immune process, the serum opsonin. Further, the resulting index can only be approxi- mate, since in counting from 50 to 100 cells which contain a number of organisms ranging from none at all to nearly 20, considerable variations must occur while enumerating different series of cells in the same slide. The experimental error is probably at least 25 per cent., or barely within the range of variations met with in disease. The method is described here partly because it has been very widely used and is still used BLOOD SERUM— AGGLUTININS AND OPSONINS 63 to some extent, but mainly because the conception of the opsonic index is acknowledged to have been a fine one and the technique of the method is instructive and of great value in experimental work. Modifications of the opsonic index. — Attempts have been made to lower the experimental errors of the reaction by modifications of the actual technique, and it may be said that, except for minor alterations adopted by individual workers, no technical changes of importance have been introduced into Wright's original method. More important alterations have been suggested in the theory and working of the reaction. It has been claimed that in localised, and particularly in tuber- culous, affections massage or other exercise of the affected part leads to a great increase of opsonin in the general circu- lation. For purposes of diagnosis the index is taken with the part at rest, and again after exercise. Any considerable fluctuations in the index are considered as evidence of infection. Other observers heat the serum with the view of destroying the general opsonin and leaving the more thermo-stable specific opsonin ; and it is claimed that with heated sera more difference can be demonstrated between a normal and an immune serum. The amount of phagocytosis induced by these heated sera is, however, small. Another modification is that known as the hsemophagocytic index. This method is simple to perform, and has the additional advantage of esti- mating the net result of alterations in the activity of the leucocytes as well as in the amount of opsonin in the serum. It was originally claimed that the phagocytes played a purely secondary part and did not differ in health or in disease, but this has been shown to be erroneous. The phagocytes in disease may be much more active than in health, or they may be less active ; nor does their activity vary with the amount of opsonin in the serum. To estimate the haemophagocytic index it is necessary to make the bacterial suspension with citrated salt solution instead of with normal saline. A volume of the patient's blood is drawn direct from the finger into a pipette, and a volume of the bacterial suspension is mixed, and incubated with it. The control normal blood is treated in the same way. After incubation the slides are made and counted as in the ordinary method. In this method the patient's own phagocytes act upon the opsonised bacteria, and 64 CLINICAL PATHOLOGY. the blood is subjected to little mechanical disturbance. It cannot be said that any of the above modifications have rendered the estimation of the opsonic index of any great practical value. On theoretical grounds the hsemophagocytic index has some advantage in that it is a measure of the phagocytic power of the blood, not merely of the amount of opsonin, and in practice it is readily obtained. CHAPTER V. THE BLOOD SERUM (continued) COMPLEMENT FIXATION TESTS THE WASSERMANN REACTION. The Wassermann Reaction. — This reaction was devised as a test for the presence in the blood of a syphilitic patient of the specific antibody to the toxin of the Spirochceta pallida. The nature and meaning of the reaction are most readily explained by a brief account of the steps in our knowledge of immunity which led to its discovery. It was shown by Bordet that if an animal of one species, for example a guinea-pig, were injected several times with the red cells of an animal of a different species, for example a rabbit, then the blood serum of the guinea-pig acquired in very high degree the property of hsemolysing, or dissolving the haemo- globin out of, the red cells of the rabbit and of no other species. That is to say : — Immunised guinea-pig's serum -f- normal rabbit's red cells = haemolysis. It has been shown also that the sera of some animals are capable under normal conditions of haemolysing the red cells of other widely different species. For example, human serum haemolyses sheep's red cells. Such a haemolysing serum is a normal or natural haemolytic serum as opposed to the serum of the injected guinea-pig, which is an immune serum. A natural serum is never so powerfully hemolytic as an immune one. It was next shown that if the immune serum were previously heated to 60° C. and then added to the red cells no haemolysis occurred : — Heated immune serum (guinea-pig) + red cells (rabbit) = no haemolysis. It was found that if a very small quantity of normal guinea- pig's serum, which of itself had no haemolytic effect, were added to the above mixture haemolysis then took place : — Heated immune serum (guinea-pig) + normal serum (guinea-pig) + red cells (rabbit) = haemolysis. p. 5 66 CLINICAL PATHOLOGY. The explanation of this apparent anomaly is that the hemo- lytic substance or hemolysin present in the immune serum consists of two bodies, both of which are necessary for the reaction. One body, the complement, is destroyed by heat, is present in any serum and is non-specific. The other body, the amboceptor, is not affected by moderate heat, is present only in immune sera (and in a natural hemolytic serum), and is specific. The hemolytic action depends upon a union of red cell with amboceptor, and then a further linking of red cell and amboceptor with complement. Bed cells to which a heated immune serum has been added combine with the amboceptor present, but are not evidently affected. Such red cells are known as sensitised red cells, since they only need the addition of complement for hemolysis to take place. It was also discovered that what is true of hemolysins is true of toxins and other similar bodies, and further that any substance of sufficient chemical complexity inoculated into an animal leads to the appearance in the animal's blood of a body of the nature of an amboceptor which is capable of uniting chemically with the substance inoculated. If the substance inoculated be a toxin the combining substance produced is called an antitoxin, and if a bacillus be inoculated a bacterio- lysin is produced. Any substance capable of producing an antibody is known as an antigen ; thus in the case of a hemo- lytic serum the antigen is a red cell. Bordet and Gengou then showed that the antibody in the serum of an inoculated animal, brought into contact with the specific antigen in the presence of complement, united with antigen and complement. They proved that this triple com- bination had taken place by adding the mixture to sensitised red cells and demonstrating that no hemolysis of the red cells occurred owing to the previous fixation of complement. The reaction was found to take place in numerous infections and to be capable of being applied as a method of diagnosis in human pathology. They found, for example, that if the serum of a patient convalescent from typhoid fever were heated (to destroy the complement, but not the antibody) and incubated with typhoid bacilli (antigen) and guinea-pig's serum (com- plement), and then added to sensitised red cells, no hemo- lysis took place, because typhoid bacilli, typhoid antigen and complement had united and no complement remained for BLOOD SERUM— COMPLEMENT FIXATION TESTS. 67 the sensitised red cells ; whereas if normal human serum (heated) were incubated with typhoid bacilli and guinea-pig's serum and then added to the red cells, haemolysis took place readily, since normal serum contains no antibody to the typhoid bacillus and has therefore no substance capable of uniting with the bacilli and the complement in the guinea-pig's serum. Consequently complement is available to combine with the sensitised red cells and hemolyse them. This reaction is known as the Bordet-Gengou reaction, or the com- plement deviation test. It is a specific test. Antibody can unite only with its specific antigen. Specific antigen and specific antibody must both be present before any combination with the (non-specific) complement can take place. The original Wassermann reaction was simply an applica- tion of the Bordet-Gengou test to the diagnosis of syphilis. In the case of syphilis the specific antigen is the Spirochceta pallida, and, since this organism cannot be cultivated on any of the ordinary media, Wassermann conceived the idea of utilising some organic extract rich in spirochetes, and for this purpose made use of an extract of the liver of a syphilitic foetus. The reaction was completely successful, since it was found that the heated serum of a syphilitic patient incubated with guinea-pig's complement and the extract of syphilitic antigen absorbed, by virtue of the syphilitic antibody, the complement from the mixture, whereas normal serum failed to do so. The test appeared to afford a specific proof of immunity to the spirochete. The meaning of the test is so far clear. It has, however, since been demonstrated that the Wassermann reaction is not an essential combination between syphilitic antigen and antibody, since the spirochete can be altogether omitted from the mixture. If an alcoholic extract of human heart muscle or even of guinea-pig heart muscle be substituted for the syphilitic liver extract, the reaction is equally reliable in as much as it is obtained with syphilitic and not with normal sera. The essential substances in the extract employed as antigen are found to be certain fatty bodies, or lipoids, such as can be extracted from normal organs. Why lipoids are capable of acting as syphilitic antigen in place of the true antigen, the spirochete, is unexplained. It has been suggested that the syphilitic virus has an affinity for the body lipoids and 5—2 68 CLINICAL PATHOLOGY. combines with them forming a toxo -lipoid, and that an antibody is produced to the toxo-lipoid which would be capable of combining with it or with lipoid. According to this explanation the reaction is a chemical combination between anti-toxo-lipoid present in syphilitic serum, lipoid acting as antigen in place of the toxo-lipoid, and complement. (It may be added that the toxophore group of a toxin is not essential for the combination of toxin and antitoxin, and that anti- diphtheritic toxin can combine with the diphtheria toxoid or non-poisonous toxin.) It has also been suggested that syphilitic serum contains some abnormal proteid derived from the destructive action of the spirochete on proteo-lipoid compounds in the body, which proteid when added to lipoid outside the body is precipitated and in its precipitation carries down complement. On this theory the removal of complement from the Wassermann mixture is a mechanical rather than a purely chemical action. "Whatever may be the explanation of the reaction, it follows that, since the spirochete may be replaced as antigen by lipoid, a positive reaction is not evidence of a specific immunity to the Spirochata pallida, but rather of a peculiar and abnormal lipoid metabolism such as might be common to more than one disease. It is further probable that a positive Wassermann reaction means not so much that a person is immune to syphilis as that he is still infected by syphilis. The reaction in diagnosis, — Since the reaction is not a truly specific one we might expect it to be present in some diseases other than syphilis, and this is found to be the case. A positive reaction is associated with certain diseases foreign to this country, including sleeping sickness, yaws, and some forms of leprosy. A positive reaction is also found occasionally, and for a brief period only, in scarlet fever. "With these exceptions — and from the point of view of diagnosis they are unimportant exceptions — a positive Wassermann reaction is very definite evidence of a syphilitic affection. As to the occurrence of the reaction in the various stages of syphilis : — In primary syphilis the reaction is nearly always positive, and becomes so at periods varying from 4 to 12 weeks after infection. A negative reaction at any date later than this in the case of a doubtful sore is strongly opposed to the diagnosis of syphilis. Since it is of the utmost importance, however, BLOOD SERUM— COMPLEMENT FIXATION TESTS. 69 to commence treatment at the earliest possible date, and preferably before the reaction has become positive, there is fortunately no necessity to rely upon the Wassermann test. In cases of any doubt the presence of the specific spirochsete is conclusive (page 142). In secondary syphilis if untreated the test is practically always positive, and a negative reaction almost contra- indicates syphilis. In tertiary syphilis positive reactions are not quite so invariable ; the test is, however, positive in from 80 to 90 per cent, of treated and untreated cases. It is perhaps more common to find a positive reaction in some tertiary lesions than in others ; a negative reaction with a thoracic aneurysm, for example, is extremely rare. In latent syphilis, that is to say in cases. of past infection with no present manifestations of the disease, the reaction is positive in only from 30 to 40 per cent. This class includes those who have been cured, and it is probable that a positive reaction in a person who has had syphilis some years previously is evidence that he is still liable to recurrence of the disease, and that a negative reaction indicates that he has been cured. In parasyphilis the reaction is positive in almost every case of general paralysis, but in only from 60 to 70 per cent, of tabetics. The serum test is of great value in the diagnosis of syphilis of the central nervous system and of parasyphilis ; it does not, however, help us to distinguish between these conditions. Further information is obtained by examination of the cerebro-spinal fluid (page 203). In both syphilis of the central nervous system and parasyphilis a lymphocytosis is present in the fluid ; but in syphilis the Wassermann test of the spinal fluid is negative as a rule, while in parasyphilis it is nearly always positive and, particularly in general paralysis, very strongly positive. In other forms of syphilis there is no alteration in the spinal fluid. In congenital syphilis the reaction is practically always strongly positive. The Wassermann test is thus seen to be of great value in the diagnosis of all syphilitic lesions ; but it must be remembered that a negative reaction sometimes occurs with lesions undoubtedly syphilitic, and that a positive reaction 70 CLINICAL PATHOLOGY. means that a patient is tainted with syphilis — it does not necessarily mean that the particular lesion for which he is under observation is syphilitic. In the case of a doubtful tumour, for example, a negative reaction is evidence against syphilis ; a positive reaction yields the valuable information that the patient has had syphilis and is probably still infected, but it does not tell us that the " tumour " is a gumma. It may be added that we are not dependent upon the Wassermann test for our diagnosis of all cases of syphilis. The majority of syphilitic lesions are sufficiently obvious on clinical grounds to make their recognition certain, particularly in the presence of a truthful history. The student at a hospital sees the reaction tested in many cases as a matter of routine, and is in danger of falling into the error of placing too much reliance on a test and neglecting the cultivation of his clinical experience. The response of the reaction to treatment. — The alterations which may take place in the reaction as the result of treatment depend partly upon the efficiency of the treatment and partly upon the stage of the disease at which treatment is being undertaken. In the primary and secondary stages of the disease the reaction alters rapidly in response to energetic treatment, and should become negative in from 6 to 12 weeks. A positive reaction becomes negative most rapidly after intravenous injections of salvarsan, and readily also after intensive mercurial treat- ment. During the treatment the reaction may be seen to change from strongly positive to partial and finally to negative. A negative reaction at this stage does not mean that the patient is cured unless it remains negative for many months, and possibly not even then, since if further treatment is omitted a negative reaction may again become positive and be followed by a relapse in the symptoms and physical signs. If the treatment is inadequate during the primary and secondary changes the reaction remains positive or becomes partial only, and, since there can be little question that a permanently negative reaction is the goal to be aimed at, the effect of treatment is reasonably controlled by the state of the reaction, which appears to be a more sensitive guide than the clinical condition of the patient. In the tertiary stage of syphilis the behaviour of the reaction is quite different, since BLOOD SERUM— COMPLEMENT FIXATION TESTS. 71 at this stage, whatever the treatment adopted, it is quite exceptional to obtain a completely negative reaction, and frequently very little change is observable in it. The behaviour of the reaction in this stage of syphilis is compar- able with the effect of treatment in the clinical condition of the patient, since it is notoriously difficult to cure the disease after tertiary symptoms have manifested themselves. In the tertiary stage lesions frequently clear up rapidly under treatment, but have the greatest tendency to recur, and a very small percentage of cases is actually cured. The Wassermann reaction is thus a valuable guide, not only in the diagnosis of syphilis, but also in estimating the effect of treatment. The value of the reaction in prognosis is less certain. It is probable that a patient who has had syphilis, and whose reaction is found to be negative a year or more after treatment has been stopped, is cured of the disease. It is probable also that patients who have had no symptoms for some years but are found to still have a positive reaction, are liable to tertiary manifestations or to parasyphilis. There is at present, however, no statistical proof of these statements, since the reaction has not been available for a sufficient period. The technique of the Wassermann reaction. — There are numerous methods of performing the reaction, and they differ very considerably from one another. Two methods are described here, and both are widely used in this country. The first method is given because it more closely corresponds to the original reaction of Wassermann, the second because it is a more simple method and at the same time a very sensitive one. The advantages and disadvantages of the two methods will be discussed at the end of the chapter. Method 1. — A modification of the original technique. — The materials required are serum tubes, small test tubes, a graduated pipette (0*1 to 1 c.c), watch glasses, saline, a centrifugal machine, etc., and in addition fresh guinea-pig's serum (complement), immune serum (rabbit to sheep), red cells (sheep), an alcoholic extract of human heart muscle (antigen), human sera (normal, syphilitic, unknown). The complement is fresh guinea-pig : s serum. It is obtained by plunging a needle attached to a 5 c.c. syringe into the animal's heart, withdrawing the blood and ejecting it into a 72 CLINICAL PATHOLOGY. centrifuge tube. After centrifuging the clear serum is pipetted off, and should be used the same day or within 24 hours. The guinea-pig is usually none the worse for the operation. The immune serum is the serum of an animal which has been inoculated with washed sheep's corpuscles. The sheep's blood is obtained from the butcher and received into sterile citrated salt solution. It is then centrifuged and washed several times with saline. About 2 c.c. of the washed corpuscles are injected beneath the skin of a rabbit, and the injection is repeated every 7 days for 3 or 4 injections or until a serum is obtained strongly hemolytic for sheep's cells. It is somewhat a matter of chance when a serum becomes strongly immune. The rabbit's blood is removed by a needle and syringe from a vein in the ear, and after standing is centrifuged. The clear serum is pipetted off into sterile capsules. The capsules are sealed and heated in water at 60° C. for 15 minutes. They are then stored, and will keep their specific attributes for several months. The red cells are those of a sheep , and are obtained and treated in the same manner as those used for injection into a rabbit. A 5 per cent, saline suspension of the final washed deposit is used for the reaction. The above materials — namely, the complement, the ambo- ceptor of the heated immune serum, and the red cells — constitute the hemolytic system. The antigen is prepared as follows : A heart is obtained from any cadaver in the post-mortem room, and slices of the muscular portions are removed with a clean knife, blotted dry, and weighed. The slices are transferred to a mortar and thoroughly ground up with absolute alcohol in the proportion of 1 gramme of heart muscle to 5 c.c. of alcohol. The mixture is transferred to a sterile test tube and heated in a water bath at 60° C. for 1 hour and then incubated for 24 hours at 37° C. The mixture is then filtered through an ordinary filter paper into a sterile bottle and stored in a closed cupboard at room temperature. Extracts obtained in this way are extremely constant in strength, and will keep without variation for some months. They should, however, be standardised before use and be constantly controlled. The human sera are obtained in the same way as for the Widal reaction (page 51). Two tubes, at least hatf -filled, BLOOD SERUM— COMPLEMENT FIXATION TESTS. 73 should be obtained from each case, and any tube which shows haemolysis in the serum after centrifuging must be discarded. The serum is pipetted off into capsules and inactivated by heat in the same manner as the immune serum. Serum is required from a non-syphilitic source, from a known case of syphilis, and from the cases to be tested. The salt solution is made in the usual manner from pure sodium chloride and distilled water. For the purposes of this reaction it should be freshly prepared and of a strength of exactly 0'9 per cent. Standardisation of the materials. — The most variable reagent is the immune serum, and this is standardised as follows : A 1 in 1,000 dilution of the serum is made with saline and into a series of tubes "1, *2 up to *9 c.c. of this diluted serum are put, and each tube is made up to "9 c.c. with saline. To each tube is added "1 c.c. of a 1 in 4 dilution of guinea-pig's serum and *5 c.c. of the 5 per cent, suspension of sheep's red cells. The tubes are incubated for 1 hour at 37° C. and then allowed to stand aside for the corpuscles to settle. A " unit " of amboceptor was present in that tube which contained the smallest quantity of immune serum necessary for complete haemolysis. Two and a half units are used for the Wassermann reaction. The standard of the immune serum having been fixed, it is usual on each occasion to fix the standard of the complement, and this is done in exactly the same way, except that a fixed quantity (2^ units) of amboceptor is added to each tube and the amount of complement is varied. Two units of complement are used for the test, and this amount is usually contained in *025 c.c. of serum or "1 c.c. of a 1 in 4 dilution. The amount of complement present in the serum is extremely constant, and it is almost unnecessary to estimate it. The antigen is standardised as follows : A series of dilutions of the alcoholic extract are made in saline, and •025 c.c. of complement added to each tube. The tubes are shaken and incubated for 1 hour; then to each tube are added 2J units of amboceptor and - 5 c.c. of the diluted red cells. The tubes are again shaken and incubated for 1 hour. The smallest amount of antigen is noted which has been able of itself to absorb the complement, and one-third of this amount is used in the reaction. 74 CLINICAL PATHOLOGY. To perform the reaction prepare a series of tubes in pairs, sufficient for 2 for each serum to be examined, 2 for the normal serum, 2 for the syphilitic serum, as well as 3 control tubes, which are to contain no human serum. Sufficient saline is placed in each tube to make the final volumes equal (i.e. from 0*7 to 1*0 c.c. of saline). Into one of each pair of tubes place the requisite quantity of antigen as well as into the first of the 3 control tubes. Into every tube except the last control tube place "025 c.c. of complement (this is conveniently diluted with saline to make up *1 c.c. of fluid). Into each pair of tubes place "1 c.c. of the appropriate serum (i.e. the inactivated human serum). After mixing incubate 1 hour at 37° C. After incubation add to all the tubes '5 c.c. of the sheep's red cells and 2J units of the amboceptor. Again shake and incubate for 1 hour at 37° C, then stand the tubes on ice for the corpuscles to settle. The 3 control tubes should show complete haemolysis in the two containing complement and none in the third tube. The normal pair of tubes should show haemolysis in both tubes. The pair of tubes containing syphilitic serum should show haemoly sis in the tube with no antigen and no haemolysis in the tube with antigen. The tubes for the serum to be examined must show haemolysis in the tube without antigen and either complete haemolysis or no haemolysis in the other tube, according as the reaction is negative or positive. The technique of this test may be further elaborated by a quantitative reaction instead of the qualitative one described above. The degree of positiveness of the test is commonly determined by diluting the serum to be tested. By using a series of dilutions of each serum the amount of antibody present can be estimated from the degree of dilution sufficient to inhibit haemolysis in the mixture of antigen and haemolytic system. In the above technique each reagent of the Wassermann test is treated separately. The antigen is obtained from human heart muscle ; the antibody is tested for in the heated human serum ; the amboceptor for the red cells is present in the heated immune serum ; the red cells are those of a sheep ; and the complement is obtained from fresh guinea-pig's serum. It may be pointed out here that the final mixture by this method contains a varying amount of immune body for sheep's BLOOD SERUM— COMPLEMENT FIXATION TESTS. 75 cells, since human serum contains a normal amboceptor for these cells, and is absent from the 3 control tubes and diluted when the quantitative test is used. These variations are not of great importance provided sufficient amboceptor is present in the mixture. When the test is made on cerebro-spinal fluid that sub- stance is substituted for the serum, but there is no need to heat the fluid, since it contains no complement. Method 2. — The Hecht-Fleming modification. — The materials required are Wright's tubes, serum tubes, sheep's blood, antigen, normal salt solution, human sera (normal syphilitic and unknown), a centrifugal machine, and an incubator. The sheep's blood is obtained from the butcher, who is provided with a sterile glass bottle containing about 30 c.c. of citrated salt solution (0'9 gramme of sodium citrate, 0*9 gramme of sodium chloride in 100 c.c. of distilled water) and instructed to bleed the animal directly into the bottle, adding about 10 c.c. of blood. The bottle is immediately inverted several times. Blood obtained in this manner will keep for several days on ice. Before use a portion of the mixture is transferred to a centrifuge tube and centrifuged at a moderate speed for about 3 minutes. The supernatant fluid is pipetted off, normal (0*9 per cent.) salt solution is added, and the tube is inverted several times. This process is repeated four times — or 5 centrifugings altogether. The supernatant fluid in the final washing must be absolutely clear. With a Wright's tube make a 10 per cent, dilution of the final deposit in normal saline. The antigen consists of an alcoholic extract of human heart muscle prepared as described under Method 1. Before use 2 dilutions of the alcoholic extract, a 10 per cent, and a 5 per cent., are made with normal saline. In making the dilutions the saline is slowly added to the alcoholic extract, and an opalescent fluid should result. The human sera are obtained in the usual manner, and should not be withdrawn more than 24 hours before use. They are unheated, and are proved to contain both com- plement and amboceptor for sheep's red cells. It is essential in each set of reactions to have both a normal and a syphilitic serum in order to be certain that the antigen is being used in 76 CLINICAL PATHOLOGY. the required strength. The 10 and 5 per cent, dilutions of antigen prepared as described above are almost invariably found capable of absorbing the complement completely from a strongly positive syphilitic serum and to have little or no action on a normal serum. An antigen which fails to act in these dilutions should be discarded. To perform the reaction.— First stage.— In a Wright's tube draw up 1 volume of normal serum, 4 volumes of normal saline, and 1 volume of the 10 per cent, suspension of sheep's red cells. Mix thoroughly on a clean glass slab. Draw up the mixture into the tube and seal the end of the tube in the flame. Remove the rubber teat. Repeat the process with the syphilitic serum and with the unknown sera, labelling each tube. Place the tubes in the incubator at 37° C. and remove at the end of half an hour. Hemolysis must be com- plete in every tube — that is to say, the tube must contain no visible deposit of red cells, and the mixture must be evenly tinged with haemoglobin. This stage of the process proves that each serum contains a sufficiency of complement and amboceptor for the haemolysis of sheep's red cells. Second stage. — Draw up 1 volume of normal serum and 1 volume of the 10 per cent, lipoid. Mix thoroughly on the glass slab. Draw up the mixture into the tube ; admit a consider- able column of air. Draw up 1 volume of the red cells. Seal the end of the tube and remove the teat. Repeat the process with the 5 per cent, lipoid. Put up two similar tubes with the syphilitic serum and with each of the unknown sera. Place in the incubator at 37° C. and remove at the end of 1 hour. During this stage the serum is in contact with the lipoid, and union takes place between complement, antigen, and syphilitic antibody in those tubes which contain the antibody. The amboceptor for the sheep's red cells takes no part in the combination. The red cell suspension is not in contact with the serum lipoid mixture, and on removal from the incubator it should be noted that no haemolysis has taken place in it. Third stage. — With a glass file nick the end of each tube and break it off. Slip on the teat and blow out the con- tents of the tube. Mix thoroughly, draw up again into the tube, and seal the end. Replace in the incubator for 30 minutes. On removal from the incubator stand all the tubes BLOOD SERUM— COMPLEMENT FIXATION TESTS. 77 in a vertical position, preferably in the ice-chest, until the red cells have settled to the bottom. Those tubes in which haemolysis has occurred show no deposit, or very little deposit, of cells at the bottom, and the supernatant fluid is strongly tinged with haemoglobin. These tubes are " negative." The tubes in which no haemolysis has taken place show a well- marked deposit of red cells, and the supernatant fluid is colourless. These tubes are " positive." Other tubes may show absence of haemolysis in the tube containing the 10 per cent, lipoid and slight haemolysis in the 5 per cent. tube. These tubes are " partial." In this stage absence of haemolysis proves that complement was removed from the mixture of serum and antigen in the previous stage — in other words, that the serum contained syphilitic antibody. The presence of haemolysis proves the presence of complement, and shows that no antibody was present in the serum capable of combining with lipoid and complement in stage 2. In performing the reaction with cerebro-spinal fluid the first stage may be omitted, since the fluid contains neither complement nor amboceptor. In the second stage take 1 volume of normal serum, 3 volumes of cerebro-spinal fluid, and 1 volume of 10 per cent, lipoid ; mix and draw up separately 1 volume of sheep's cells as before. Repeat with 5 per cent, lipoid and incubate for 1 hour. In this process the normal serum provides complement and amboceptor and the spinal fluid, if syphilitic, the antibody. The third stage is performed in the usual manner. A control tube may be put up which contains saline in place of the spinal fluid. If any serum in stage 1 fails to haemolyse the sheep's red cells, the serum must be heated to 60° C. for 15 minutes to destroy any complement that may be present. In stage 2 1 volume of the heated serum is mixed with 1 volume of normal serum and 1 volume of lipoid. The remainder of the test is performed as above. One of the objections made to this modification of the Wassermann test is that human sera frequently fail to haemolyse sheep's red cells, and some observers have found that from 30 to 40 per cent, of human sera fail in this manner. Such findings arise from errors in technique, of which the most probable are the keeping of the sera more than 24 hours before use and the inadequate washing 78 CLINICAL PATHOLOGY. of the red cells. If a comparatively small trace of sheep's serum is incubated with human serum in the presence of sheep's red cells no haemolysis takes place, owing to an inter- action between the foreign sera. If clue precautions are taken to avoid these errors it will be found that only about 1 per cent, of human sera fails to hasmolyse sheep's cells. If it be necessary to use the serum more than 24 hours old the serum should be removed from the red cells after centri- fuging and stored in a capsule on ice. The serum may thus remain active for several days. Diluted Serum and Eed Cells. 1st Stage. Serum and Lipoid. Eed Cell Suspension. 2nd Stage. Clear Fluid. Deposit of Eed Cells. 3rd Stage — Positive. Complete Hiemolysis. No Deposit. 3rd Stage — Negative. ]? IG . io. — The Wright's Tubes in the Three Stages of the Eeaction. A comparison of the two methods.— The main advan- tage of the first method is that each component of the reaction — the complement, the amboceptor, and the antigen — is accurately measured. The disadvantages are that the technique is involved and laborious ; that the human sera in the process of heating to destroy the complement lose a part of the antibody, and the reaction therefore loses in sensitive- ness ; and that the ultimate mixture contains a considerable variety of sera from different animals, which interact to an unknown extent and probably lead to some loss of comple- ment on that account. In practice there can be no doubt of the value and accuracy of the method. BLOOD SEEUM— COMPLEMENT FIXATION TESTS. 79 The main advantages of the second method are simplicity and the use of unheated sera. The disadvantages are numerous, but entirely theoretical. It has been objected that the amount of complement in human sera may vary, and that a positive serum with excess of complement might give a negative reaction, whereas a negative serum with a shortage of complement might give a positive result. There is no evidence of any such gross variations in the amount of com- plement present in fresh human sera, and the amount of complement in guinea-pig's serum is so constant that it is commonly not estimated by those who prefer the original method. In a large number of undoubtedly normal sera I have never seen a positive or even partial reaction. In syphilitic cases the Hecht-Fleming method gives a higher percentage of positive reactions, and the reaction takes longer to become negative under treatment than with the original method. Quantitative estimations of the reactions are most accurately made by altering the amount of the human serum, that is, of the syphilitic antibody, and this is best done by the original method. Similar estimations can be made by varying the amount of antigen, as is. done in the Hecht-Fleming method, and the results are sufficiently graded for all clinical purposes. Both methods have their adherents, and both are of great value in clinical medicine. The second method has been subjected to considerable criticism on theoretical grounds, but has been found entirely satisfactory in practice, and owing to the simplicity of the technique is described here in full. The complement fixation test in other diseases.— Before the test described above was applied to the clinical diagnosis of syphilis it had been demonstrated in numerous other infections by Bordet and Gengou. The test is applicable in cholera, typhoid, whooping cough, and other diseases, but is not constant during the infection, and is only strongly positive during convalescence. The test is considerably used in the diagnosis of gonorrhoeal infection in America. The antigen employed is derived from a mixture of several strains of gonococci. In human tuberculosis no satisfactory diagnostic test of this nature has yet been published, though several methods are under consideration. The difficulty lies in the preparation of a satisfactory antigen. In hydatid disease the test may be used as a elinical method 80 CLINICAL PATHOLOGY. of diagnosis. The antigen is here the fluid from a hydatid cyst, and if this be substituted for the lipoid extract the test for the presence of hydatid infection may be carried out in the same manner as the Wassermann test for syphilitic infection. On theoretical grounds it might be expected that the com- plement fixation test would be applicable in all diseases in which the specific infecting body or antigen is known; in practice the test is at present almost entirely confined, with the exception of hydatid infections, to the diagnosis of syphilis. Both in tuberculosis and in gonorrhoea the test is still under consideration. The test has been used in a similar manner to the agglutina- tion test as the specific proof of an infecting organism. The bacillus isolated by Bordet and Gengou from cases of whooping cough resembles closely the influenza bacillus, but by means of the complement fixation test they have been able to prove the specificity of their bacillus. The serum of patients convalescent from whooping cough unites with the Bordet- Gengou bacillus and fixes complement, but does not unite with the influenza bacillus. CHAPTEE VI. THE PARASITOLOGY OF THE BLOOD. The cultivation of bacteria from the blood. — In health the blood as obtained from the peripheral circulation is sterile, and the demonstration of organisms in it during life is of pathological significance. A migration of organisms from the tissues, and particularly from the intestinal tract, into the circulation frequently takes place shortly before death. Blood cultures should be made in the following conditions : — Infective endocarditis ("Progressive," "Ulcerative," " Malignant " endocarditis) differs from the simple acute and the simple chronic endocarditis in several particulars. On clinical grounds the infective form may be diagnosed by the prolonged and intermittent pyrexia often associated with rigors, by the variability of the murmurs, and by the casting off of emboli, which may lodge in the spleen, kidneys, lungs, or the larger arteries or veins. The main pathological dis- tinctions of this disease consist in the presence of organisms in the blood and the nature of the cardiac lesions, which differ from those of simple endocarditis in being associated with an actual loss of substance or ulceration of the cardiac valves or walls. The complete proof of an infective as opposed to a simple endocarditis rests during life upon a demonstration of the causative organism in the blood. In simple chronic endocarditis the blood is sterile, and a positive blood culture means that the condition has become infective. Unfortunately in a considerable percentage of cases of infective endocarditis the organisms cannot be recovered by the ordinary methods, and a negative blood culture is very little evidence against the infectivity of the process. The most common variety of infective endocarditis is that which occurs in rheumatic subjects. After two or more attacks of rheumatic fever have crippled the cardiac valves the disease may change into the ulcerative variety. The organisms obtained from the blood in these cases are almost p. 6 82 CLINICAL PATHOLOGY. invariably streptococci, which differ in many respects from the ordinary Streptococcus pyogenes. The exact role of these streptococci is at present not certainly known. It is held by some that rheumatic fever is a streptococcal infection, and that if the resistance of the individual fails, the local lesion may progress to ulceration and the organisms invade the blood-stream in overwhelming numbers. Others believe that rheumatic fever is a disease of unknown aetiology, and that the streptococci found in the blood in ulcerative endocarditis are secondary invaders which have attacked the crippled valves and produced ulceration in them. . Less commonly ulcerative endocarditis may arise quite independently of rheumatic infection, and may be associated with organisms other than streptococci, as the pneumococcus, or more rarely the gonococcus. The pneumococcus may be present in the blood in association with a typical ulcera- tive endocarditis, and may be found as a sequel to lobar pneumonia. Primary lesions other than cardiac. — Organisms may get into the blood from numerous peripheral lesions and give rise to a condition which has been known as septicemia. In such cases recent vegetations may be found on the cardiac valves, just as localised areas of inflammation accompanied by collections of organisms may occur in other parts of the body. Actual ulceration of the valves and of the heart wall may also occur. This class of infection is best considered apart from the cases of ulcerative or infective endocarditis, in which the cardiac lesion is primary and predominant. These infections include puerperal septicaemias, in which the organisms gain entrance through the genital tract and may follow numerous surgical conditions, such as whitlow, urethritis, or suppuration in the middle ear. The organism most frequently obtained from the blood is a streptococcus. The coccus usually grows readily, can be cultivated in the great majority of the cases, and has all the characters of the ordinary Streptococcus pyogenes. Organisms less commonly obtained are the pneumococcus, and rarely, in cases of general blood infection following a urethritis or cervicitis, the gonococcus. General diseases. — In certain general infections the causa- tive organism can be obtained from the blood at some time during the course of the disease. Pneumonia, Malta fever, THE PARASITOLOGY OF THE BLOOD. 83 and typhoid fever are among the fevers which have been com- paratively recently recognised as general diseases associated with characteristic local lesions. The value of the results obtained from blood cultures. — -In infective endocarditis a positive blood culture is definite diagnostic evidence of the progressive nature of the cardiac lesion ; it is also very strongly suggestive of a fatal termination in the near future. A single negative result is no evidence against the presence of infective endocarditis. Vaccines may be prepared from the organisms obtained, but it is now recog- nised by most observers that vaccine treatment of ulcerative endocarditis is practically worthless, and if any other mode of treatment in any way capable of arresting the disease were available vaccines would be gladly abandoned. In local lesions associated with clinical evidence of a general infection the cultivation of organisms from the blood confirms the diagnosis, and is naturally of serious import. A consider- able proportion, however, of such cases, in which the Strepto- coccus pyogenes is obtained from the blood, recover, and a vaccine should be prepared and given as soon as possible, since there is reason to believe that vaccines not only do good, but, in conjunction with surgical treatment of the local lesion, they may be the essential cause of recovery. In typhoid fever the cultivation of the blood in the first week of the disease is of the greatest value. The early diagnosis of typhoid is difficult on clinical grounds alone, and the agglutination test does not become positive before the end of the first week. The bacillus may be isolated from the blood within the first day or two of the attack in almost every case, and may be tested either by cultural methods or by the action of a known typhoid serum upon it. In estimating the significance of organisms obtained from the blood it must be realised that the bacteria present in the culture tubes do not necessarily come from the circulation. Skin contaminations are not extremely infrequent in skilled hands. They are almost the rule, if great care is not taken with the technique. The organisms most frequently obtained in contaminated cultures are staphylococci, particularly S. alius, diphtheroid bacilli, and bacillus subtilis. The mode of performing a blood culture. — The materials required are a sterile syringe and needle, culture media, a 6—2 84 CLINICAL PATHOLOGY. sterile solution of citrated saline, sterile swabs and a towel, a bandage, an alcoholic solution of iodine, and a paint-brush. The syringe should be an all-glass instrument capable of holding 10 c.c. The needle should be a moderately stout one, and must have a sharp point. It is as well always to be provided with two needles and to test the permeability of both before sterilising. The syringe is taken to pieces, and each piece is lightly wrapped in cotton wool to prevent bumping. It is then placed in a flat, round glass dish provided with a cover and of such a size that it can be conveniently transported after sterilisation. Dish and syringe are placed in the steriliser or in an ordinary saucepan filled with water, and boiled for at least half an hour. The needles wrapped in cotton wool should be dropped into the water during the last 5 minutes of the boiling. When the water is cool fit the syringe together, place in the glass dish and fit the cover on, taking care to touch only the external surfaces of the syringe with the hands, which have previously been washed with soap and water. The culture medium requisite for most purposes consists of ordinary beef broth. At least 4 tubes should be made ready. The sterile citrated salt solution consists of normal saline with 0*9 per cent, of sodium citrate added. Only a few cubic centimetres are required, and these should be contained in a small, shallow flask. The iodine solution is the same as that used before ordinary surgical operations and consists of a 2 per cent, solution of iodine in rectified spirits of wine. The blood is obtained as follows :— Before commencing make sure that everthing is within easy reach, and if possible obtain the help of an intelligent assistant. The patient should be lying in bed with the arm selected supinated and drawn well away from the side, but resting on the bed or on a pillow, and with the face turned towards the opposite shoulder. Tie a bandage tightly round the arm in such a way as to compress the main vessels and tell the patient to clench his fist. Choose the largest vein about the bend of the elbow ; this is almost invaribly the median-basilic. The vein can be readily seen and felt in almost all subjects, but occasionally in young well-nourished women it is possible only to feel the vein. If the arm is (edematous the vein may be neither seen nor felt. THE PARASITOLOGY OF THE BLOOD. 85 and in such circumstances it is advisable to expose it as for an ordinary venesection. When the vein has been rendered prominent, place a piece of waterproof sheeting under the arm and paint a considerable area of the skin over and round the vessel with the iodine solution. Wash the hands throughly and surround the patient's arm with a sterile towel. Take the syringe with the needle firmly attached and draw up about 1 c.c. of the citrated salt solution. The object of this solution is to prevent the blood clotting in the needle. Pass the needle slowly and steadily through the skin into the vein, holding the syringe with the needle pointing in the opposite direction to the blood flow and the barrel of the syringe as nearly parallel as possible with the patient's forearm. Directly the needle enters the lumen of the distended vein the blood flows into the citrate solution and the syringe must then be held quite still. Withdraw 10 c.c. of blood and remove the needle and syringe. Immediately press a sterile swab upon the puncture mark and hold it there until the bandage has been released. Divide the blood among the 4 broth tubes, placing 1 c.c. in the first tube, 2 c.c. in the second, 3 c.c. in the third, and 4 c.c. in the fourth. It is found that by varying the proportions of blood and medium in the culture tubes a growth of the organisms is more certainly obtained, and it not infrequently happens that the growth only takes place in the tube containing the least blood. Before leaving the patient bandage a piece of sterile gauze over the puncture wound, and let it be removed the next day. The culture medium employed is commonly broth, but this must naturally be varied with the nature of the organism sought for. In the case of suspected gonorrhoeal infection it is advisable to use blood serum slope cultures. A useful medium to employ for the isolation of typhoid bacilli is sterile ox bile, which inhibits by virtue of its salts the growth of organisms other than members of the typhoid-coli group. The media should be incubated at 37° C. for 24 hours, when a sub-culture is made from each broth tube on to an agar slope and the tubes are again incubated. The majority of organisms grow in from one to two days, but some of the streptococci found in cases of infective endocarditis grow very slowly, and all cultures should be kept at least 7 days before they are finally pronounced to be sterile. Even if no 86 CLINICAL PATHOLOGY. visible growth be seen in the broth it is advisable to make and examine films and to sub-culture at intervals on to solid media. The organisms present, having been obtained in pure culture, should be examined as to their nature by the ordinary methods (Chapter XL). Other bacteria which may exceptionally be obtained from the blood are the tubercle bacillus, influenza bacillus, the bacillus coii, and the anthrax bacillus. The isolation of the tubercle bacillus is too uncertain to be of clinical value. The anthrax bacilli may in the terminal stage of anthrax septicaemia in the human subject be extremely numerous, and may be actually demonstrated in film preparations. In no other infection is the demonstration of bacteria in blood films within the range of ordinary probability, and it should not be attempted. The parasitology of the blood in tropical diseases. — Persons who have lived in tropical or sub-tropical climates may on their return to this country still harbour in their blood parasites with which they have become infected. Some acquaintance with the more important blood parasites is therefore necessary in making a clinical diagnosis. The follow- ing brief description should be supplemented by reference to the larger text-books on tropical medicine : — Malaria. — Ague is now practically non-existent in England, although it was until comparatively recently endemic in the fen districts, and members of the anophelinse, the mosquitoes responsible for the spread of the disease, are still to be found there. Those who have lived in malarial countries on their return to England are apt to look upon any febrile condition as malarial, and it indeed appears that many who have been infected over long periods are subsequently liable to consider- able rises of temperature from comparatively trifling causes. It is very exceptional, however, to demonstrate the parasite in the blood after more than a year's residence in this country. Patients recently returned from infected countries may harbour the parasites in considerable numbers. In all suspected cases it is advisable to withhold quinine until the blood has been examined in order that the diagnosis may be confirmed and the type of organism determined. The malarial parasites are three in number : the quartan, the tertian, and the benign tertian. Owing to the frequency PLATE V. , j^. : ;v; v •■"." j PLATE V. Benign Tertian. Quartan. Sub-tertian "Rings." Sub-tertian "Crescents. Malarial Parasites. (Leishman's Stain.) THE PAKASITOLOGY OF THE' BLOOD. 87 of mixed infections the temperature chart is not as a rule a sufficient guide to the nature of the organism, which must be identified by means of the microscope. The quartan and the benign tertian forms sporulate in the peripheral circulation, and are the two parasites most easily confused. The quartan is feebly amoeboid, its pigment granules are coarse, and the parasite commonly fills the red cell without distending it. The rosette contains 8 to 10 segments. The benign tertian is the parasite most commonly met with : it is actively amoeboid and contains fine pigment granules ; it only partially fills the red cell, which is almost always enlarged, frequently shows polychromatophilic degeneration and commonly contains numerous chromophilic granules known as Schuffner's dots. The rosette contains 15 to 26 segments. The fresh blood should always be examined in order to observe the activity of the parasite and the dancing movements of the pigment granules. It is advisable to use a j^-inch objective, and if the blood is examined immediately a warm stage is unnecessary. The rosette forms are not commonly met with in this country. The great majority of the parasites in a preparation made with Leishman's stain show an irregularly-shaped blue body containing a small knot of purple- staining chromatin and black pigment granules. The malignant tertian parasite is a more serious infection, responds less readily to quinine, is frequently associated with a very grave anaemia, and may be complicated by blackwater fever. This parasite sporulates in the internal organs and the forms present in the peripheral circulation are readily distinguished, since the commonest appearances met with are the so-called signet rings and crescents. In Leishman-stained preparations the " signets " show as small delicate blue rings with a knot of purple chro- matin at one spot on their circumference. The crescents are blue crescentic bodies containing a central cluster of black pigment granules. The crescents appear at first sight to be lying free in the blood, but on closer inspection a narrow rim of the cytoplasm of the red blood corpuscle can be made out, often bridging the concavity of the crescent. The malignant tertian parasites are commonly very scanty and require a prolonged search before they can be demonstrated. The best method of looking for all forms of malarial parasites is to obtain the fresh blood and to make films in the ordinary 88 CLINICAL PATHOLOGY. way. The films should be stained by Leishman's stain. A prolonged search may be necessary, and if the parasites cannot be found by the usual methods it is advisable to make the films as thick as possible, and when dry to hsemolyse them by dipping them in tap-water until no more haemoglobin comes out. They should then be stained in carbol thionin for 3 minutes, washed in water and blotted dry. Trypanosomiasis. — Trypanosome infections in man are rarely met with and never arise in this country, but the organisms are so widely spread and so fatal to man and animals that a very short account of them may be given here. Try- panosomes are to be found in the blood of a large variety of animals, and in many of them appear to produce no ill effect, while in others they cause disease and often a heavy mortality. In man trypanosomiasis is the cause of sleeping sickness, a disease which is incurable, has almost depopulated vast areas of country, and is still extending into districts previously free. The more important trypanosomes are the following : — T. lewisi, the rat trypanosome. T. evansi, which attacks camels, elephants, etc., and is the cause of the disease known in India as " Surra." T. brucei, which attacks horses and bovines and produces the disease called " Nagana " in Africa. This parasite is found also in the native antelopes, which appear to be immune to its poison and to act as reservoirs for the infection of the domestic animals. T. brucei is spread by a tsetse fly, Glossina morsitans. T. gambiense is the cause of sleeping sickness in man, and is spread by another biting fly, Glossina patyalis. In the early stages of the disease the trypanosomes are present in the blood and more numerously in the lymphatic glands and may then cause few symptoms. It is only in the late stages that the organisms gain access to the central nervous system, are found in the cerebro -spinal fluid, and produce the symptoms of the disease. It may be mentioned that the most constant early symptom of sleeping sickness is insomnia. In order to demonstrate the presence of the trypanosomes in a suspected case the blood should be examined both in the fresh state and in ordinary films stained by Leishman's stain. The parasite is, however, frequently scanty in the peri- pheral circulation, and it may be necessary to withdraw THE PAKASITOLOGY OF THE BLOOD. 89 several cubic centimetres of the blood from a vein, mix them in citrated salt solution, centrifuge, and examine the deposit. Usually the lymphatic glands are enlarged, and most frequently the cervical glands, and the simplest method is to puncture the most prominent gland with a hypodermic needle and syringe and make films from the small quantity of fluid obtainable. By this method trypanosomes can usually be demonstrated with ease. In the later stages the cerebrospinal fluid may be removed by lumbar puncture, centrifuged, and the deposit examined. An excess of lymphocytes is present in the fluid in addition to the parasites themselves. If parasites cannot be found by any of these methods, the conclusive proof of absence of infection rests upon the inoculation of susceptible animals with the patient's blood. The trypanosome as seen in the fresh blood is actively amoeboid and provided with a free flagellum at its posterior extremity. In stained preparations the following points may be made out. Near the anterior rounded extremity is a small, deeply staining round spot, the blepharoblast. Posterior to the blepharoblast is a vacuole, and posterior to this again is the nucleus, which is commonly situated near the centre of the trypanosome. The undulating membrane can be seen arising from the blepharoblast, winding along the free border of the parasite and terminating in the flagellum at the posterior extremity. The identification of the different species of trypanosomes must be left to the expert. Leishmania. — Leishmania is in reality a variety of try- panosomiasis. The mature trypanosome, however, is in this instance absent from the human tissues and is represented by a developmental form known as the Leishman-Donovan body. The disease produced is known as kala-azar or black fever, is almost invariably fatal, and is accompanied by considerable pyrexia and marked enlargement of the spleen. The diag- nosis of the disease from malaria and other causes of splenic enlargement rests upon the demonstration of the parasites. The organisms may be searched for in the blood, in which they may be found within the leucocytes and usually the large hyaline cells. The parasites, however, are commonly scanty in the peripheral circulation, and it may not be possible to find them even after a prolonged search. The most practical method is to perform puncture of the spleen, a proceeding 90 CLINICAL PATHOLOGY. which has in the past been attended by fatal results owing to the wounding of considerable blood vessels with a large needle. If puncture of the spleen is performed with the ordinary hypodermic syringe armed with the usual fine needle, the operation is practically devoid of risk and the minute quantity of splenic fluid obtained is quite sufficient for diag- nostic purposes. Films should be made from the fluid and stained with Leishman's stain. The organism is found for the most part within the splenic cells and appears as a small, rounded body containing a round nucleus and a small, deeply staining rod-shaped micronucleus or blepharoblast. The organism can be cultivated outside the body in citrated blood at 20° C, and flagellated trypanosomes are produced from the Leishman-Donovan bodies. It is probable that the organism usually passes through its developmental stage in a bug, and is by this agency inoculated from one human being to another. Similar bodies are present in cases of " Oriental sore " and in other varieties of European splenomegaly. Spirochetosis — The only spirochaBte which can be demon- strated in man in the circulating blood is that of relapsing fever, known as the spirochceta recurrentis or the spirillum Obermeieri. There is every reason, however, to suppose that the European form, the spirochceta recurrentis, differs from the Egyptian, the African, the American and the Asiatic varieties. The disease is accompanied by febrile attacks of 5 to 7 t days' duration, followed by periods of apyrexia and one to two relapses. The spleen is usually enlarged. Eelapsing fever is still common in Russia and other parts of Europe, but has become practically non-existent in this country, although cases are still to be met with in Ireland. The mortality rate is under 15 per cent. The spirochaBte is considerably coarser than the syphilitic organism, and the spirals are less regular ; it has a length of about 12 fi and is actively amoeboid. The fresh blood during the febrile stages shows the organisms in large numbers ; they are also readily recognised in stained films. Filariasis. — The most common embryo of the nematode worms which may be found in the blood of man is that of F. iioctuma. The parent worms live in the lymphatics of the limbs or trunk, and pass their young into the lymphatic stream and so into the blood. The parent worms by blocking the lymphatic circulation may give rise to elephantiasis, or, in the PLATE VI. r v m Tr / panosomes - Leishman-Donovan Bodies. Camel s Blood. (Lehman's Stain.) (Leishman's Stain.) Spirilla of Eelapsmg Fever. Filaria and sheath CJ^Z S t ^ HEemolysed Blood Film. (LeishmansStam.) (Hematoxylin.). PLATK VI. THE PARASITOLOGY OF THE BLOOD. 91 rare cases in which they are located in the bladder, to chyluria. In the majority of cases, however, they give rise to no symptoms. The embryos appear in large numbers in the peripheral blood at night, and during the daytime retire to the heart and large vessels. The embryos in the blood are enclosed in a sheath from which they cannot escape, but within which they can move. The embryos are sucked from the blood by the mosquito (the culex fatigans), and in its stomach get rid of their sheath. They subsequently bore their way through the stomach wall of the mosquito and pass into the thoracic muscles, where they undergo further development. The worm then works its way into the proboscis of the mosquito, and so passes again to man. The filariae of man are represented by three main species, F. nocturna, F. diurna and F. pcrstans. The embryos should be looked for in the fresh blood and in stained preparations made from comparatively thick films. In the case of F. nocturna the blood should be examined between 7 o'clock at night and 7 o'clock in the morning. The embryos are easily recognised, and should be sought for under a low power (e.g., f-inch objective) of the microscope. For the differentiation of the various species the student should refer to works on tropical medicine. CHAPTER VII. THE CHEMICAL AND PHYSICAL EXAMINATION OF THE BLOOD. The spectroscopic examination of the blood. — In order to examine the blood in this way all that is necessary is to prick the patient's thumb and squeeze 3 or 4 drops of blood into a clean beaker containing about 10 c.c. of distilled water. A portion of the mixture is then transferred to a test tube and diluted until it is of a pale pink colour. The test tube is then examined with a direct vision spectroscope. The normal spectrum of the blood is of course that of oxyhemoglobin, and the two characteristic absorption bands are placed between the D and E lines. The band nearest the D line is darker, narrower, and more sharply defined than the other band. On adding a few drops of ammonium sulphide to the test tube, inverting the tube several times and allowing it to stand for a few minutes, the two absorption bands become merged into the one band of reduced hemoglobin. Carbonic oxide poisoning may occur after exposure to coal gas or to the fumes of charcoal stoves. The colour of the blood in these cases is brighter than normal, and has a characteristic cherry-red appearance. The diagnosis of the condition is made certain by an examination of the spectrum. The absorption bands of carboxyhsemoglobin are two in number, and are situated slightly nearer the violet end of the spectrum than those of oxyhemoglobin. The difference in position of the carboxyhernoglobin and the oxyhemoglobin bands is, how- ever, so slight as to be appreciated with difficulty when working with a small spectrum. The nature of the spectrum is definitely determined by adding ammonium sulphide and finding that no alteration takes place in the bands, which remain distinct and separate since the carbonic oxide combination with hemoglobin is more stable than that of oxygen. Methaemoglobinsemia may be produced by certain drugs, particularly phenacetin and antipyrin, even when taken in medicinal quantities by susceptible patients. The same condition may arise among workers in aniline dyes and PLATE VIL C D -E T) \65 60 1 55 50 ^H JS 60 II III 1 ^^^ 50 //'■ 65 60 | 1 | I | | 1 | 1 | | | ^ 50 fif 60 | 55 50 1 IT I I I I i ill 1 1 i I | i i iii ' 1 Iii J g fi liTiinfi ii i f 1 1 ■! | j i I | I ; 1 | | Mill 1,1,! ^^^^R ■ ^H KSfl^H .. 65 SO , , ' , 55 . , ■50 , !///■ 65 60 55 50 ^^^^^^^■flsoMty^x-yi ■ ki , i i f i i f i 5p xl 1 Oxyhemoglobin. Hemoglobin. Carboxyhremoglobin. Methaemoglobin. HaBmochromogen in alkaline solution. Hematin in acid alcohol. Acid HsBmatoporphyrin. Alkaline Hemalxmorphyrin. Urobilin. Uroerythrin. D E h Absorption Spectra. (From Hoppe-Seyler-Thierfelder's " Handbuch der Physiologisch-und- Pathologisch-Chernischen Analyse.") EXAMINATION OF THE BLOOD. 93 nitro-glycerin factories from the inhalation of nitrobenzol com- pounds, and may follow poisoning by chlorate of potash and by pyrogallic acid. The patients are markedly cyanosed, and the colour of the blood in severe cases is distinctly brownish. It may be added that in a considerable proportion of cases of hemoglobinuria the hemoglobin is present in the urine as methemoglobin. Methemoglobin contains the same amount of oxygen as oxyhemoglobin, but in different combination ; it can be produced artificially by adding a few drops of potassium ferricyanide to diluted blood and warming gently. It is well, in cases of doubt, to make an artificial solution of this nature for purposes of comparison. The characteristic absorption band of the spectrum is a narrow, sharply denned band in the red (between C and D). In dilute solution other bands apjiear, including two bands corresponding to those of oxyhemoglobin. On adding ammonium sulphide to the methemoglobin solu- tion, the band in the red disappears at once, and the two bands of oxyhemoglobin more slowly merge into the single band of hemoglobin. Sulph-haemoglobinsemia is an extremely rare condition, accompanied clinically by considerable cyanosis. There are very few recorded cases, and there is reason to believe that all of these are not genuine. Sulph-hemoglobin can be artificially produced by the addition of a small volume of sulphuretted hydrogen, and it has been suggested that the origin of the condition should be sought in the intestinal tract. The spectrum of sulph-hemoglobin gives a band in the red similar to that of methemoglobin, but nearer the violet, as well as the two bands of oxyhemoglobin. On adding ammonium sulphide the band in the red persists, while the two bands of oxyhemoglobin slowly merge into one. The Chemical Examination of the Blood. Lipaemia. — By lipemia is meant the presence of fat in readily demonstrable amount in the blood. The condition is a rare one, and has been described in a variety of affections including tuberculosis, alcoholism, and nephritis. The disease most frequently associated with lipemia, however, is diabetes, and particularly that variety of diabetes which attacks young subjects. The condition has been diagnosed clinically by direct observation of the fat droplets circulating in the retinal 94 CLINICAL PATHOLOGY. vessels. Lipsemia can be recognised by withdrawing blood in a serum tube in the same manner as for a Widal reaction and, after allowing to stand for half an hour, centrifuging at a moderate speed. The blood obtained in this way has a most characteristic appearance, the serum loaded with fat forming an opaque, turbid layer above the red cells. It is necessary to prove that the turbidity of the serum is due to fat. A few drops of the serum should be placed on a slide with a cover- slip over them and examined with the microscope for the presence of fat droplets. In addition, films should be made on slides, fixed in formalin vapour for 15 minutes, stained with Scharlach E. for from 12 to 21 hours, dipped in 75 per cent, spirit for a few seconds, washed in distilled water and mounted in Farrant's medium. The fat droplets are stained a bright red. In certain other conditions, and particularly in chronic nephritis with cedema, a serum may be obtained from the blood milky in appearance and strongly opalescent. The serum separates rapidly and may be mistaken for a fatty serum. The opalescence in these cases is not due to free fat, but to an unknown substance of a proteid nature. A similar opalescence may be present in other body fluids, and when found in fluid withdrawn from the peritoneal cavity has to be distinguished from the veiy much rarer condition of chylous or fatty ascites. Glycogensemia (iodophilia). — The iodine reaction in the blood consists in the presence of iodophil granules within the polynuclear neutrophil leucoc} T tes. There has been considerable dispute as to the nature of these granules, but the consensus of opinion would seem to be that they are composed of glycogen either in combination or lying free within the cells. A positive iodophil reaction may occur under a variety of conditions, but is very constantly present in all tox&emias, and particularly in septic infections accom- panied by suppuration. The reaction has been largely used as a clinical method of diagnosing the presence of pus, but owing to the wide varietj^ of causes capable of producing the condition, as well as to the varying degrees of intensity of the reaction itself and the consequent difficulty of interpreting the results, a positive reaction is of no great assistance. In cases of doubtful suppuration a negative iodophil reaction is strongly opposed to the presence of pus. EXAMINATION OF THE BLOOD. 95 The reaction is performed as follows : a blood film is made in the ordinary way on a cover- slip and dried in the air ; the film is then mounted in and simultaneously stained by the following solution : — Iodine . . . . . . .1 gramme Potassium iodide . . . . .3 grammes Distilled water 100 c.c. Gum acacia, sufficient to make a mixture of about the consistence of Farrant's solution. The smallest possible quantity of the fluid as a mountant should be used in order to avoid opacity. Preparations made in this way will keep for several weeks. On examining the preparations with an oil immersion lens two kinds of reaction are seen, an extra-cellular and an intra- cellular. The extra-cellular reaction consists of purple-brown amorphous fragments lying free in the plasma, and is of little significance since it is present in normal blood. The intra- cellular reaction is practically confined to the polynuclear neutrophils and varies from a diffuse brown staining of the cytoplasm due to the presence of innumerable minute iodophil granules, to the appearance of coarse, intensely stained, purple-brown granules in the periphery of the cells. The polynuclears of normal blood stained by iodine show only a faint lemon-yellow colour in the cytoplasm. The red cells stain a faint orange and show no variation in normal or in pathological blood. Cholsemia. — By cholremia, in this connection, is simply meant the presence of bile constituents in the blood. In most conditions associated with jaundice the amount of bile pigment in the blood is commonly sufficient to colour the skin and conjunctivae to such a degree as to render any special examination unnecessary. The pigment, however, can be demonstrated in the serum long before definite jaundice can be recognised on clinical grounds. The amount of bile which may be present in the blood in some cases of cirrhosis of the liver, pernicious anaemia and congenital family cholaBmia can commonly only be detected by a chemical examination of the blood. Such small amounts of pigment are also rarely met with in apparently normal individuals as a periodic phenomenon. To test for the presence of bile pigment in the blood 96 CLINICAL PATHOLOGY. withdraw a sample of blood in a serum tube, stand for a time and centrifuge. The presence of bile is rendered quite evident by the bright yellow colour of the supernatant serum. The colour of the serum is hardly recognisable by artificial light and should always be examined by daylight, a precaution which applies equally to the clinical examination of a jaundiced person. The coloration of the serum by bile pigment can be confounded with that of a serum tinged with haemoglobin, as may occur if the blood is carelessly withdrawn and centrifuged. The bile colour is more closely simulated by the greenish-yellow pigment present in the serum of nearly all cases of pernicious anaemia. A certain percentage of these cases are in addition actually jaundiced. The presence of bile pigment should always be confirmed by a chemical test such as Gmelin's. The serum is pipetted off and allowed to soak into as concentrated an area as possible of a clean filter paper ; a drop of fuming nitric acid is then placed in the centre of the serum area and the play of colours — green, blue, red and yellow — looked for in the rings which form at the junction of acid and serum. Uric acid in the serum. — The presence of uric acid in the blood, in excess, can be demonstrated by the following time-honoured experiment. A few cubic centimetres of serum are placed in a watch-glass and made distinctly acid with 28 per cent, acetic acid. One or two threads of cotton are left soaking in the mixture, which is allowed to evaporate at room temperature. Crystals of uric acid deposit on the threads in about 24 hours. The reaction is most commonly successful in cases of gout, but is of no particular clinical significance, since it may fail to appear in the blood of obviously gouty patients and may be present in a considerable variety of other conditions. The exact estimation of the uric acid in the blood has no serious application in medicine. The specific gravity of the blood. — The specific gravity of the normal blood is about 1*060, and except in cases of dropsy varies directly with the percentage of the haemo- globin. The estimation of the specific gravity is of very little clinical importance, but has been made use of in the past largely as a method of estimating the haemoglobin. The simplest method of taking the specific gravity is to prepare a EXAMINATION OF THE BLOOD. 97 mixture of chloroform and benzol of a specific gravity of l - 060, and add a drop of blood to the mixture. If the blood (which does not mix with these liquids) sinks to the bottom, add chloroform ; if the drop rises to the top, add benzol until a mixture is obtained in which the drop of blood remains stationary in the body of the liquid. The specific gravity of this mixture is the specific gravity of the blood. The alkalinity of the blood. — The degree of alkalinity of the blood is fairly constant in health and varies considerably in an important group of morbid conditions which includes diabetic coma and the toxaemias of pregnancy. The estima- tion of the alkalinity of the blood has not, however, come into general clinical use, partly because no simple and at the same time accurate method has yet been devised. For this pur- pose Wright's method or one of its modifications may be used, but it must be acknowledged that little information of any clinical value is to be obtained from such methods. The following technique may be adopted. Obtain a sample of blood serum in the ordinary way. Have ready a series of small bottles containing standard solutions of N N N N sulphuric acid ranging from -^-^-^ . . . — . To each o I'D 1 o bottle of acid add 1 or 2 drops of dimethyiamidoazobenzol. In a Wright's pipette take up 1 volume of the serum and 1 volume of one of the acid solutions. Mix thoroughly together in a white porcelain dish. If the resulting mixture is yellow repeat with a stronger acid until a mixture is obtained with a distinctly orange-red tinge. That strength of acid is taken as the equivalent of the alkalinity of the serum which last gave the yellow colour. The index of alkalinity is recorded as the fraction of the equivalent acid solution. For . N example, if — acid neutralises the serum the index is OI66. In normal cases the average is 0*170. In estimating the alkalinity by this method the blood should be withdrawn 3 hours after a meal, and the serum tubes and pipettes used should have been soaked in hydrochloric acid to remove all alkali, repeatedly washed in distilled water and finally dried in an oven at 120° C. The estimation is rapidly performed, but the recognition of the neutral point in the reaction is difficult when dealing with p. 7 98 CLINICAL PATHOLOGY. such small quantities, and the experimental error is consider- able. The oxygen content of the blood. — The estimation of the oxygen content of the blood, and from it the total volume of the blood in the body, has been rendered possible by the researches of Haldane and Lorraine Smith. The method which they employ is beyond the scope of ordinary clinical pathology, and, moreover, depends upon an inhalation by the patient of a volume of carbonic oxide gas, a proceeding which is certainly not devoid of risk in unaccustomed hands. The method may be briefly indicated here. The patient is made to inhale a measured volume of carbon monoxide and a few minutes later a sample of his blood is taken and the extent to which the haemoglobin has become saturated with the CO is estimated. From the amount of gas inhaled and the extent to which the haemoglobin has become saturated in the sample the quantity of CO capable of being taken up by the whole of the patient's blood can be calculated. For example, if 100 c.c. of CO were inhaled and the sample is one-fifth saturated, the blood would have been 100 per cent, saturated by 500 c.c. and the capacity of the blood for CO, and similarly the oxygen capacity of the blood would be 500 c.c. The estimation of the total volume of blood in the body can be farther determined by comparing the colour of the patient's blood with the colour of an equal sample of ox blood, the oxygen capacity of which has been previously determined. If, for example, the patient's blood has the same colour as a sample of ox blood every 100 c.c. of which has been found capable of absorbing 20 c.c. of oxygen, and the total oxygen capacity of the patient's blood is 500 c.c, since 20 c.c. of oxygen can be taken up by 100 c.c. of his blood and his total blood can take up 500 c.c. of oxygen, his total blood measures 100 X 500 KAA ^r or 2,500 c.c. The total volume of blood in health has been found to vary between 3,000 and 4,500 grammes, or from one-thirtieth to one-sixteenth of the body weight. In chlorosis the total oxj-gen capacity of the haemoglobin remains normal, while the total volume of the plasma is greatly increased ; consequently the percentage of haemoglobin as estimated in the usual way is low, but is actually only EXAMINATION OF THE BLOOD. 99 relatively diminished in proportion to the bulk of blood. In pernicious anaemia, on the contrary, the haemoglobin is actually diminished. The most marked increase in the total volume of blood occurs in Osier's disease, or splenic poly- cythsernia, in which the blood volume may reach three times that of the normal. In this disease, as already mentioned, the numbers of red cells to the cubic millimetre may be double the normal, so that the actual number of red cells in the whole body may be 6 times the normal amount. It has been indicated that no purely chemical examination of the blood has at the present any wide application in clinical medicine. Certain methods, however, which have been described in previous chapters might properly have been included here. A differential leucocyte count, for example, depends upon the chemical affinity of the cell and its granules for different dyes. Such a widely used test as the Wassermann reaction might also be considered as a chemical process. It is further probable that other and more strictly chemical methods of examination will come into general use in the near future. No description has been given here of the estimation of the various ferments in the blood, but a considerable amount of experimental work in this direction has recently been per- formed, and the relation between carcinoma and the amount of trypsin in the blood has been made use of as a method of diagnosis. The variations in the amount of this ferment in carcinoma are, however, small, and since the causes of such variations are by no means confined to carcinoma, it may be said that the method has little clinical value at the present. 7—2 SECTION II. BACTERIOLOGY. CHAPTEE YIII. Introductory — Table of Classification. CHAPTEE IX. The Cocci — The Gram -positive Bacilli. CHAPTEE X. The Grani-negative Bacilli — Spirilla — Streptothricese — Hyphomycetes. CHAPTEE XI. Bacteriological Methods— General and Special. CHAPTEE XII. Vaccines — An ti- sera. CHAPTEE XIH. Preparation of Culture Media — Staining Eeagents. CHAPTER VIII. INTRODUCTORY — TABLE OF CLASSIFICATION. Before considering the application of bacteriology and bacteriological methods in the diagnosis and treatment of disease it is necessary to describe as briefly as possible the general means at our disposal for the investigation of the chief pathogenic bacteria. For the identification of bacteria we are dependent upon a study of their morphology, staining properties and cultural characters, their pathogenicity to animals, and upon certain special methods of investigation. Morphology. — A study of the morphology of pathogenic organisms gives much important information, but mainly of a preliminary kind. Very few bacteria can be even approxi- mately identified from their appearance alone. Micro-organisms are primarily classified according to their shape. A round dot is a coccus or micro-coccus ; a rod is a bacillus ; a twisted spiral a spirochete. The shape may also give more detailed information, since some cocci are not absolutely round, but may have their opposed surfaces flattened as in the case of the gonococcus, or may have pointed ends as the pneumococcus. Diphtheria bacilli may present racquet or club-shaped extremities of a very characteristic appearance, while tubercle bacilli stain in an irregular or beaded pattern. Further information is obtained from the grouping of bacteria, either as they appear in the tissues or as they are obtained from culture media. Cocci which characteristically occur in pairs, such as the pneumococcus or gonococcus, are classed as diplococci, those with a chain arrangement as streptococci, and those which appear in groups or clusters as staphylococci. The bacilli are commonly not differentiated in this manner ; some bacilli, however, such as that of anthrax, tend to grow in chains and may be called strepto-bacilli. Others, such as the Morax Axenfeld bacillus (the cause of one 102 CLINICAL PATHOLOGY. variety of conjunctivitis), occur in pairs and are known as diplo-bacilli. The classification of organisms in this manner, according to their grouping, depends upon a knowledge of the characteristic behaviour of the bacteria, and this can only be determined by an investigation of their appearance in body tissues as well as by their mode of growth in various culture media. A paired coccus seen in a film of pus may be a diplococcus, a streptococcus, or a staphylococcus. It is necessary to isolate the organism in pure culture and examine it when growing in a liquid medium such as broth, and on a solid medium such as an agar slope. Diplococci occur mainly in pairs in all media. A streptococcus grows in long chains in broth, and frequently only in short chains, or even small clumps on agar. Staphylococci show their clump arrange- ment best on agar, and frequently appear in pairs or even very short chains in broth. The size of organisms is on the whole of little assistance in identification, since the same organisms growing under favourable conditions may be very appreciably larger than when the surroundings are unfavourable. A colon bacillus, for example, may be so short and stout as to be almost mistaken for a coccus, or it may be long and thin. A coccus may vary greatly in size, and may in some instances, such as in the case of the pneumococcus, and more rarely the strepto- cocci, be so elongated as to resemble a bacillus. At the same time with some bacilli, such as the anthrax bacillus, the individual members are very commonly stout and long. The motility or non-motility of organisms affords a further clue to their identification, since some organisms, such as the diphtheria bacilli, have no power of spontaneous movement, while other organisms are actively motile. The relative motility of organisms in the same group, such as members of the spirochete family and bacilli of the coli-typhoid group, is of considerable assistance in differentiation. It must be recognised, however, that different strains of the same organism, and even different sub-cultures of the same strain, may show marked variability in movement. The colon bacillus, for example, is as a rule sluggishly motile, but may on occasion move as briskly as strains of the more constantly motile typhoid bacillus. The motility of some organisms depends, in part at least, on the presence of flagella, INTRODUCTORY— TABLE OF CLASSIFICATION. 103 for the demonstration of which special stains are required (page 163). Capsules are present around some organisms, but not others. The presence of capsules may be made use of in identifying such an organism as the pneumococcus, which is capsulated in preparations made from the body tissues, but loses its capsule when cultivated on artificial media. The demonstration of capsules requires a special method of staining (page 162), and great caution must be exercised in recognising capsules in films stained with the ordinary dyes, since there is a tendency for clear areas to appear in the immediate vicinity of the organisms owing to a shrinkage of the fluid constituents of the pus from the bacteria, the clear empty areas having the appearance of capsules. It is, however, rarely necessary to investigate specially the presence or absence of capsules. Spores are confined to certain bacilli, and the presence of spores is of great value in differentiation, since the majority of pathogenic bacilli never produce them. The spore-bearing bacilli do not, however, always show spores, and may do so only when grown in the appropriate media. The anthrax bacilli commonly are devoid of spores in preparations made from the pustule, while in broth cultures spores only may be found and no bacilli. The situation of the spore is of further assistance, since it may be central or, as in the case of the tetanus bacillus, terminal. Spores are readily recognised in films stained with the ordinary dyes, and may be further identified by special methods (page 162). Staining properties. — The staining properties of bacteria are in effect micro-chemical tests of their composition, and for this reason are of great importance in their identification. The majority of micro-organisms stain with the ordinary dyes, such as methylene blue, carbol fuchsin, and carbol thionin ; the minority require special staining processes for their differentiation. The less important and lowly pathogenic mouth spirochetes, for example, take fairly readily the ordinary stains, whereas the spirochete of syphilis is unstained by them, and can by this method be differentiated. A most important staining method in use for the identification of organisms is that of Gram. Gram's method depends upon the use of iodine as a mordant. All or nearly all organisms are stained by gentian-violet, and are again decolorised if 104 CLINICAL PATHOLOGY. washed in alcohol. If, however, the organisms after being stained with gentian-violet are exposed to Gram's iodine solution, the stain is fixed in some organisms so as to be no longer dissolved out in the spirit, but not into other organisms. Those bacteria which remain coloured are called " Gram- positive," those which lose their stain in the spirit are known as "Gram-negative." The majority of the cocci are Gram- positive, and of the bacilli Gram-negative. Another very important staining reaction is that known as the Ziehl-Neelsen method, and is used for the identification of the tubercle bacillus. The tubercle, leprosy, and smegma bacilli can be stained with hot carbol fuchsin, and when thus stained resist decolorising with acids, whereas other organisms lose the stain again when exposed to acids. The bacteria which retain the stain are therefore known as "acid fast," and can by their staining reactions be further differentiated one from another, since the tubercle bacillus retains the carbol fuchsin after exposure to 25 per cent, sulphuric or nitric acid; the leprosy bacillus is decolorised by this strength of acid, but remains coloured in 12 per cent, acid ; while the smegma bacillus is acid fast, but, unlike the tubercle bacillus, is not " alcohol fast," that is to say, it gives up the carbol fuchsin in methylated spirit. Cultural characters. — The identification of the great majority of organisms necessitates a study of their behaviour and mode of growth in artificial media outside the body. Some bacteria do not grow at all on any media with which we are acquainted ; others require special media and fail to grow on those in common use. The majority grow readily on all the ordinary media. To some media substances are purposely added which prevent the growth of one class of organism and allow the growth of another class ; bile salts, for example, inhibit the growth of cocci, but permit the growth of bacilli of the coli-typhoid group. The optimum temperature for the growth of the majority of pathogenic organisms is that of the human body, namely, 37° C. Exceptional organisms, such as the glanders bacillus, grow best at a somewhat higher temperature. The temperatures within which bacteria will grow and the temperatures at which they cease to live form a part of the complete investigation of their natural history. The time taken for a visible growth to appear in media is INTRODUCTORY— TABLE OF CLASSIFICATION. 105 usually from 12 to 24 hours. A few organisms grow more slowly, and in the case of the tubercle bacillus little growth is visible in less than 10 days. Most bacteria are capable of growing under both aerobic and anaerobic conditions, but prefer the former. Very few patho- genic organisms are strict anaerobes, the most important being the tetanus bacillus. By a strict anaerobe is meant an organism incapable of growing in the presence of oxygen, and by an anaerobic culture is meant a culture tube placed in an atmosphere from which the oxygen has been removed. A brief account follows of the more usual media employed for the cultivation of organisms and the changes which may take place in them as the result of bacterial growth. Broth is the most universal of all media, and in addition forms the basis of numerous others. Broth is nothing more than a solution of beef extract, with sodium chloride and peptone added. The acidity of the original broth is estimated and sufficient soda is added to make the mixture alkaline to litmus and of a certain acidity to phenol-phthalein (page 181). The majority of organisms growing in broth produce in it a general turbidity, and after a time the deposit of a more or less felted mass of bacteria at the bottom of the tube. Some organisms, such as the cholera vibrio and certain non- pathogenic air bacilli, form also a thin pellicle on the surface of the medium. The streptococci, on the other hand, leave the bulk of the medium as clear as it was before inoculation, and form a stringy granular deposit, both floating free and attached to the sides of the tube. These are the appearances obvious in an inoculated tube ; in addition certain organisms, such as the colon bacillus, have the property of producing indole from the peptone present in the medium, and this has to be tested for by adding yellow nitric acid (containing nitrites), when a rose-pink colour of a nitroso- indole body diffuses through the medium. The cholera vibrio produces both indole and nitrites, so that the pink colour is produced on the addition of pure nitric or sulphuric acid only, a reaction which is known as the cholera-red reaction. On examining therefore an inoculated broth tube after incubation one looks for a general turbidity of the medium, a pellicle on the surface, a deposit at the bottom or a clear medium with a granular deposit down the side, and then 10(5 CLINICAL PATHOLOGY. adds a few drops of yellow nitric acid to test for indole formation. Agar is the most commonly used solid medium, just as broth is as a liquid medium. Agar itself is a carbohydrate derived from the stems of certain Chinese seaweeds, and agar media are prepared by adding this substance to broth. The medium is put up either in the form of " slope," " stab," or " plate " cultures. The slope culture tubes are made by pouring a small quantity of melted agar into a test tube, and allowing it to harden in a slanting position. The stab cultures are tubes filled or partly filled with the medium, and are inoculated by passing an infected platinum wire into the heart of the medium. They may be used for growing organisms under anaerobic or partially anaerobic conditions. The plate cultures are made by pouring the agar into flat, round, shallow dishes, covered with a loosely fitting lid, and known as Petri dishes. The majorhVy of organisms grow well on agar, and one is able to tell from the type of colony produced the class of organism present. Bacilli of the coli-typhoid group grow in a continuous whitish streak with laterally spreading edges up the surface of an agar slope, and in large rounded, opaque colonies, with thin crenated margins and heaped-up centres on plate cultures. Staphylococci produce large, round, opaque colonies with sharp edges, the colonies being white, lemon- coloured or yellow according to the variety of staphylococcus present. Streptococci and pneumococci grow in tiny round translucent colonies barely visible to the naked eye. Gonococci, meningococci, influenza and diphtheria bacilli are among the more important organisms which grow in somewhat similar so- called " pin-point " colonies. On examining an inoculated agar culture, therefore, one looks at the type of colonies present, whether they are large or small, white or coloured, translucent or opaque, whether the margins are rounded or crenated, and whether the colonies are discrete or grown together to form a continuous streak. In the case of stab cultures one looks to see if the growth is more abundant at the surface where there is more oxygen, or in the depths of the medium where oxygen is scanty and anaerobic bacteria grow more readily. Gelatin media consist of broth with sufficient gelatin added to produce a solid medium at room temperature. Since INTRODUCTORY— TABLE OF CLASSIFICATION. 107 gelatin media are liquefied at 37° C. it is necessary to incubate the tubes at or a little above room temperature, that is from 18 to 22° C. Gelatin is put up in slope, stab and plate cultures in the same manner as agar. The most important feature of gelatin as a medium is the fact that some organisms in their growth are able to liquefy it, while others do not. The coli- typhoid group of bacilli do not liquefy gelatin, and can thus be differentiated from such bacilli as proteus and pyocyaneus, which do. Some organisms, such as the pneumococci, which are capable of growing on the majority of the usual media, do not grow on gelatin. In stab cultures a few bacteria, for example anthrax bacilli, send out characteristic lateral processes radiating from the line of the stab. A gelatin medium, therefore, should be incubated at a relatively low temperature and should be examined for the presence and nature of the growth, and in particular for the presence or absence of liquefaction. Litmus milk consists of fresh sterilised milk rendered slightly alkaline with soda and coloured blue with litmus solution. Litmus milk is a most valuable medium, partly because the majority of organisms grow in it abundantly and partly because of the various changes which they may set up in it and by which they may be differentiated. Organisms growing in milk may produce no change in it or may render it more alkaline or, more commonly, may either simply produce an acid reaction in it or both acidify and clot it. Other organisms may not only acidify and clot the milk, but may eventually decolorise the litmus and, further, may peptonise the clot and render it liquid again. The bacillus enteritidis sporogenes first clots the milk, and then by virtue of the gas produced by it blows the curd up the sides of the tube, leaving the whey at the bottom. Litmus milk, therefore, may be unchanged, or may be rendered more alkaline, or may be acidified and remain liquid, or may be both acidified and clotted. Litmus carbohydrate broth. — A series of media may be made with broth to which litmus is added and 1 per cent, of a variety of carbohydrates. The purpose of these media is to test the capability of organisms to break up the carbohydrates present and to produce from them an acid or an acid and gas. The production of acid is shown by the change of colour of the 108 CLINICAL PATHOLOGY. litmus medium from blue to red. In order to observe the formation of gas it is convenient to fill a small tube with the medium and sink it upside down in the culture tube so that the gas may collect in the small inverted tube. Numerous carbohydrates are employed in making the media ; those in fairly common use are glucose, lactose, mannite, salicin and raffinose. In examining the media after inoculation look for the same changes that are to be found in ordinary broth, such as the presence of a general turbidity or of a clear medium with a granular deposit, and in addition observe the colour of the litmus and the presence or absence of gas in the small inverted tube. The production of an acid reaction is not necessarily accompanied by the evolution of gas. Neutral red broth consists of broth with a dye called neutral red added. The medium is a useful one for distinguish- ing organisms, and particularly bacilli of the coli-typhoid group. The colon bacillus alters the red colour of the broth to a distinct yellow and also produces in the medium a green fluorescence ; the typhoid bacillus produces no change other than the general turbidity of its growth. MacConkey's bile salt medium. — This is a solid medium commonly used in plate cultures for the purpose of isolating colonies of the coli-typhoid group from mixtures of organisms. The medium has as its basis agar, and in addition sodium- taurocholate, lactose and neutral red. The presence of the bile salts inhibits the growth of the great majority of organisms other than members of this group. The neutral red gives a different colour reaction with those organisms which ferment the lactose to those which do not. The medium is there- fore extremely useful for the isolation of bacilli from such sources as the urine and fseces, since the cocci and other non- pathogenic bacteria do not grow on it, while the colon bacillus grows in bright red, and the typhoid bacillus in whitish yellow colonies. Special media. — While the above media are commonly used for organisms of ready growth, a minority of pathogenic bacteria do not grow at all on them, or grow very poorly. Numerous special media have been devised for such organisms, and a few are in common use. Blood serum media are essential for the growth of some bacteria, of which the most important is the gonococcus. The blood serum can be used alone in the INTRODUCTORY— TABLE OF CLASSIFICATION. 109 form of slope cultures after inspissating the serum in steam, or it may be mixed with agar. The simplest medium of this nature is made by smearing a drop of sterile blood over the surface of an agar slope. A valuable medium also is " Nasgar," which consists of nutrose, ascitic fluid (or blood serum) and agar. The essential elements of the blood serum may be derived from a variety of sources, such as hydroceles, ovarian cysts and ascitic fluids. The serum is most readily obtained from a large animal, such as an ox or a sheep, and for the purpose of growing the diphtheria bacillus the former source is prefer- able, since this bacillus, whose growth on ordinary agar is apt to be crowded out by the growth of other organisms, multiplies rapidly on ox serum and outgrows the common cocci. The tubercle bacillus is an example of another organism which will not grow on the ordinary media, but will grow in broth or on agar to which glycerin has been added. Another useful medium for the growth of the tubercle bacillus is made from eggs, and is perhaps the most commonly used medium for this purpose : both the white and the yolk of the egg are used and slope cultures of the solidified medium are put up. Another special medium may be mentioned, consist- ing of agar to which glycerin and oleic acid have been added, and which is used for the cultivation of the acne bacillus. Animal inoculation. — Animal inoculation is resorted to for purposes of testing the pathogenicity of an organism, or for isolating it, or for determining its nature. The animals most commonly employed are guinea-pigs, mice and rabbits. A few examples only may be given here of the occasions on which animals should be used. In suspected cases of diphtheria organisms may be isolated which exactly resemble the genuine bacillus in appearance, in staining reactions and in cultural characters, yet may differ from it in the very important circumstance that they are non-virulent and produce no toxin. In cases of doubt the only reliable test is to inoculate a guinea-pig with a suspension of the bacteria. The innocuous or so-called diphtheroid bacilli do not affect the animal ; the genuine diphtheria bacilli kill it within 36 hours. The test may be further elaborated by inoculating a second animal with the bacilli after previous injection of anti-diphtheritic serum, no ill effect resulting. The virulence of streptococci 110 CLINICAL PATHOLOGY. or pneumococci is best tested on mice or rabbits, since these animals are more susceptible than guinea-pigs. The identification of the tetanus bacillus is completed by the production of tetanic spasms following the inoculation of the suspected organism in mice. The glanders bacillus is rarely met with in human pathology, and its identity should be con- firmed by the intra-peritoneal inoculation of a male guinea- pig, a characteristic suppuration resulting in the tunica vaginalis. Body fluids suspected to be tuberculous, yet in which no tubercle bacilli can be detected microscopically or by cultures, may be proved to be infected by means of guinea-pig inoculation. The guinea-pig is highly susceptible to the human tubercle bacillus, and the smallest dose injected into the leg leads to tuberculosis of the nearest lymphatic gland within 10 days, and to death from generalised tuberculosis in from 4 to 6 weeks. The tubercle bacillus can be identified and, if necessary, recovered in culture from the lesions in the guinea-pig. Special Methods. — Certain other methods of identifying organisms have been described in the section on the blood. These include the agglutination tests, in which a known serum is mixed with a suspension of an unknown bacillus, a method most frequently used for the rapid identification of the typhoid bacillus. A similar method, less commonly employed, is the complement deviation test as elaborated by Bordet and Gengou. Another method, which is used for the identification of the cholera vibrio, is that known as Pfeiffer's reaction. A mixture is made of a suspension in broth of the organism to be tested and anti-cholera serum and the mixture is injected into the peritoneal cavity of a guinea-pig. After a few minutes the peritoneal fluid is examined, and if the spirilla injected were cholera spirilla they are found to lose their motility, break up into globules and disappear ; if they were not cholera spirilla they are found to be unaffected. A control experiment in which normal serum replaces the anti-cholera serum should always be performed upon another animal. The reaction can also be made in vitro by mixing the vibrios, the anti-cholera serum and fresh guinea-pig serum, and incubating in the form of a hanging drop for from 1 to 2 hours. If bacteria, isolated and identified by the methods indicated INTRODUCTORY— TABLE OF CLASSIFICATION. Ill above, are classed primarily on the basis of their morphology and staining reactions, and secondarily according to their cultural characters, they will be found to fall into groups of which the individual members resemble each other, not only in their laboratory characteristics, but also in the nature of the lesions which they produce in the human subject. In the accompanying table the pathogenic bacteria are divided into cocci, bacilli, spirilla, streptothrices, etc., and sub- divided into those which are Gram-positive or Gram-negative, acid fast or non-acid fast. The Gram-negative bacilli are further sub-divided into groups according to their cultural characters. It is not intended that a table condensed into a page should replace the larger text-books of bacteriology, but it is con- venient for the student to have some scheme into which he can work the more detailed knowledge subsequently acquired. In many excellent bacteriological text-books organisms closely allied in their staining and cultural properties may be widely separated in the text, with the disadvantage that all the methods by which the bacteria have to be identified must be learned for each organism instead of for each group of organisms. The most characteristic special reactions for the identification of the individual members of the groups are further indicated in the table, an amplification of which forms the basis of the following chapters. The division of bacteria into groups in this manner has certain justifications other than convenience, since it indicates to some extent the origin of bacterial species. There can be little doubt that at one period of the world's history all pathogenic bacteria were saprophytes, that is to say, lived upon their hosts without harming them and pro- duced no highly specialised and deadly toxin. To each group of pathogenic bacteria in the table could be added a non- pathogenic prototype closely resembling the other members of the group in morphological, staining and cultural characters. As examples may be given the practically non-pathogenic staphylococcus albus of the skin among the Gram-positive cocci, the hay and butter bacilli of the acid-fast group, the diphtheroid bacilli, and the harmless mouth spirochetes. It must not be supposed that the acquirement of toxins by bacteria has been directed solely against humanity. From the same non-pathogenic prototype can often be traced those •ranias pocqq Xji^x -noi;.XBd 'mpain p3ioads .13}3jd ptre pooo + otbi-q aq^ uuq; jfpttojs 9.10m a\oj§ : dd\\ pjooooo^dans 30 s9iuo[oo CD ^ ass. ." - PI p c y 5 03 T 4 _ C 03 » «* ri ^ B S 2^ 5 c5 rt c S9}uip£q -OqXBO A\3J ^JipiOB saraojoo ^niod -uid ^naoiqsnuiq. O O CO /2 ' 5 'S Pi ^ ~ ^ QJ , , ^ r* O ^ i>»*a * >> oj £ a> "8 •— m "o to 03 Pi o o S-. 'rt ^ n CO DO SO j7 , D 3 B o P? — o> "2 -2* a a + P4 A o £ o ci T3 c3 I— I o cS J-l O ^ 2 &o B 03 +J CO a CO 0) v. ^ a c O 32 •S a-o n « ROci Oh £ & cTQ to ES 4^ d> a : roteus pocyan M hi hi 1— 1 Ph "lO^Sfflfi !! _C0 CD ^ ^ co c3 rf-5 a .w op." _« "^ CO CD CD i -M l - , 'Ebo . cd ^ 3 .„ o W Ph w 3 <* .3 js it 11 3 y o O O o ^ _^ ~- g K 02 ^ 3 ^ go-go ».g s-> -5 o o ST-a •S2 -2 -o o -^ S ^ g H L, ^ (O 08 -rt to j£ Ph P4 114 CLINICAL PATHOLOGY. organisms which cause similar diseases in animals, and which have retained many of the characteristics of the group they belong to. For example, the gonococcus will only live and produce disease in the human subject, but there is a contagious urethritis found in bulls producing the so-called granular vaginitis of cows, of which the causative organism is a coccus and Gram-negative. The tubercle bacilli are also distinctive for man, bovines, birds and fish. Although these allied bacteria closely resemble each other in so far as our com- paratively crude methods permit investigation, it must be recognised that each member is a distinct and established species. The rapid multiplication of micro-organisms might lead us to expect a rapid variation of species, but since bacteria multiply by the simple process of splitting in two the minimal opportunity for variation exists, and such varia- tion as does take place is largely evolved out of minute alterations in the composition of the bacterial toxin. Con- sequently we should not expect to change human tubercle bacilli into the bovine or avian species by passing them through numerous members of the appropriate animals over intervals of a few years, and as a fact the change does not occur. The establishment of species has taken place by natural selection in the course of ages. It is well to recognise also that the most virulent of human bacteria are still capable of maintaining in the tissues a harm- less saprophytic existence. The pneumococcus may and often does exist in the mouth, and less frequently the typhoid bacillus ' in the gall bladder, without causing disease. The presence of the essential organism is not the only factor in the production of the disease, and it should be evident that the isolation of bacteria from the body can have little meaning or significance apart from the clinical investigation of the nature of the lesion present. PLATE VIII. >* * s 7 ! PLATE VIII. Staphylococci. In Pus from Abscess of Neck. (Carbol-thionin.) Streptococci. In Pus from a case of Cellulitis. (Carbol-thionin . ) Gonococci. From a Urethral Discharge. (Carbol-thionin.) Tubercle Bacilli. In Sputum. (Carbol Fuchsin and Methylene Blue.) Diphtheria Bacilli. Culture on Blood Serum. (Loffler's Methylene Blue.) Anthrax Bacilli. Culture on Gelatin. (Ccubol-thionin.) Tetanus Bacilli. Anaerobic Culture on Agar. (Carbol Fuchsin and Methylene Blue.) Influenza Bacilli. Tn Pus from Knee Joint. (Carbol Fuchsin.) CHAPTER IX. the cocci — the gram-positive bacilli. The Gram-positive Cocci. This group of organisms comprises the common pyogenic cocci, and gives rise to a number of morbid conditions which have in common a comparatively brief incubation period, an acute onset, the local production of pus, a usually rapid recovery, and a' tendency to leave the patient predisposed to future attacks. The Gram-positive cocci include the staphylococci, the pneumococcus, and the streptococci. The staphylococci (Plate VIII.). — The staphylococci grow in groups or clusters, an arrangement best seen in films pre- pared from solid media and least obvious in broth culture. In preparations made from pus the organisms are usually in pairs with occasional clumps, and are readily taken up by the phagocytes. It is a common elementary error to suppose that numerous diplococci within a polynuclear cell are necessarily gonococci. On slope or plate cultures the staphylococci grow in large, round, opaque, discrete colonies with clean-cut edges. In broth they produce a general turbidity. Litmus milk is usually acidified and clotted. Gelatin is liquefied, and a green fluorescence is produced in neutral red broth. The majority of the litmus carbohydrate media are acidified, but no gas is produced by these or any other cocci. These organisms therefore as a group grow readily in all the usual media and produce active changes in them. The staphylococci are divided, according to the nature of the pigment they produce when growing on solid media, into S. aureus, S. citreus, S. dibits. The colour of the pigment is a fairly constant feature, and is most obvious in a recently isolated coccus, but may not appear until the culture medium has been exposed to direct sunlight for some hours. The 116 CLINICAL PATHOLOGY. power of pigment production may be lost in old cultures, and it is probable that there is little racial difference between the varieties of staphylococci obtained from human lesions. As a general rule, S. aureus is the most virulent of the staphylo- cocci and the most active in culture media. S. citreus occupies an intermediate position and is comparatively seldom met with. S. albus is usually the least virulent and the least active in culture media. White staphylococci are frequently obtained from the skin which are practically non-pathogenic and very inactive in the various media, often producing no change in litmus milk. The most characteristic lesion produced by the staphylococci is the formation of a local abscess. The most virulent lesion is the acute suppurative epiphysitis or osteomyelitis of children, the causative organism being commonly S. aureus and less frequently a streptococcus, pneumococcus, or one of the other staphylococci. The staphylococci have a particular affinity for the lymphatic tissues, and often give rise to metastatic abscesses in the lymph glands. The condition known as lymphangitis is practically always produced by S. aureus. Boils, carbuncles, and the suppurative lesions engrafted upon acne are among the local conditions caused by staphylococci. The organisms may further be spread from a local source by the blood- stream and give rise to abscesses in the tissues and joints, a condition known as pyaemia. The staphylococci are also very commonly found as secondary invaders of tissues affected by some other agent, and particularly by the tubercle bacillus. The pneumococcus (Frankel's diplococcus : Diplococcus lanceolatus) (Plate X.). — The pneumococcus occupies an intermediate position between the staphylococci and strep- tococci. It is an encapsulated diplococcus, which loses its capsule in cultures but regains it after inoculation into an animal. The coccus is commonly not rounded, but shaped like the triangular blade of a spear, the bases of the triangles being opposed to each other in a single pair. Not infrequently one member of the pair is lance-shaped and the other round. In preparations made from liquid media the organisms may be found in short chains of 4 or 6 members, but the great majority are in pairs. In films of pus the diplococci are sometimes found agglutinated into clumps of considerable THE COCCI— THE GRAM-POSITIVE BACILLI. 117 size. It is extremely rare to find them taken up by the phagocytes in any numbers. The appearance of Gram-positive, extra-cellular, encapsulated, lance-shaped diplococci in pus is very suggestive of the pneumococcus ; but the diagnosis of the organism should never rest upon film preparations alone, since innumerable mistakes have thus been made. The cultural characters must be investigated also. The organism grows fairly readily on the ordinary media, but seldom so freely as the other members of this group. It grows best on agar or serum agar and in milk. It does not grow on gelatin. On agar it produces minute round translucent colonies. Milk is acidified and usually clotted after 2 to 3 days. A general turbidity, together with a less obvious granular deposit, is produced in broth. Few only of the carbohydrate media are acidified, the most constant being dextrose and raffinose. If the pneumococcus is inoculated subcutaneously into a mouse the animal dies of a rapid septicaemia, and films made from the heart blood after death are found to be swarming with diplococci. The most characteristic lesion produced by the pneumococcus is lobar pneumonia, and the organism is found in the sputum, the lung tissue, and the blood. The common comiDlication of pneumonia, an empyema, is almost always due to the pneumococcus, which can be readily demonstrated in the pus. The organism may also produce an arthritis (usually of the large joints), a peritonitis, a salpingitis (particularly in young girls), and a meningitis. Pneumococcal arthritis and peritonitis may arise as sequels of lobar pneumonia, or much more commonly independently of it. These affections are more frequent in children than in adults, often give rise to com- paratively little general disturbance, and are of favourable prognosis. Pneumococcal meningitis is a somewhat rare affection, is usually secondary to suppuration in the middle ear, and runs a rapidly fatal course. The pneumococcus is a rare cause of conjunctivitis, but is often associated with serpiginous ulcer of the cornea. A particularly virulent form of infective endocarditis may be set up by the pneumococcus, usually as a sequel of lobar pneumonia, but it is a rare com- plication. The organism may be found in the oral cavity of healthy persons, and can frequently be isolated from the sputum 118 CLINICAL PATHOLOGY. in cases other than lobar pneumonia. A diagnosis of pneu- monia from the bacteriological examination of the sputum is most unsatisfactory. The streptococci (Plate VIII.).— The streptococci grow in chains, an arrangement best seen in liquid media. The number of cocci in a chain varies from 10 to over 100. In preparations made from pus short chains are usually numerous, but the majority of the organisms may be in pairs, and many of them are found within the phagocytes. In cultures some of the cocci may show elongated bacillary forms. On solid media the streptococci grow in small round translucent " pin- point " colonies. Broth shows a granular deposit on the sides of the tube with a few white granular colonies floating in an otherwise clear medium. Gelatin is not liquefied, and as a rule few of the carbohydrate media are acidified. There are in all probability a considerable number of different types of streptococci, the complete differentiation of which is beyond the scope of this book. In human pathology the most important type, and the one most frequently met with, is the Streptococcus pyogenes. Two other less constant types of streptococci have been named S. salivarius and S.fcecalis. The >S'. pyogenes aj)pears as a rule in moderately long chains of 30 to 40 cocci. Litmus milk is acidified, but never clotted. Neutral red broth in anoerobic culture is unaltered. Lactose, dextrose, and salicin are acidified. Inoculation into a mouse is followed by rapid septicaemia and death. The most characteristic lesions produced by this organism in man are the acute spreading inflammations of the nature of cellulitis. Erysipelas, which at one time was considered to be caused by a special organism, the Streptococcus erysipelatis, may be due to any of the streptococci and very rarely to the pneumococcus ; by far the most common causative organism is, however, S. pyogenes. The difference between erysipelas and cellulitis is largely one of situation, the former condition being an acute inflammation of the skin, the latter of the subcutaneous tissues. S. pyogenes may in addition cause acute inflammation in almost any part of the body, and the infec- tions set up by it are usually of a serious nature. The lymph glands present a very feeble barrier to the spread of the organisms, which not infrequently gain entrance into the general circulation, and may be recovered from it in blood THE COCCI— THE GRAM-POSITIVE BACILLI. 119 cultures. Puerperal septicaemia is a typical example of local infection by the S. pyogenes with a general dissemination in the blood-stream. Infective endocarditis in the acute and virulent variety which follows a local infection is commonly produced by this organism. S: salivarius, or, as it is sometimes called, S. brevis, appears as a rule in short chains of 8 to 12 members. Litmus milk is acidified and usually clotted. A green fluorescence is often produced in neutral red broth under anaerobic conditions. The coccus is non-pathogenic to mice. S. salivarius is commonly present in the normal saliva, and a closely allied type known as S. anginosus, which grows in longer chains, is a frequent inhabitant of the throat and very constantly present in the angina of scarlet fever. S. Salivarius is found in lesions of a usually milder type than those associated with S. pyogenes. It is the type of organism most frequently obtained in blood cultures from cases of chronic infective endocarditis, such as result from old rheumatic infections. The organism known as the Diplo- eoccus rheumaticus, and believed by some to be the cause of acute rheumatic fever, is probably identical with S. salivarius. This streptococcus is also often associated with severe pyorrhoea alveolaris, and may be a factor in the production of some toxic lesions which accompany an infective condition of the mouth, particularly the variety of osteo- arthritis which mainly affects the hands and is so fre- quently preceded by a local inflammatory lesion in the mouth or elsewhere. S.fiecalis grows in short chains, clots milk, alters neutral red broth, and acidifies the majority of the carbohydrate media, including litmus mannite. It is non-pathogenic to mice. The organism is commonly present in the fasces, but is not found with any frequency in human lesions. It may be isolated from sinuses or from spreading inflammations in association with the gut, and is found less commonly in cases of infective endocarditis. The Gram-negative Cocci. The organisms of this group present certain features in common. The cocci grow slowly and with difficulty or not at all on the ordinary media. They grow well 120 CLINICAL PATHOLOGY. on media containing blood. The colonies produced on solid media are of the streptococcal type. In the bocly the organisms show a marked preference for certain special localities, such as the urethra, the meninges, and the nasal cavities. The diseases produced are on the whole chronic with a marked tendency to relapse, and, except in the case of the meningococcus infections, rarely terminate fatally. Micrococcus eatarrhalis. — This coccus is the least important member of the group, and may be regarded as a more or less normal inhabitant of the nose and throat. In film preparations it appears as a fairly large rounded diplo- coccus, and is often found within the phagocytes. It grows feebly on agar in small colonies when first isolated, but more abundantly in sub-cultures. Growth is best obtained on blood serum media. No acidification of the carbohydrate media is produced. The organism is often associated with the common " in- fluenzal" catarrh, and may be obtained in such cases in pure culture. It may also be isolated from the urine in some cases of cystitis. Micrococcus melitensis. — This coccus is the causative organism of Malta fever, and may be isolated from the blood or the urine of infected persons. The diagnosis may be confirmed by agglutination tests performed with the serum on the coccus in the same manner as for the Grun- baum-Widal reaction of typhoid fever. The disease is spread by the drinking of goat's milk, a widely used beverage on the Mediterranean coasts. The cocci are present in large numbers in the milk, and the sera of the infected animals give positive agglutination tests. Malta fever is a disease attended with practically no danger to life, but it runs an extremely protracted course, and it may be one or more years before the patient is able to again follow his ordinary occu- pation. Since the discovery of the causative organism and the path of infection, the disease, formerly an endless source of danger to the British troops in Malta, has been almost eliminated. The coccus grows very poorly, if at all, at 20° C, and at body temperature grows slowly on agar, the small colonies taking about three days to reach maturity. Litmus milk is rendered slightly more alkaline. The coccus in hanging-drop preparations THE COCCI— THE GRAM-POSITIVE BACILLI. 121 is definitely motile, and has been described as flagellated by some observers. In films made from the media it appears in pairs and small clumps. The meningococcus (Diplococcus intracellularis menin- gitidis) (Plate X.). — The meningococcus in films of pus obtained from thecerebro-spinal fluid appears as a diplococcus; some of the organisms are extra-cellular, but the majority are within the phagocytes. The cocci are usually rounded, but occasional pairs with flattened opposed surfaces may be seen, and rarely a polynuclear cell may be found distended with such pairs, the organisms thus closely resembling the gonococcus in appearance. Fortunately it is practically never required to distinguish between the two organisms, owing to the diverse situations in which they are found. Meningococci and gonococci, however, are readily differentiated by their cultural characters. The meningococcus will grow on the ordinary media; but the first culture is usually obtained with difficulty, and frequent sub-cultures at one-day intervals are necessary before a ready growth results on agar or in broth. It is advisable to make the first cultures on to nasgar or some other blood serum medium and into milk. The coccus grows in pin-point colonies on nasgar in 24 hours, and as a rule grows freely in milk without changing the appearance of the medium. The coccus is usually agglutinated by the serum of meningitis cases up to dilutions of 1 in 50 or over. The meningococcus is the causative organism of cerebro-spinal meningitis, both in its epidemic and sporadic form, and can nearly always be obtained in pure culture from the cerebro- spinal fluid during life. The organism has also been found in the nose and throat, and is probably spread from these situations. The isolation of the organism from a nasal discharge is a much more difficult matter, owdng to the prevalence of other Gram-negative cocci of the catarrhalis type. The organism cannot be identified in film preparations, and the chief cultural points of distinction are the failure of the meningococcus to grow at 20° C. its more delicate colonies on nasgar and its behaviour in the carbohydrate media. Serum agglutination tests should also be performed. The gonococcus (Plate VIII.). — The gonococcus is characteristically a diplococcus in which the individual members of each pair have the opposed surfaces flattened. 122 CLINICAL PATHOLOGY. In a film of pus the great majority of the cocci are found within the phagocytes, and a typical field shows large numbers of empty polynuclear cells, together with one or two only of the polynuclear or epithelial cells crowded with diplococci. A film of pus in which the comparatively few cells which are phagocytic are distended with flattened Gram-negative diplo- cocci, particularly if obtained from a urethral or cervical discharge, is sufficiently characteristic to practised eyes to ensure the diagnosis of a gonorrheal infection. It must be remembered, however, that errors in the technique of using Gram's stain are common, and that an intracellular diplo- coccus is by no means necessarily a gonococcus. It is unfortunate that, owing to the difficulty of isolating the gonococcus in culture, the diagnosis has often to be made from film preparations, and in cases of any doubt the beginner should be far more cautious than is customary in expressing an opinion. The gonococcus will not grow at all on the ordinary media, but requires some medium containing the essential elements of blood serum, such as nasgar or serum agar. Perhaps the best medium upon which to grow the organism is an agar slope over which has recently been smeared with a platinum wire a drop of sterile blood obtained by pricking the thumb. On this medium the gonococcus grows well, but somewhat slowly, the small " pin-point " colonies taking from 24 to 48 hours to reach maturity. Owing to the slow growth of the organism it is likely to be crowded out by any other bacteria that may be present in the pus. The gonococcus rapidly dies out in culture tubes unless frequent subcultures are made. The most common infections produced by the gonococcus are intragenital — that is to say, a urethritis in the male and a urethritis, or more commonly an inflammation of the vagina and cervix uteri, in the female. The diagnosis of the nature of an acute urethritis in the male is easy ; the diagnosis of gonorrhoea in the adult female requires a special examination. A swab taken at random in the usual manner from the vaginal secretion is quite useless. Films made in this way, even from the normal vagina, are found to be inundated with colon bacilli, cocci, and other organisms, so that it is a profitless labour to search through them for occasional gonococci. The films should be made from the interior of the urethra or, after the THE COCCI— THE GRAM-POSITIVE BACILLI. 123 passage of a speculum, from the cervical canal, care being taken not to touch the walls of the vagina. The gonococcus is the commonest cause of pyosalpinx, but can very rarely be found in the pus obtained at operation. Films made from the pus show, as a rule, numerous phagocytes but no cocci, and cultures prepared from it remain sterile. The infective vaginitis of little girls differs in some clinical respects from the gonorrhoea of adults, in that it is extremely contagious and may spread through a children's ward. It has an unduly long incubation period, and is very exceptionally followed by the ordinary sequelae, such as arthritis. Films made from the vaginal discharge of these cases are found to be swarming with organisms indistinguishable from gonococci. Among the complications of gonorrhoea in which the gono- coccus may be detected are conjunctivitis (including the most frequent variety of ophthalmia neonatorum), arthritis, and rarely a general septicaemia, with or without an infective endocarditis. Commonly included among the cocci must be mentioned a group of organisms known as Sarcinae (Plate IX.). These are non-pathogenic, and are met with most frequently as con- taminations in culture tubes from the skin or from the air. In film preparations they appear in small irregular clumps, or more characteristically in little packets of 2, 4, or 8, the individual members dividing at right angles to each other. Their opposed surfaces may be flattened. The colonies on agar are usually large, round and opaque, with a polished and often pigmented surface. Another common contamination of culture tubes may be noticed here, namely, the bacillus subtilis, which can be recognised by its usual tendency to abundant spore formation and the pellicle produced by it on the surface of broth media. The Gram-positive Bacilli. These may be divided into those which are acid-fast and those which are not acid-fast. (1) The acid-fast bacilli.— This important group of organisms includes a considerable variety of bacilli some of which are mainly saprophytes ; others are pathogenic to animals and not to man ; while the few more particularly 124 CLINICAL PATHOLOGY. considered here are highly pathogenic to human beings. The organisms resemble each other in that they do not grow on the ordinary media, and when growing on special media their rate of growth is extremely slow. The lesions produced by them resemble each other in their nodular or tubercular characters and in their chronicity. Although the bacilli are all acid-fast, they can be further divided by their staining reactions, since some are more acid-fast than others, and some, though acid-fast, are decolorised by alcohol, that is to say, are not alcohol-fast. The bacilli have a tendency to produce long thread-like, beaded forms, particularly in cultures, and are no doubt fairly closely related to the streptothrices, some of which are acid-fast (page 145). The tubercle bacilli (Plate VIII.).— The tubercle bacilli can be recognised in films made from human lesions and stained by the Ziehl-Neelsen method (page 156). They appear as thin, curved and beaded red rods, and are almost entirely extracellular. In preparations made from the ordinary sources such bacilli may be regarded for clinical purposes as tubercle bacilli, but it must be remembered that an acid-fast bacillus is not necessarily the human tubercle bacillus, even if found in human tissues. The different species of tubercle bacilli can be differentiated by their behaviour in culture and by animal inoculation. These bacilli may be divided into those which are pathogenic, or possibly pathogenic, to man and animals, and those which are not. The pathogenic bacilli include the following : — The human tubercle bacillus is by far the most important of this group of organisms, and is responsible for the great majority of all human tuberculous lesions, including nearly all cases of pulmonary tuberculosis. The human tubercle bacillus will grow on blood serum, or on media to which glycerin has been added, such as glycerin agar or glycerin broth, as well as on inspissated egg medium. Growth is very slow, and little is to be seen in less than 10 days ; later it becomes wrinkled, greyish and scaly. Inoculated into animals the bacillus is found to be highly virulent to guinea- pigs but to have comparatively little virulence for rabbits and calves. The bovine tubercle bacillus is responsible for a proportion of the glandular and arthritic tuberculous THE COCCI— THE GRAM-POSITIVE BACILLI. 125 affections, particularly in children, and much less frequently for pulmonary tuberculosis. Owing to the extreme frequency of tuberculosis among cows and the common presence of bovine bacilli in milk, this source of infection for the human subject is no doubt a wide one ; at the same time the typical human infection is pulmonary tuberculosis, and the spread of tuberculosis in its most important aspect is by the human sputum. The bovine bacillus on serum grows more slowly than the human bacillus, taking from 2 to 3 weeks to produce a thin greyish film, which is neither wrinkled nor pigmented. It is highly pathogenic to calves and rabbits. The avian tubercle bacillus appears to be practically non-pathogenic to man, and since tuberculosis is extremely rare among birds in the wild state, though common among those kept in captivity, the bacillus can scarcely be regarded as a serious factor in the spread of human tuberculosis. The avian bacillus is readily recognised by inoculation experi- ments, since it is highly virulent to pigeons, but in guinea-pigs produces only a local lesion. The bacillus of fish tuberculosis does not grow at body temperature, and is non-pathogenic to mammals. The non-pathogenic tubercle bacilli embrace a number of acid-fast organisms, which differ from the pathogenic bacilli in their far more rapid growth on artificial media. They are practically non-pathogenic to animals, and on inoculation into the highly susceptible guinea-pig give rise to a small local lesion only. These bacilli include Rabinowitch's butter bacillus, Moeller's timothy-grass bacillus, and a variety of similar organisms found in dust, manure, and other substances. These bacilli rarely if ever cause confusion in human pathology. The smegma bacillus. — This organism belongs properly to the group of non-pathogenic tubercle bacilli, but is considered separately because of its human distribution and the liability to confusion with the virulent tubercle bacillus. The smegma bacillus is found in the genital secretions, and in film preparations appears as a shorter, stouter, and less beaded rod than the tubercle bacillus. The smegma bacillus can be readily differentiated by its staining reactions, since after treatment with 25 per cent, acid it is decolorised by immer- sion in methylated spirit for 1 minute. It is, therefore, 126 CLINICAL PATHOLOGY. acid-fast but not alcohol-fast. The smegma bacillus has been grown with difficulty on serum and glycerin- agar media. For the purposes of clinical pathology, the demonstration of bacilli which are strongly acid- and alcohol- fast in the human tissues is sufficient for the diagnosis of a tuberculous lesion. The only organism likely to be confounded with the human tubercle bacillus is the bovine bacillus, and for practical diagnosis the two may be considered as identical. In tuberculosis of the lung and of the urinary tract the bacilli are commonly found in large numbers in the sputum and urine. In the pus obtained from tuberculous sinuses and abscesses or from tuberculous joints the bacilli are almost always extremely scanty. The same is true of tuberculous body fluids, whether peritoneal, pleural, or cerebro-spinal ; also of the skin tuberculides. In the faeces the bacilli may be present in large numbers in cases of tuberculous enteritis and in very small numbers in pulmonary tuberculosis. In all situations, other than the urine or the sputum, it is necessary to adopt special processes for the demonstration of the organisms, and these will be described in a subsequent chapter. The lepra bacillus. — The leprosy bacillus closely resembles in appearance the human tubercle bacillus, but the following distinctions can be made out. The bacilli are very numerous in any leprous lesion, whether in the pus from a breaking-down focus, or from a sinus, or in microscopic sections of lepra nodules. Many of the bacilli are seen within the cells, and certain elongated cells are found containing a number of bacilli arranged like a bundle of cheroots. Further, the lepra bacillus is not so acid-fast as the tubercle bacillus, but is decolorised by 25 per cent, sulphuric or nitric acids, retaining the stain only when treated with acids of half this strength. The lepra bacillus does not grow on the media usually employed for the tubercle bacillus, and until recently had never been cultivated. Cases of leprosy are extremely rare in this country, and all are imported. The diagnosis can in nearly every case be confirmed by making film preparations of the nasal secretion. The majority of leprous individuals have a chronic crusted nasal discharge containing the bacilli in large numbers, so that it is rarely necessary to excise a suspected nodule for microscopic examination. THE COCCI— THE GRAM-POSITIVE BACILLI. 127 In spite of the prevalence of the bacilli in the nasal secretion the disease appears to have very little direct infectivity, and it is held by certain authorities that some intermediate host, such as a biting insect, is necessary for the spread of the disease. (2) The non-acid-fast bacilli. — This small group of Gram- positive non-acid-fast bacilli consists of four members only, one of which is practically non-pathogenic to man. Each member of the group possesses some important property not usual among human parasites ; thus two members produce powerful extra cellular toxins, two are strict anserobes, and two form spores. One member of the group only, the diphtheria bacillus, has any wide distribution in human pathology. The diphtheria bacillus (the Klebs-Loffler bacillus) (Plate VIII.). — The appearance of the diphtheria bacillus in films prepared from the tissues, and more particularly from cultures, is very characteristic. The bacilli are arranged in small groups, and the members of each group have a peculiar angular relation to each other, so that the groups resemble the fingers of the hands crossed or a Chinese character. The individual bacilli are thin, curved and beaded, staining alternately in light and dark areas. They are non-motile. Some bacilli may show bulbous or racquet-shaped extremities, and these so-called " involution " forms are common in cultures more than 48 hours old. The bacilli are readily identified from their size, shape, beading and arrangement, after staining with Loffler's methylene blue. The bacilli grow well on the ordinary media, but it is preferable to make the first culture on ox serum, since the diphtheria bacillus grows more readily and rapidly on this than the ordinary bacteria present in the throat. On solid media the bacilli grow in colonies of the streptococcal type, but they are slightly more heaped up and opaque. A general turbidity is produced in broth, and if a broth culture which has been incubated for several weeks is filtered free from bacilli the filtrate contains in large amount a powerful toxin, which, unlike the majority of bacterial toxins, is extracellular, or, in other words, is not intimately bound up with the bodies of the bacilli. An acid reaction is produced within 24 hours in litmus dextrose broth, and the acidity passes off again in a few days. Gelatin is not liquefied. If a suspension of the bacilli is introduced into the leg of 128 CLINICAL PATHOLOGY. a guinea-pig death results in about 36 hours, and iiost mortem a small greyish necrotic membrane is found at the seat of inoculation. The stomach is greatly dilated. Haemorrhages are present in the supra-renals, and the cardiac muscle on microscopical examination is found very extensively affected by a fine fatty degeneration. If a second guinea-pig is inoculated with the bacilli or their toxins together with anti-diphtheritic serum no ill effects result. The diphtheria bacillus is the pathogenic type of a large number of bacilli, the non-pathogenic members being known as "diphtheroid" bacilli. The diphtheroid bacilli are very widely distributed in the human body, and it is essential to have some knowledge of them owing to the extremely close resemblance they bear to the diphtheria bacillus itself. The diphtheroid bacilli may be divided into those which can be distinguished from the Klebs-Lofner bacillus on morphological grounds, those which cannot be so distinguished but which have cultural differences, and those which are both morphologically and culturally identical. Hofmann's bacillus can be distinguished on morphological grounds by its appearance in film prepara- tions made from a culture. The bacilli are distributed in groups, the members of which have a tendency to a parallel rather than an angular arrangement. They are short and rarely curved, and are not truly beaded, but stain deeply at each end, displaying a pale band across the middle. There is some doubt as to the ability of the Hofmann bacillus to produce disease or as to whether it is capable of conversion into the diphtheria bacillus, and it is wiser to treat patients from whom this bacillus is isolated as infective, particularly if a lesion in any way suggestive of diphtheria is present. Bacilli are commonly met with in the conjunctival sac (in which situation they have undeservedly obtained the name of Xerosis bacilli), in the skin, in old sinuses, and less frequently in the nose, which are morphologically identical with the diphtheria bacillus, differing from it only in a greater tendency to produce club-shaped forms. The niajorhVv of these bacilli can be differentiated on cultural grounds, since they merely maintain an existence in liquid media and produce little or no growth in them. They do not acidify dextrose broth. Other bacilli, which are frequently isolated from the urethra THE COCCI— THE GRAM-POSITIVE BACILLI. 129 and less often from any of the above situations, resemble the diphtheria bacillus exactly both in their morphology and in their cultural characters. They differ, however, in one very important respect — they produce no toxin, and are consequently non-pathogenic to guinea-pigs. In practical pathology, if a bacillus is obtained from the throat or larynx of a person whose clinical condition suggests diphtheria, and if it is found to resemble the diphtheria bacillus in appearance, the patient should be isolated and given antitoxin. In the case of nasal diphtheria the full cultural and morphological characters of the bacilli should be ascertained, since diphtheroid bacilli are common in this situation, and greater attention should be paid to the clinical condition than to the bacteriological findings. In patients convalescent from diphtheria it is more practical to let them mix again with their fellows, if they are free from tonsillar or nasal discharge, than to confine them on purely bacteriological grounds. In sus- pected diphtheritic lesions in unusual places, such as the con- junctiva, surface wounds, the vagina, etc., the morphological and cultural characters of the bacillus are insufficient for diag- nosis. In all cases of doubt or of exceptional importance (such as a sporadic case of diphtheria in a school) the bacilli must be isolated and animal inoculations performed. The routine bacteriological examination of the throat is therefore of great assistance in suspected cases of diphtheria, but the results should be accepted with reserve in other situations, and should not be regarded as proven until a guinea-pig has been inoculated. The successful isolation of the bacilli from the throat is largely dependent upon the care with which the culture is taken. The necessary apparatus consists of a blood serum culture tube and a second tube containing a straight piece of stout copper wire, around one end of which has been firmly twisted a piece of absorbent wool. Wool and wire must be sterile. In the case of a refractory child have the arms, legs, and body wound in a blanket and the child's head held by a nurse. Choose a good light, depress the tongue with a spatula, pass the cotton wool swab on to the membrane and rub it firmly into the lesion. Withdraw without touching the tongue or cheeks. Rub the swab over the surface of the serum and replace in its own tube without burning it. Incubate for p. 9 130 CLINICAL PATHOLOGY. 12 hours. Examine films stained with Loffler's methylene blue from both swab and medium. The acne bacillus is an organism of low pathogenicity belonging to this group. In film preparations it resembles the diphtheria bacillus in its grouping, but is smaller and is not beaded, being in appearance not unlike Hofmann's bacillus. It grows with difficulty on the ordinary media except in sub-culture, and the first culture is best planted on oleic acid glycerin agar, and grown under anaerobic conditions. The acne bacillus is obtained in consider- able numbers from the depths of acne comedones. When suppuration occurs a white staphylococcus is usually present in addition. The tetanus bacillus (Plate VIII.).— Tetanus bacilli when growing in the tissues commonly have few, if any, spore - bearing forms. On artificial media the majority of the bacilli contain spores. The bacillus under these conditions is comparatively slender, and the delicate rounded spore is situated at one extremity. Other spores are seen lying free among the bacilli. The bacillus is feebly motile and is provided with flagella. The tetanus bacillus usually maintains its existence at the depths of septic wounds, and for this reason is difficult to identify and isolate. One method of isolation presumes the existence of spores which are very resistant to heat, and on this supposition a series of melted agar tubes are inoculated at varying temperatures in the hope that other organisms will be destroyed and the tetanus spores survive. The culture tubes must be grown under strictly anaerobic conditions. In agar and gelatin stab cultures the bacillus sends radiating spikes into the media, and the gelatin is slowly liquefied. Such cultivation of a bacillus with terminal spores is extremely suggestive of the tetanus organism; the positive proof, however, rests with animal inoculation. Subcutaneous inoculation of the bacilli into a mouse produces tetanic spasms in 24 hours and death m 3 days. The tetanus toxin is an extremely powerful one, and, like the diphtheria toxin, is extracellular. The anthrax bacillus (Plate VIII.).— Anthrax infections in man are extremely rare and occur in two main forms. The more usual variety is a local infection of the skin, arising among hide porters and workers, and known as the malignant pustule. THE COCCI— THE GKAM-POSITIVE BACILLI. 181 Recovery from this form under treatment is usual. The other variety is known as wool-sorters' disease, and in this the local infection is in a bronchus, extension taking place through the bronchial glands. This form is invariably fatal. In films made from the clear fluid of the bleb of a malignant pustule occasional polynuclear cells are found, and large numbers of long, stout bacilli, with sharply cut ends, are seen. The majority of the bacilli are arranged in long chains, each chain being contained within a delicate capsule. In films made from old cultures the great majority of the bacilli are seen to contain central spores ; not infrequently spores only are found and no bacilli. The bacillus grows readily on all the ordinary culture media, the most characteristic growths being found on agar plates and in gelatin stabs. On agar plates the colonies examined with a magnifying glass appear like wreathed coils of hair, an appearance produced by the long-twisted strands of bacilli. In gelatin stab cultures growth occurs along the line of the stab, and branches out from this in spikes radiating into the medium. The spikes nearest the surface are the longest, so that the growth looks like an inverted fir tree. Liquefaction of the gelatin begins at the surface on about the second day of the growth. The bacillus aerogenes capsulatus. — This bacillus is of little pathological importance, but may give rise, usually in association with other organisms, to emphysematous gangrene and to gas-containing abscesses. It is more commonly found post mortem as a gas-producing putrefactive organism. The bacillus is a strict anaerobe growing in capsulated chains on the ordinary media. It produces abundant gas in the carbohydrate media, and does not appreciably liquefy gelatin. 9—2 CHAPTEE X. THE GEAM-NEGATIVE BACILLI — SPIRILLA — STREPTOTRICHEjE — hyphomycetes. The Gram-negative Bacilli. The great majority of bacilli being Gram-negative, it is convenient to further subdivide them into a number of groups, mainly according to their similarity in cultural characters. Group 1. This group consists of four members, two of which produce acute specific fevers; two are common causes of eye affections. There is great morphological and cultural similarity between the members. The whooping-cough bacillus (the Bordet-Gengou bacillus),. — A great variety of organisms have been described as the essential cause of pertussis. The bacillus referred to here is that isolated by Bordet and Gengou and proved by them to be the causative organism. The bacillus, which is found in considerable numbers in the sputum in the early stages of whooping-cough, is a minute one closely resembling the influenza bacillus, but in culture has less tendency to produce involution forms, and on subculture grows more abundantly. The bacillus is agglutinated by the sera of patients convalescent from whooping-cough, and its specificity has been further demonstrated by complement fixation tests. The influenza bacillus (Pfeiffer's bacillus) (Plate VIII.). — In films made from the sputum the bacilli appear as tiny rods, often in clumps of considerable size, and many of them are found within the phagocytes. The organisms are present also in many of the complications of influenza, and may be obtained in pure culture from empyema pus and from joint fluids. A purulent exudate in which the polynu- clear cells contain minute Gram -negative bacilli, and in addition PLATE IX. B. Pestis. Cholera Vibrio. In Smear from Spleen. (Carbol-thionin.) Broth Culture. (Carbol-thionin.) B. Coli. Actinomyces. Broth Culture. (Carbol-thionin.) Filaments in Sputum (Gram's Stain. Spiroehaeta Pallida.* Spirilla and Fusiform Bacilli (Vincent). Film from Chancre. (Giemsa's Stain.) From Septic Mouth. (Carbol-thionin.) Oidium Albicans. Sarcinse. From Agar Culture. (Carbol-thionin.) From Agar Culture. (Carbol-thionin.) * The sharpness and regularity of the spirals are to a large extent lost in the process of reproduction. PLATE IX. ' ) ~ ) i ', '. \ I Li » - , N 1 u J V ■0>,V^ ^ ) ^ f ' 1 ' J v>-« V, "* „ j i S. s ' e > V -s '» *' ~ ' £ i ... 'V <" GRAM-NEGATIVE BACILLI— SPIRILLA, ETC. 133 clumps of the same bacilli lying free, is an exudate caused by one of the bacilli in this group. In cultures the influenza bacillus will grow only on media containing serum, and preferably also haemoglobin, the best medium being an agar slope streaked with fresh blood. The colonies are very small and very translucent. While there is no doubt that this bacillus, described first by Pfeiffer and Kitasato, is the causative organism of some influenzal epidemics, it must not be expected to recognise it in all cases of so-called influenza. The diagnosis of influenza is often a haphazard one, and in some undoubted epidemics there has been a failure to discover Pfeiffer's organism; moreover, there is possibly a variety of different organisms associated with a number of similar clinical conditions. The Koch- Weeks bacillus. — This bacillus is by far the most common cause of acute contagious conjunctivitis. The organisms are almost identical in appearance with the influenza bacillus, and are found in large numbers in films made from the conjunctival discharge. The cultural properties of the bacillus also closely resemble those of Pfeiffer's organism, but the more favourable media are serum or ovarian agar. The Morax-Axenfeld bacillus. — This bacillus is the cause of diplo-bacillary or angular conjunctivitis, a common but less acute form of contagious conjunctivitis than the preceding. The bacillus appears as a fairly large, broad bacillus with rounded ends, mainly in pairs, but also in short chains. It is found in the conjunctival secretion, and in a fair proportion of cases in the nasal discharge also. It can be cultivated on blood serum media, and grows in colonies of the streptococcal type. Group 2. This small group consists of two members only, which differ in many respects from the bacilli of the preceding group. Both organisms are met with but rarely in the human pathology of this country. B. Mallei. — The bacillus of glanders mainly affects horses, and very exceptionally produces disease in man. Infection in man starts from a local abrasion on the skin or the nasal mucous membrane and spreads by the lymphatics, giving rise to an acute or chronic pysemic condition, with secondary 134 CLINICAL PATHOLOGY. abscesses in the tissues, lungs, or joints. The bacilli are slender, curved rods, staining faintly, and often in a beaded manner, with the ordinary dyes. In film preparations they are mainly extracellular. The organism grows on the ordinary media, but somewhat slowly. On agar and blood serum growth appears as a shiny, greyish streak in two days. On rjotato a membranous growth is formed, which by the eighth day becomes a reddish brown colour. Inoculation into the abdominal cavity of a male guinea-pig is followed by peritonitis, swelling of the testicles, and a purulent exudate into the tunica vaginalis. The appearance and staining reactions of the organism, the growth on potato, and the effect of inoculation into a guinea-pig should all be investi- gated before making a diagnosis. B. Pestis (Plate IX.). — The bacillus of plague is mainly spread to man from the rat by the intermediary of the rat flea. The disease in man is of three main types — the bubonic, in which the lymphatic glands are affected, the pulmonary, which almost invariably terminates in a fatal septicaemia, and the septicaemia. The bacilli are found in large numbers in films made from gland pus or sputum, and stain deeply at each end and faintly in the centre, each bacillus having the appearance of a diplococcus. This polar staining is best seen if the films are first fixed in absolute alcohol. B. pestis grows on the ordinary media, forming a streak on agar. Gelatin is not liquefied. Broth shows a granular deposit, and the bacillus grows in chains. The disease can be reproduced in mice and guinea-pigs by inoculation. Group 3 a. The third group of Gram-negative bacilli comprises a con- siderable variety of organisms, the great majority of which are morphologically indistinguishable from one another. The group can be subdivided on the basis of one important cultural characteristic, namely, the manner of growth on gelatin. The bacilli of the first division include all the important organisms of the coli-typhoid group, and do not liquefy gelatin. The bacilli of the second division are of less pathological significance, and all liquefy gelatin. Group 3a contains the typhoid and paratyphoid bacilli, the dysentery bacilli, and the colon bacilli. GRAM-NEGATIVE BACILLI— SPIRILLA, ETC. 135 B. Typhosus. — The typhoid bacillus is an actively motile organism provided with numerous flagella. Rapid growth is readily obtained on all the usual cultural media. The more important cultural characters are the following. On agar slopes a streak is produced which is rather more translucent and has less tendency to lateral spread than in the case of the colon bacillus ; the differences, however, are not pronounced. In broth a general turbidity is produced, but no indole. In litmus milk a faint acid reaction is set up, but no clotting takes place. No gas is produced in any of the litmus carbohydrate media, but litmus dextrose is acidified. Neutral red broth is unchanged. Yellow colonies are produced on MacConkey's neutral red bile-salt lactose agar medium. The bacillus can be identified by its appearance, staining reactions and cultural characters, and the identification is preferably confirmed by making use of the agglutination test with a known typhoid serum. Typhoid fever is spread either by direct contact, such as may occur in nursing a patient, or more commonly by an infected water supply. The disease is in the first instance a general blood infection, and the bacilli may be readily isolated from the blood in the first week of the disease and during a relapse. Later the bacilli are found in the faeces, and less commonly in the urine. The bacilluria which may complicate the late stages of typhoid fever is usually set up by the colon bacillus, and far less commonly by the typhoid bacillus. The bacilli may be obtained from the gall bladder many years after the attack, and are present in the bone abscesses which may follow the attack. The bacilli may persist in the fgeces, and less commonly in the urine, many years after the primary infection and without injury to the host. Persons thus infected are known as " typhoid carriers," and are a grave source of danger to the community. The laboratory diagnosis of typhoid fever can be made in the first week of the disease by means of a blood culture. A few cubic centimetres of blood are taken into broth or into ox bile, incubated for 12 hours, and plated out on MacConkey's medium. The yellow colonies are then picked off and tested culturally and by the serum reaction. At the beginning of the second week the Grunbaum- Widal test becomes positive in the patient's serum and the, bacilli may be isolated from the feces. 136 CLINICAL PATHOLOGY. Prophylactic inoculation against typhoid fever with dead cultures of the bacilli is advisably given to those compelled to live in countries where the disease is rife. Persons thus inoculated should not abate the customary precautions taken with regard to their water supply, since they are not immune against a heavy infection. The paratyphoid bacilli. — The paratyphoid bacilli form a small group of closely-allied organisms which can be recog- nised generally by their cultural characters, specifically only by agglutination tests. Four members of the group may be mentioned here : — B. paratyphosus A ; B. paratyphosus B ; B. suipestifer ; B. enteritidis (Gaertner). The first two bacilli produce a disease which cannot be distinguished on clinical grounds from typhoid fever. Paratyphoid infections, however, commonly run a milder course than typhoid, are perhaps more contagious, are to be suspected when patients apparently suffering from typhoid fail to give positive Widal reactions, and are definitely diagnosed on the agglutination tests with the appropriate bacilli, and on the isolation of the organisms from the blood or f&ces. The paratyphoid B bacillus is the organism most commonly met with in this country, while numerous cases of infection by the A bacillus have been recorded from America and India. B. suipestifer and B. enteritidis (Gaertner) are commonly associated with meat-poisoning epidemics, and produce a disease with an incubation period of a few hours, followed by acute gastro- intestinal symptoms of comparatively short duration. The organisms as a group have the following cultural characters. Acid and gas are produced in litmus dextrose and litmus mannite. Litmus lactose is unchanged. No indole is formed. Litmus milk becomes acid in the first 24 hours and then strongly alkaline. Neutral red broth is turned yellow. B. ■paratyphosus A differs from the other members of the group in its behaviour in litmus milk, in which it produces a permanent acidity but no clot. The bacilli can be differentiated one from the other by care- fully performed absorption tests with the sera of infected persons on the lines indicated in a previous chapter. Such tests are rarely required in clinical pathology. Suspected cases of typhoid fever which give in their sera negative or partial agglutination reactions with B. typhosus should be GEAM-NEGATIVE BACILLI— SPIKILLA, ETC. 137 tested upon a known culture of B. paratyphosus B. The organism should also be sought for in the blood and in the fseces, and by its milk reaction it can be distinguished cultur- ally from the A bacillus. Cases of acute food poisoning should in the later stages of infection be tested for agglutinins against Gaertner's bacillus and the organism sought for in the blood or fseces in the acute stage, as well as in the suspected food. The dysentery bacilli. — The dysentery bacilli form a small group of closely-allied and widely-spread organisms, of which the main varieties are the B. dysentcrice of Shiga and the B. dysenteries of Flexner. These organisms are the causes of bacillary dysentery, such as is met with in tropical and in temperate climates. They are also associated with some forms of ulcerative colitis in this country, and have been found in outbreaks of asylum and other varieties of dysentery, as well as in epidemics of infantile enteritis. The bacilli are practically non-motile, and do not appear to be provided with flagella. Their chief cultural characters are as follow : — No gas is produced in any of the carbohydrate media. Litmus glucose is acidified, and litmus lactose is unchanged. Litmus mannite is acidified by the Flexner organism, but not by the Shiga bacillus. No indole is formed. Milk is rendered first acid and then alkaline. Neutral red broth is unchanged. In suspected cases of dysentery the bacilli should be sought for in the stools, and the patient's serum should be tested by ordinary agglutination methods upon the bacilli isolated and upon known strains of dysentery bacilli. In a positive serum reaction the bacilli are agglutinated somewhat slowly in dilutions of the serum up to 1 in 50. The colon bacilli (Plate IX.) — The Bacillus coll communis, like other members of this group, has certain typical characters, but occasional varieties are met with presenting differences of minor importance. The typical bacillus only will be con- sidered here. The colon bacillus has few flagella, and is only sluggishly motile, though active strains are occasionally met with. The organism can be identified by its cultural characters, never from its morphological appearances alone. The main cultural reactions are as follow. Acid and gas are produced in the 138 CLINICAL PATHOLOGY. great majority of the carbohydrate media, including dextrose, lactose, and mannite. Indole is produced in broth. Milk is acidified and clotted. On agar slopes a thick greyish white streak is formed with spreading edges ; on agar plates large circular colonies appear with heaped-up centres and crenated margins. (The appearances on solid media are almost iden- tical for all members of the colon-typhoid group, except that the typhoid growth is slightly more delicate, while the other organisms occupy an intermediate position.) The colour of neutral red broth is changed to a canary-yellow, and a green fluorescence is produced in the medium. Eed colonies are formed on MacConkey's bile salt medium. The colon bacillus is a normal inhabitant of the large intestine, but in abnormal situations it produces suppuration and disease. The more important affections associated with this organism are those connected with intestinal lesions, as, for example, the general peritonitis which follows a perforated appendix. The colon bacillus frequently gains entry into the urinary tract, particularly of females, where it may lie latent, producing no symptoms, or may give rise to severe suppura- tive nephritis, pyelitis, or cystitis. The bacillus is also found in diseased processes in the gall bladder (often in association with calculi), in the bile passages, and in numerous other parts of the body. It has even been isolated in pure culture from the cerebro -spinal canal. It is rarely isolated from the general circulation, except shortly before death. The pus in which the colon bacillus is found is frequently most offensive, owing to the fact that since these lesions are so often in communication with the gastro-intestinal tract there are present in addition to the colon bacilli certain long, thin, delicate saprophytic bacilli which normally inhabit this tract, and are capable of producing the most virulent odour. It is this odour to which the surgeon is apt to call attention as typical of the bacillus coli, which may, however, be absent. Serum reactions with the bacillus are unsatisfactory, since an appreciable increase in agglutinin is not commonly present, and the sera of infected persons rarely agglutinate the bacillus in dilutions of more than 1 in 10. Note. — The student is scarcely expected to remember in detail the various cultural characters of the organisms in this group (Group 3a). He should, however, remember the main GRAM-NEGATIVE BACILLI— SPIRILLA, ETC. 139 distinctions between the typhoid and colon bacilli. A reference to the bacteriological table affords a simple guide to these characters. The bacilli are placed from above downwards in order of their virulence, their specificity, their motility, and their properties of producing agglutinins. The cultural characters proceed in the opposite direction ; thus the colon bacillus alters almost every medium in the greatest possible manner, the typhoid bacillus has the least effect upon the media, and the other organisms occupy an approximately intermediate position. Group 3b. The members of this group, all of which liquefy gelatin, are not of great pathological importance. They may be described as bacilli which usually lead a saprophytic existence, but may on occasion, either alone or more commonly in association with other organisms, produce disease. Only three members of the group are considered here. B. Proteus- — This bacillus is represented by a number of closely-allied organisms, which differ somewhat in their cultural characters. The bacilli in appearance are indis- tinguishable from the colon bacillus, and are very sluggishly motile. In cultures they produce acid and gas in several carbohydrate media, but as a rule do not change lactose. A yellow colour with a marked green fluorescence is rapidly pro- duced in neutral red broth. Milk is unchanged by some members of the group and acidified by others. The colonies on MacConkey's neutral red bile-salt lactose agar are yellow. Abundant indole is produced in broth. B. proteus is commonly found in the fseces and in the urine. In septic infections of the urinary tract the organism may be recovered in pure culture, or more commonly in association with the colon bacillus. The presence of B. proteus in a carefully taken catheter specimen of urine is suggestive of some underlying organic lesion such as tuberculosis, malignant disease, or calculus, since a primary infection with this organism, unlike a primary infection by the colon bacillus, is most unusual. B. Pyocyaneus. — This is an actively motile bacillus with a tendency to spontaneous agglutination in hanging-drop prepara- tions. The organism is readily recognised in cultures by the bright green pigment which it produces. Broth is turned a 140 CLINICAL PATHOLOGY. vivid green (without fluorescence), and on agar the green colour diffuses into the substance of the medium. Acid and gas are produced in the majority of the carbohydrate media. B. pyocyaneus most frequently occurs as a secondary infection, often in association with lesions connected with the intestinal tract, as in ischio-rectal abscess or in general perito- nitis secondary to a gangrenous appendix. The bacillus may also be associated with cerebral abscess, a spreading cellulitis, or a local abscess. The pus in these infections is alleged to be of a sky-blue colour, a tint which is far from obvious. The lesions associated with B. pyocyaneus are often somewhat intract- able, and a general peritonitis accompanied by this bacillus is almost invariably fatal. After inoculation into the peritoneal cavity of a guinea-jiig a virulent and rapidly fatal peritonitis follows. The bacillus of malignant (Edema. — This bacillus is commonly somewhat larger than the other members of this group, is sluggishly motile, and forms centrally situated spores. The organism grows readily, but only under anaerobic conditions. It produces abundant gas in the carbohydrate media. The bacillus is widely distributed in nature, and may be found in many samples of garden earth. It rarely gains entrance into the human body, but may obtain a footing in a septic wound and set up a virulent spreading gangrene, associated with the formation of blebs and the production of gas. The Spieilla. The spirilla form an important group of organisms which, owing to the inclusion of the cholera vibrio, differ widely from each other. The cholera vibrio is an organism which cul- turally resembles the members of the coli -typhoid group, and, being merely a curved rod and not a true spiral, might reason- ably be classed among the bacilli. The properly spiral organisms, which include the spirochete of syphilis, have numerous points of resemblance, and are evidently possessed of a higher organisation than the bacilli. They are included by many authorities among the protozoa. The cholera vibrio (Comma bacillus) (Plate IX.). — The cholera spirilla are small, actively motile, flagellated curved GEAM-NEGATIVE BACILLI— SPIRILLA, ETC. 141 rods, having a tendency to lie with their long axes in the same direction. The spirilla grow readily on all the ordinary media. They liquefy gelatin, and the young colonies on gelatin plate cultures are irregularly granular and look like "fragments of broken glass." In broth a pellicle is produced on the surface of the medium, and both indole and a nitrite are formed from the peptone, so that a pink colour (the cholera- red reaction) appears on the addition of a few drops of sulphuric acid only. Litmus milk is unchanged. The identification of the organism from similar vibrios should be completed by testing the lytic action of immune serum upon it on the lines indicated in Chapter VIII. , page 110. In the human body the organisms are confined to the intestine, and in the acute cases the watery stools appear to form an almost pure culture of the organism. Numerous other varieties of spirilla have been described, and are of particular interest in that they may be mistaken for the cholera vibrio. Metchnikoff has recorded a similar vibrio found in the intestines of fowls dead of gastro-enteritis. Einkler and Prior described another present in infantile diarrhoea. Vibrios distinct from the cholera spirillum have been found also in the mouth, and in the water supply of numerous towns. It is evident, therefore, that considerable caution should be exercised in the identification of the genuine vibrio, particularly in the absence of an epidemic. The Spirochaeta Pallida (Treponema Pallidum) (Plate IX.). — In the fresh exudate examined under the dark ground illumination (page 161) the spirochete of syphilis appears as a slowly-moving, extremely delicate organism with numerous regular spirals, which do not straighten out during rest. On occasion small refractile granules (the granular bodies of Leishman) may be seen to be shot out from the spirochete into the surrounding medium. In Indian ink preparations (page 160) the spirochete has a very similar appearance, but has of course no movement. The spirochsete is left unstained by the ordinary methods, and is best stained by Giemsa's dye after preliminary fixation with absolute alcohol (page 160). The appearance in stained preparations is very characteristic. The Spiroclueta pallida stains pink, in contradistinction to the majority of other spirochetes, which stain a bluish red. The minute delicate spirals are numerous — from 10 to 20 — and very 142 CLINICAL PATHOLOGY. regular. The spirochete is usually present in considerable numbers in both primary and secondary lesions. It has been very rarely demonstrated in tertiary syphilis and never in parasyphilis. The Spirochceta pallida cannot be cultivated on the usual media. Inoculation of fluid containing the organisms into one of the higher apes is followed by a local chancre and later by secondary manifestations. Inoculation into the lower monkeys or into the testicle of a rabbit is followed by a local sore only, which tends to heal spontaneously. The early diagnosis of syphilis by the demonstration of the spirochete in the primary chancre before the serum reaction has become positive is of the utmost importance, since the cure of the disease depends so much upon the promptitude with which treatment is commenced. The success of the demonstration rests largely upon the care with which the material to be examined is obtained. The surface of the chancre should first be washed over thoroughly with sterile salt solution and an attempt made by vigorous squeezing of the edges of the ulcer to cause a clear or blood-tinged serum to exude from the depths of the base. If the chancre is very painful or firmly encrusted over, the surface should be well swabbed with 4 per cent, eucaine and a further attempt made to obtain the proper fluid, aided, if necessary, by a scraping of the chancre base with the edge of a glass micro- scope slide. The fluid is transferred in a platinum loop to a slide, and both stained and fresh specimens should be pre- pared (page 160). The greatest care should be taken to avoid infecting one's own fingers in performing this examination and to wash immediately and thoroughly if the fingers touch the chancre. A negative examination is not sufficient contra-indication of syphilis, but in skilled hands negative results are unusual. In cases of doubt a second examination should be made, and if enlarged glands are present in the groin the most prominent should be punctured with a hypodermic needle, and the minute quantity thus obtained often yields a positive result. The recognition of the Spirochceta pallida is positive evidence of syphilis, provided the identification of the organism is correct. Numerous other varieties of spiral organisms are to be found in the body, particularly in the region of the mouth, the anus, and much less frequently the male urethra and the vulva. GRAM-NEGATIVE BACILLI— SPIRILLA, ETC. 143 Particular care, therefore, should be exercised in the examina- tion of the majority of extragenital chancres. The main points in the identity of the syphilitic organism are : the com- parative lack of motility on the dark ground illumination, the failure to stain by the ordinary dyes and the rose-pink colour when stained by Giemsa's dye, the delicacy of the spirochete, the large number of its spirals (from 12 upwards should be counted) and their regularity. The more important of the spirochetes which can be confused with S. pallida are men- tioned below. The Spirochete of Yaws (S. pertenuis) is morpho- logically identical with S. pallida. Spirochseta Refringens, etc.— 8. refringens is a sapro- phytic organism, and is frequently associated with S. pallida. It is long, coarse, and has about six irregular spirals. It stains, though somewhat faintly, with the ordinary dyes. S. Gracilis occupies an intermediate position, so far as appearance goes, between S. pallida and S. refringens. S. Dentium is a minute spirochete, of about half the length of S. pallida, and is commonly found in the mouth. It stains with the ordinary dyes. Many other names have been given to these saprophytic spirilla, some of which have been cultivated, and a useful study of some of them can easily be made by examining films made from any case of ulcerative stomatitis, or even from a comparatively normal mouth, by the methods used in the diagnosis of the S. pallida. The spirillum and fusiform bacillus of Vincent (Plate IX.). — Among the spirochetes which have achieved notoriety either as producing disease or as saprophytes the organisms of Vincent must be mentioned. These organisms consist of spirilla which are usually large, irregular, and with few spirals, resembling S. refringens, and less commonly minute and delicate like S. dentium. The spirilla are asso- ciated in film preparations with large, coarse, fusiform bacilli. Both organisms stain with the ordinary dyes and do not grow on the ordinary media. The spirilla and bacilli are fairly constantly present in septic mouths. They are extremely numerous in certain forms of ulcerative stomatitis and tonsilitis, often associated with sloughing and membrane formation, particularly in children. The diphtheria bacillus 144 CLINICAL PATHOLOGY. is absent in such cases, though other organisms, such as streptococci, are commonly present. The condition is known as Vincent's angina. Spirochaeta Obermeieri (S. recurrentis). — This organism has been considered in the chapter on the parasitology of the blood. StreptotrichejE. Actinomyces (Ray fungus) (Plate IX.).— This class of organism, which doubtless embraces a number of closely- allied species, rarely attacks man, and is found more commonly among the domestic animals. Under the microscope the parasite is found to consist of three parts: — (1) Long, thin filaments enclosed in a sheath and often beaded : the filaments are interlaced and may show branching; they are Gram-positive. (2) Coccus-like bodies, formed from the filaments, and which have been considered to represent gonidia ; they also are Gram-positive. (3) Clubs or elongated pear-shaped bodies radiating from the centre of an actinomycotic colony ; they are usually Gram-negative. Actinomyces grow on all the ordinary media, and on glycerin-agar growth becomes visible about the fourth day, and later forms tough, nodular, granulated colonies of a brownish colour. Gelatin is liquefied. Clubs are not present in cultures. In the human subject actinomyces may attack the mouth or tongue, spreading from thence to the glands of the neck, or may effect a lodgment in the intestine, and not infrequently in the appendix, and spread from thence to the liver and other parts of the body. In the examination of pus from suspected actinomycotic lesions the little colonies which are visible to the naked eye should be sought for. They are readily recognised as round or oval greenish-yellow granules of about the size of a pin's head, more or less translucent and definitely resistant to the touch. If a granule is squashed on a slide and stained by carbol thionin or by Gram's method the beaded filaments can be certainly recognised. The clubs, which are common in lesions of the ox, are very rarely seen in man. Cultures are not strictly necessary for diagnosis, and are often difficult to obtain owing to the presence of other organisms. The GEAM-NEGATIVE BACILLI— SPIRILLA, ETC. 145 granules should be picked out with a sterile needle and repeatedly washed in sterile salt solution before planting on glycerin-agar. Other varieties of streptothrices have been described and isolated in lesions from human beings which differ in their cultural characters, and particularly in the colour of their colonies, on solid media. Some are acid-fast, and the majority of them produce caseous nodules when inoculated into guinea-pigs. These streptothrices are rarely met with, but may produce pulmonary lesions such as can be mistaken on clinical grounds for tuberculosis, and on pathological grounds may escape identification if the beaded threads are acid-fast. Suspected cases should be carefully examined both micro- scopically and culturally, and the sputum should be inoculated into guinea-pigs with a view to recovering the organism in pure culture. Madura disease is comparatively common in India and in other tropical countries. It is a granulomatous con- dition of the foot set up by a streptothrix culturally distinct from actinomyces. The growth is pigmented, and the colour may be either pale pink or black. The mycelial filaments and the granules may readily be detected in film preparations made from colonies scraped out of the skin. Hyphomycetes. The more important of the hyphomycetes are in human pathology met with in diseases of the hair and skin. A brief description only can be given here. The organisms are again referred to in the section on the skin. Ringworm. — Two species of parasite are met with. The microsporon Audouini is the common cause of ringworm among children, and appears in the form of numerous small spores and scanty mycelial threads situated outside the affected hairs. Trichophyton megalosporon endothrix is less fre- quently met with; the spores are large and the parasite is situated within the hair shaft. The majority of such moulds can be grown on agar media and preferably on Sabouraud's maltose agar medium. p. 10 146 CLINICAL PATHOLOGY. The demonstration of the parasites in hair can be performed as follows : — Eemove with a pair of fine forceps a few of the bent and stunted hairs. Place in a watch-glass and soak in ether for a few minutes. Mount on a slide in 10 per cent, caustic potash beneath a cover-glass and examine with a J-inch objective for the oval refractile spores. To make a permanent specimen : — After cleansing in ether fix on a slide with a little melted paraffin. Stain by Gram's method up to the stage of decolorising in methylated spirit, which should be completed by immersion in absolute alcohol. Then wash in xylol, and scrape off the remainder of the paraffin. Drain off the xylol and mount in Canada balsam. Favus is due to a parasite, the Achorion schoenleinii, which consists of a tubular branching mycelium with a few oval spores. Pityriasis versicolor is caused by the Microsporon furfur, which consists of abundant mycelium, spores and hyphse and is readily cultivated. To examine for these parasites scrapings can be taken of the affected skin scales and mounted in potash or stained with gentian violet in a similar manner to the examination of hairs for ringworm. Aspergillus niger is an instance of a non-pathogenic mould which may on rare occasions infect the tissues, and has been found widely distributed throughout the lungs. The organism produces a sooty growth on potato. Thrush (Plate IX.) is caused by the Oidium albican?, an organism classed by some among the moulds, by others among the blastomycetes or yeasts. It grows like a yeast in culture, showing large oval budding cells, and in scrapings from the throat coarse mycelial threads are seen, together with as a rule the yeast-like cells. The Protozoa. — The majority of these have been described in the chapter on the parasitology of the blood. Others, such as the Entamoeba histolytica of tropical dysentery, are con- sidered under the examination of tbe fceces. CHAPTER XL bacteriological methods — general and special. General Methods. The general apparatus required for the majority of bacteriological examinations consists of — Slides and cover-glasses. Platinum wire. Forceps. Culture tubes and crates. Incubator. Staining reagents. The slides and cover-glasses used require no special pre- paration, but are conveniently kept in methylated spirit in wide-mouthed glass jars until required for use, when they are wiped dry with a clean cloth. After use the slides can be placed in lysol for an indefinite time and subsequently cleaned and used again. The platinum wire should be a moderately stout piece about 4 inches long. The wire should be fixed into one end of a glass rod about 9 inches in length by the simple procedure of heating the end of the rod to red heat in the blow-pipe flame and plunging the platinum wire into it while hot. The free end of the platinum wire should then be bent round upon itself to form a small oval loop. The returning end of the wire should exactly meet the straight wire and should not overlap it. An untidy platinum wire is often responsible for untidy and con- sequently both inaccurate and dangerous bacteriological work. The forceps should be of the ordinary straight variety used in dissection or operation work. It is convenient also to have a pair of catch forceps of the pattern known as Cornet's. The culture tubes should be of the kind described in a former chapter, and can be bought ready for use. If the amount of bacteriological work required is considerable, it is very much more satisfactory and economical to prepare the media in the laboratory. The methods of making the media will be 10—2 148 CLINICAL PATHOLOGY. described subsequently. The essential media consist of broth and agar slopes, and these are sufficient for taking the primary cultures in most cases. For the complete investigation of organisms other media are necessary. After inoculation the tubes are preferably placed in basket wire crates lined at the bottom with cotton wool. The crates can be dispensed with and ordinary glass beakers or even old coffee tins may be made use of instead. The incubator should be one capable of maintaining a constant temperature of 37° C. Reliable incubators pro- vided with self-regulating capsules are supplied by several firms. A less expensive ap- paratus consists of a copper chamber fitted with a water- jacket and warmed by a gas-jet, the heat within the chamber being controlled by the size of the jet. Such an apparatus is liable to vary with any considerable fluctua- tions in the room temperature. "When occasional bacteriological investigations only are required, a wide-mouthed "Thermos" flask can be readily adapted. The variety of flask made to hold soups or stews, and costing 30s., is the most suitable, and can be fitted with an amateur copper wire framework capable of hold- ing 4 culture tubes. The flask is filled about two-thirds fall with water at 40° C, a temperature which in 18 hours will have dropped to 35° C. Such an incubator is sufficiently reliable for the cultivation of diphtheria bacilli, or indeed the majority of readily-growing pathogenic organisms. The staining reagents requisite for all ordinary bacterio- logical work are few in number. The majority of them can be bought ready made up in solution, and the mode of pre- paration of each is given subsequently. The general procedure to be followed when investigating the bacterial content of pus or of any body fluid is as follows ; — (1) Make films of the pus. Fig. 11. — Bacteriological Incubator. BACTERIOLOGICAL METHODS. 149 ((a) with some simple dye such as carbol- (2) Stain themj thionin. ( (b) by Gram's method. (3) Examine the films. (4) Put up cultures in both liquid and solid media. (5) Incubate at 37° C. for from 12 to 24 hours. (0) Examine the culture tubes with the naked eye and with a hand-glass. (7) Make films from the cultures. Stain and examine them. (8) If the organism is in pure culture, subculture it from the solid medium into the appropriate media. If a variety of organisms are present plate out from the culture into Petri dishes. (9) After incubation of the sub-cultures for 24 hours (or longer if necessary) examine them, note the changes which have occurred in them, and make film pre- parations from them. (1) To make films from pus. — Clean a slide. Sterilise the platinum wire by heating it to a red heat in a Bunsen flame. Let the wire cool in the air. Take up a loop of the pus and spread it evenly by a circular motion so as to make a thin round film about the size of a shilling in the centre of the slide. Sterilise the platinum wire. Dry the film by waving it in the air above the flame. Fix by passing it three times rapidly through the flame. (2) To stain the films.— (a) With a simple stain. This should always be done as a routine. The examination of films made from the original fluid yields information which may be altogether missed in the investigation of the culture tubes. Also an indication is obtained of the nature of the organism and consequently of any special stain that may be necessary, as well as of the appropriate media upon which to make the cultures. The best general stain is carbol-thionin. The advantages of this stain are that it brings out the majority of organisms and the cells present, and that it is almost impossible to either under- or over- stain the films. To stain with carbol-thionin.— Cover the entire slide with the stain ; do not merely place a few drops of the stain on the film itself. Leave for approximately 3 minutes. 150 CLINICAL PATHOLOGY. Wash in tap water. Blot dry. Mount in Canada balsam. (The mounting of any film which is to be examined with a Y^-inch objective is unnecessary unless a permanent specimen is required, since the cedar oil placed directly on the film clears it.) In place of carbol-thionin, dilute methylene blue may be used ; the staining process is identical. (b) By Gram's method. — It is not necessary to make use of this method in all cases. The less experience the student has in the examination of bacteriological films the more frequently he should take advantage of the information given by Gram's stain. To stain by Gram's method. — Take 3 c.c. of aniline oil and 17 c.c. of distilled water in a measure glass. Shake vigorously for 5 minutes. Filter. The filtrate must be clear. Take 3 c.c. of a saturated solution of gentian violet in absolute alcohol and 7 c.c. of the aniline water. Mix. Filter the aniline gentian violet on to the slide. Stain for 5 minutes. Pour off the stain and rapidly dip the slide in tap water. Drain off the water. Cover the slide with Gram's iodine. Leave for 30 seconds. Wash in methylated spirit and continue to wash until the blue colour ceases to run from the film. Blot dry. Countersfcain with carbol-fuchsin diluted with tap water to about the colour of red ink. Stain for 2 minutes. Wash in tap water. Blot dry. Mount. The principle of the stain is as follows. Aniline gentian violet stains the great majority of all organisms. If the film is then washed in spirit the stain comes out again. But if the film, after staining, is treated with Gram's iodine the gentian violet is fixed in some organisms so as to resist subsequent decoloration by spirit, but not in other organisms. The counter-stain with carbol-fuchsin is not part of Gram's method, but is used to display those organisms which have lost their colour in the spirit. " Gram-positive " organisms are consequently coloured violet and " Gram-negative " organisms red. The distinction between these colours is BACTERIOLOGICAL METHODS. 151 more obvious by daylight than by artificial light. No fixed time is given for the decoloration stage in spirit, because the time occupied depends largely upon the thickness of the film. It must be recognised that it is possible to wash the colour out of a "Gram-positive" organism or 'to leave the colour in a " Gram-negative " one if the washing is too long or too short. The alcohol process is finished directly the blue colour ceases to run from the film, and this point is readily determined if a small quantity of clean methylated spirit is reserved for the final dipping of the slide. (3) To examine the films.— Use a No. 2 eye-piece, a Y^-inch objective and, when available, daylight. Look for the kind of cell present. In the great majority of such films these will be polynuclears, with an occasional large hyaline cell and possibly a few epithelial cells. Examine for the presence of bacteria, and note whether they are cocci or bacilli, whether they are arranged in pairs, clumps or chains, and whether they are mainly within the cells or outside them. If more than one variety of organism is evidently present observe which variety appears to pre- dominate. If no bacteria are seen in the films the causative organism may still be recovered in the cultures. (4) To put up cultures. — If the pus is taken directly from the body it is commonly necessary to make both films and cultures at the same time. In such cases the culture media suitable to the suspected organism are chosen. If the pus is received in a sterile receptacle in the laboratory it is preferable to examine the films before putting up the cultures. In the majority of cases two tubes should be inoculated, and these should be a broth tube and an agar slope. It is often convenient to add a litmus milk tube or to substitute it for the broth tube, since some organisms, and in particularly the pneumococcus, grow more readily in milk than in broth or even than on agar. When organisms such as the gonococcus or meningococcus, which do not grow at all, or grow poorly, in the primary cultures on agar and broth, are suspected the appropriate blood-containing media must be used. To inoculate the tubes. — Hold the test tube containing the pus and the culture tubes between the thumb and first finger of the left hand. The tubes should not be held upright, 152 CLINICAL PATHOLOGY. but in a slanting direction, since dust and organisms may fall into them. Sterilise the platinum loop in the flame. Allow it to cool. Place the glass handle between the first and second fingers of the left hand. Take a pair of forceps in the right hand and with a screwing motion twist out the cotton-wool plugs from the tubes. Place each plug between the third and little fingers of the left hand. Put down the forceps on the bench. Take the platinum wire in the right hand and pass it into the tube of pus. Take out the wire, pass it into the agar slope tube and spread it gently over the surface of the medium. Dip the wire again into the pus, then into the broth tube, and shake off the pus into the broth. Be careful not to touch the sides of any of the tubes with the wire. Sterilise the wire and lay it down. Pick up the forceps. Take each wool plug separately and light it in the flame. Put the plug, still alight, into the tube. Do not blow the plug out ; it will cease to burn as soon as it is pressed well home in the tube. With a blue glass-pencil mark each tube with the date and a distinguishing number. (5) To incubate the tubes. — Place the tubes in a wire crate, taking care not to knock them on the edge of the crate, and place in the incubator at 37° C. till the following day. From 12 to 24 hours is sufficient time in which to obtain a naked-eye growth of the majority of organisms, but the cultures should not be considered sterile before the lapse of at least 4 to 5 days. Exceptional organisms, such as the tubercle bacilli, show little or no growth before the tenth day. (6) To examine the culture tubes. — Remove the tubes from the incubator and look at them to see if any growth has taken place. Note the character of the growth in broth and the size, shape, colour, and density of the colonies on the agar slope. Examine the agar slope further with a hand lens. (7) To make films from the cultures. — In the case of a liquid medium hold the tube in the left hand, sterilise the platinum wire, pass it into the left hand, and allow it to cool. Remove the wool plug with forceps and with the wire take BACTERIOLOGICAL METHODS. 153 up a considerable loop of the broth (or milk) culture. Pass the wire back into the left hand. Ignite the plug and replace it in the tube. Make as thick a film as possible exactly in the centre of a clean slide. Sterilise the wire. In the case of a solid medium first place a small drop of tap water on the centre of the slide, then proceeding as above, take up in the wire a small particle of the growth and rub it in the water, making as thin a film as possible. Allow the films to dry. Fix them in the flame. Stain with carbol- thionin. If necessary make additional films and stain by Gram's method. Examine the films under the microscope. (8) To make sub-cultures. — It is advisable in the majority of cases to " plate out " from the broth culture, but this may be dispensed with if an examination of the original films and of the cultures reveals only one variety of organism. Particular attention is to be paid to the nature of the colonies on the agar slope, and if these appear identical with each other the culture may be presumed "pure." When the original cultures are pure, subculture from the agar slope into those media which give characteristic reactions with the suspected organism. In most cases it is advisable to subculture into litmus milk, neutral red broth, a selection of the litmus carbohydrate broths, and on to gelatin slopes. To make the sub-culture hold the agar tube and two or three of the tubes to be inoculated in the left hand. Sterilise the wire. Allow it to cool. Scrape off a colony, or portion of a colony, from the agar slope. Shake off the growth in a liquid medium or rub it over the surface of a solid one. Sterilise the wire. Replace the plugs. Label each sub-culture with the name of the medium (particularly in the case of the carbohydrate media), the date, and the name or number of the case. A variant of the above procedure consists in taking a single colony from the agar slope and inoculating it upon a second agar slope, incubating till the next day, and then subculturing from the second slope into the media. By this method a pure culture is practically assured, but an extra day is required. When the original cultures are " mixed " it is necessary to separate the different organisms, and this is done by means of plate cultures in Petri dishes. A minimum of two plates 154 CLINICAL PATHOLOGY. should be used, and if the organisms are varieties of cocci both plates should contain agar. If organisms of the colon group are suspected prepare one agar plate and one plate containing MacConkey's neutral red medium. The plate cultures are pre- pared as follows : — Place " stab " culture tubes in a tall beaker filled with hot water to above the level of the medium in the tubes. Bring the water to the boiling point over a Bunsen burner. When the media are completely liquid turn out the gas and leave for 5 or 10 minutes. Have ready sterile Petri dishes with well-fitting lids. Take a melted stab culture tube from the beaker. Twist out the wool plug with forceps. Pass the mouth of the test tube through the flame (to sterilise the outside of the glass). Lift ivp the lid of the Petri dish just enough to insert the mouth of the test tube. Pour the con- tents of the test tube into the dish. Replace the lid and rotate the dish so as to spread the medium evenly over its bottom. As soon as the medium is set, place the dish upside down in the ice-chest (or failing that at room temperature) until the dish feels quite cold. To use the plates : — Take the incubated broth culture tube in the left hand. Sterilise the platinum wire. Take one loop of the broth culture. Put back the broth tube in the crate after replacing the plug. Lift up the lid of the agar plate the minimum distance. Rub the platinum wire repeatedly across the agar medium in a succession of parallel streaks. Replace the lid of the dish. Lift the lid of the second agar or the MacConkey plate and repeat the process without recharging the platinum loop. Place the plates in the incubator upside down in order to prevent the water of condensation w T hich collects in the lid of the plate from dropping on the medium surface. Do not breathe on the plate w T hile filling it or while inoculating it. After from 12 to 24 hours' incubation of the plate cultures examine them with the naked eye and with the hand glass. Observe the types of colony present in those parts of the plates in which the colonies are discrete. Note the colour, shape and size of the colonies. Make film preparations if necessary. Subculture from each variety of colony on to agar slopes. Incubate the sub-cultures and the following day pass them through the various media. (9) To examine the sub-cultures. — Note the naked-eye changes which have occurred in each tube at the end of BACTERIOLOGICAL METHODS. 155 24 hours. Examine film preparations from one or two of the tubes. The complete changes will probably not have taken place in the media after only 1 clay's incubation, but with many organisms a diagnosis can be made at this stage. Indole formation in the broth tube may require at least 3 days' incubation, and so may the production of a green fluorescence in neutral red broth. The complete coagulation of milk, the liquefaction of gelatin, and the production of an acid reaction in the carbohydrate media may require some days. It is advisable, therefore, to postpone the indole test as long as possible and to incubate all the tubes for from 5 to 7 days before discarding them. Special methods. — The preceding sections deal with the routine bacteriological examination of pus or other body fluids. The majority of the following methods are special only in the sense that a special organism is deliberately sought for. Nearly all the methods are in common use. Further informa- tion as to the organisms may be found under the description of each in a previous chapter. Tuberculosis. — The methods of demonstrating the tubercle bacillus differ with the nature of the suspected material. In the case of sputum it is advisable to collect a mixed sample of the matter expectorated in the 24 hours. When the sputum is scanty or the tubercle bacilli are few in number, the early morning sputum should be particularly examined. Quite young children commonly swallow the sputum, and it may be impossible to obtain sufficient for examination. In such cases the bacilli may be demonstrated in the fseces. Having obtained the sputum, empty it into a wide-mouthed bottle provided with a well-fitting cork. Add five times the volume of 1 in 20 carbolic acid. Shake thoroughly for about 5 minutes. Stand till the next morning. (The object of the carbolic is to separate the mucoid from the purulent part of the sputum and to a less extent to clump the tubercle bacilli.) After standing, pour off the layer of mucus, which has separated off at the top of the bottle, together with the supernatant fluid, leaving the deposit of pus. Centrifuge the deposit. Shake the sediment on to a glass slide held in the left hand. Take a second glass slide in the right hand, holding each slide at its extremity. Press the top slide with a to-and- fro movement several times firmly over the under slide until 156 CLINICAL PATHOLOGY. an even film of sputum is spread over both slides. Wipe the backs of the slides and dry the films. To stain the films : — Filter carbol-fuchsin (full strength) into a large-bore test tube. Bring it carefully to the boiling- point, constantly shaking the tube and keeping its mouth pointed away from the face. Cover the slides with the boiling stain. Stain for 7 minutes, and during that time pour off the stain twice and add fresh boiling stain. After 7 minutes pour off the stain, dip the slide in water and then place in 25 per cent, sulphuric acid. (It is advisable when dealing with two slides to leave one staining in the fuchsin while the other is being decolorised in the acid.) When the slide is decolorised place it in tap water. The red colour will probably return, in which case replace in the acid and then return to the water. Eepeat the process until the pink colour fails to return after immersion in water. .Leave in fresh water for 2 minutes. Counterstain in 1 per cent, methylene blue, diluted four times with tap water, for 2 minutes. Wash in tap water. Dry and mount. This method is known as the Ziehl-Neelsen process. In the preliminary staining with fuchsin all organisms and cells are stained red. After the acid everything is decolorised except the tubercle bacillus. The final washing in water brings a brighter red colour into the tubercle bacillus. The counter-stain renders other organisms and the cells blue. The slides should be examined under the oil immersion lens, and at least a quarter of an hour should be spent on them before the tubercle bacillus is pronounced to be absent. When tuberculosis of the lung is strongly suspected on clinical grounds and the bacilli cannot be demonstrated by this way, the " antiformin " method may be used. "Anti- formin " can be prepared by making a stock solution of equal parts of liquor sodse chlorinate (B. P.) and 15 percent, sodium hydrate. A 20 per cent, dilution of this in tap water is shaken with about one-fourth its bulk of sputum and left until all the gas has been evolved and a fairly homogeneous solution has resulted. The mixture is then centrifuged at a high speed. Films are made from the sediment and stained in the usual way. The effect of the antiformin is to dissolve almost everything BACTERIOLOGICAL METHODS. 157 in the sputum except the tubercle bacillus, the capsule of which is not acted upon. Cultures on Dorset's egg medium can be made from the sediment after washing it free from antiformin with distilled water, or animal inoculations may be carried out. In the case of urine, collect the deposit of the urine passed during the 24 hours. Treat the deposit with carbolic acid and prepare films in the same manner as with the sputum. The advantage of investigating the deposit obtained in this way rather than that from a catheter specimen is that the bacilli are less likely to be missed, since they are not necessarily passed with every sample of urine. The disadvantage is that smegma bacilli are likely to be present. The smegma bacillus can be disregarded if the films are passed through alcohol. In the case of all urinary pus, therefore, after decolorisation in acid, wash in water and then soak in methylated spirit for 2 minutes. Then wash in water and counterstain in methylene blue. The examination of urinary slides should be made with particular care, since numerous erroneous diagnoses of tubercle bacilli have been made. The mistake lies as a rule in mis- taking, not the smegma bacillus, but the bacilli of the colon group, which may be present and stained red. The colon bacillus is in no sense acid-fast, but it is not infrequent to find that urinary crystals, or even large epithelial cells, have held the fuchsin stain in the acid, and that the stain diffuses out over a small area in the tap-water stage and stains the bacilli in that area a bright red. Such bacilli are seen to be lying on a red background, and the mistake is thus easily recognised. The slide must be again decolorised. The red tubercle bacilli must be seen to lie on a blue background. The possibility of this error is a very real one, and a clump of such diffusion-stained bacilli is to be seen beautifully figured, and labelled as tubercle bacilli, in a standard text-book. In the case of faeces tubercle bacilli are commonly very scanty except when actual ulceration of the gut is present. It is advisable to take a small quantity of the faeces and treat it direct with antiformin. Films are made from the centrifuged deposit and stained in the same manner as for sputum. In the case of tuberculous pus, such as may be obtained from a caseous gland, a tuberculous joint, etc., the bacilli are sometimes 158 CLINICAL PATHOLOGY. numerous, but in the great majority of cases are extremely scanty. Such material is best treated at once with antiformin, and scanty bacilli can frequently be demonstrated in this way. Antiformin gives better results with tuberculous pus or tissues than with sputum. The detection of tubercle bacilli in pleural, peritoneal, and cerebro- spinal fluids is dealt with in the section which treats of these exudations. The investigation of urine, faeces, and sputum for other organisms than the tubercle bacillus is considered under the appropriate headings. The cultivation of tubercle bacilli is rarely successful if attempted direct from the human lesion owing to the slow growth of the bacillus and the frequent presence of other readily growing organisms ; nor is it often necessary to cultivate the bacillus, since in the great majority of cases it can be recognised in film preparations. When a culture of the organism is desired, the sputum, urinary deposit, or caseous pus is first inoculated into a guinea-pig and the cultures are made from the resulting lesion in the animal. By the use of antiformin animal inoculation may be avoided and the cultures can be made on Dorset's egg medium from the sediment left after centrifuging the solution. The sedi- ment should be washed two or three times with sterile water before inoculating the tubes. It must be confessed that the antiformin method of treating sputum is often disappointing, and that if the bacilli are not found in the carbolised sputum they are not likely to be found or grown after treatment with antiformin. The inoculation of animals is still occasionally necessary in order to prove the tuberculous nature of a body fluid in which the bacilli can neither be found nor cultivated, or to identify the exact nature of the acid-fast organisms present. The material for inoculation is preferably injected into the thigh of a guinea-pig, but if the bulk is too great for intra- muscular injection, the inoculation is made into the peritoneal cavity. About 10 days after injection into the thigh the inguinal gland becomes palpably enlarged. In from 2 to 3 weeks the spleen becomes infected, and the animal dies of generalised tuberculosis from G to 8 weeks after inoculation. The injections are made with strict aseptic BACTERIOLOGICAL METHODS. 159 precautions, and preferably two animals are inoculated. After killing the animal the bacilli are readily identified in the more advanced lesions, which show the typical gross and micro- scopical changes of tuberculosis. Diphtheria. — The taking of swab and culture for the identification of the diphtheria bacillus and the differentiation of this organism from similar bacteria have been described under the heading of the diphtheria bacillus. The examination of the swab and culture is conducted as follows : — Twelve hours incubation at 37° C. is sufficient to obtain a growth of the organism. After incubation examine the blood serum tube with the naked eye and with a hand- glass. Clean two slides and place a drop of water in the centre of each. On one slide make a thin film from the most suspicious colonies on the serum slope. On the other slide make as thick a film as possible by rubbing the swab in the drop of water. Dry and fix the slides. Pour on Lofiier's methylene blue and leave it for 3 minutes. Wash in tap water. Blot dry and mount. Lofner's methylene blue consists of an alcoholic solution of the dye with potassium hydrate added. The beading of the bacillus is more satisfactorily displayed by means of this stain than with the ordinary dyes such as carbol-thionin. The identification of the bacillus on morphological grounds is a matter requiring practice, and no real additional informa- tion is gained by more elaborate staining methods, although the student is advised to confirm his opinion with a Gram preparation. Neisser's method of staining is still in common use, and those who prefer brown bacilli with blue dots to alternating pale and dark blue organisms should use this method. The differential staining is no real criterion of the virulent as opposed to the diphtheroid organisms, but is a help to those unacquainted with the morphology of this group of bacilli. The method of staining is as follows : — Make a mixture of 2 parts of a solution composed of Methylene blue powder, 1 gramme ; Absolute alcohol, 50 c.c. ; Glacial acetic acid, 50 c.c. ; Distilled water, 1,000 c.c. ; 160 CLINICAL PATHOLOGY. and 1 part of the solution Crystal violet, 1 gramme ; Absolute alcohol, 10 c.c. ; Distilled water, 300 c.c. Stain in this mixture for 2 seconds. Wash rapidly in water. Counterstain for 3 seconds in the following solution : — Cresoidin, 1 gramme (dissolved in 300 c.c. of warm water and filtered). Wash in water. Dry and mount. The body of the bacillus is stained brown and the granules blue. Syphilis. — The method of obtaining the material for examination has been described in the section on the SpirocJiceta pallida. The fluid is examined in the following ways : — (a) The Indian ink method. The most suitable variety of Indian ink is that known as " chin chin liquid pearl." The bottle must not be shaken. Place at one end of a clean slide a small drop of the ink. Take a platinum loop of the secretion and thoroughly mix it with the ink. With a second slide make a thin film of the mixture in the same manner as if making a blood film. When dry examine direct with the oil immersion lens, using artificial light. The spirochetes are seen as white refractile threads on a brownish-black background. (b) By Giemsa's stain. Make on clean slides two or three films of the secretion with a platinum loop. When dry cover with absolute alcohol and leave for 15 minutes. Make up from the stock Giemsa's stain a mixture of 10 c.c. of distilled water and 14 drops of the stain. Pour off the alcohol and cover with the stain for 45 minutes. Wash in a rapid stream of distilled water. Dry and examine with a -^-inch objective. The Spirochceta pallida stains a rose-pink colour, other spirochetes and bacteria blue. The difference in shade between the syphilitic and other spirochetes is frequently BACTERIOLOGICAL METHODS. 161 far from obvious, and greater reliance is to be placed on the morphological points of distinction. (c) The dark ground illumination (the ultra-microscope). A special condenser (the paraboloid condenser) is required for this method of investigation. It is fitted into the collar pre- pared for the ordinary condenser. A " stop " is also used which fits into the inside of the j^-inch objective. Special thin slides and cover-glasses should also be obtained. The illuminant must be a powerful one, and should either be a small arc or a Nernst lamp. The microscope should be vertical. A drop of the fluid is placed on the centre of the slide, and if only a small quantity is available saline should be added. The cover-glass is carefully let down on to the slide so that all aiu bubbles may be avoided. A drop of cedar oil is placed on the upper surface of the condenser and another on the under surface of the slide. The condenser is racked up until the two drops coalesce. A third drop of oil is placed on the upper surface of the cover-glass, and the objective is focussed after first directing all the available light through the preparation. Instead of an immersion lens the J-inch objective and a high eyepiece may be substituted. The spirochetes show as shining white refractile bodies on a black background. A great advantage of this method is that the motility of the organisms can be studied. The bacterial investigation of viscera. It is occasionally necessary to investigate the bacterial content of a closed viscus, such as a cyst or a pyosalpinx. The procedure is as follows : — Place the specimen on a clean plate. Heat a metal spatula to red heat in the flame and smear it firmly over the surface of the specimen. With a sterile knife cut through the seared surface into the centre of the specimen. Hold the edges of the cut apart with a pair of sterile forceps. With a platinum loop make films and cultures from the pus or from the fluid in the viscus. The bacteriological examination of viscera post mortem is conducted in the same manner. In cases of general infection, and particularly in typhoid fever, the causative organism may p. 11 162 CLINICAL PATHOLOGY. be obtained in pure culture from the spleen juice. Owing to the rapid emigration of organisms from the intestinal tract into the viscera at the time of death post-mortem bacterial findings should always be accepted with reserve. Anaerobic cultures. By an anaerobic culture is meant the incubation of an organism in an atmosphere free from oxygen. The simplest method of obtaining such an atmosphere is by means of Buchner's tubes. These tubes consist of long, wide-bore test tubes constricted a few inches from the bottom in such a way that an ordinary test tube passed into the Buchner's tube can rest upon the constriction. The tube is supplied with a well-fitting rubber cork. To put up the anaerobic culture : — Shake pyrogallic acid powder into the Buchner's tube so as to fill the space between the bottom of the tube and the con- striction. Pour in 15 per cent, caustic potash to the level of the constriction. Kapidly slide in (do not drop in) the inoculated culture tube, with wool plug in place. Bapidly fix in the rubber cork of the Buchner's tube. Plaster round the junction of the cork and the tube with soft paraffin. The alkaline pyrogallic solution absorbs the oxygen from the interior of the Buchner's tube and of the culture tube. Special staining processes. Capsules. — Cover the films for 3 minutes with 1 per cent, acetic acid. Drain off the acetic acid and dry. Stain for 10 seconds in aniline gentian violet (see under Gram's stain). Wash in water and examine in water. If the capsule is stained too deeply wash in 1 per cent, acetic acid. Dry and mount. This method of staining is not infrequently unsuccessful and is rarely called for. Spores. — Fix the film after drying by passing many times through the flame. Stain with boiling carbol-fuchsin, giving several changes, for 10 minutes. Dip in acid alcohol (99 c.c. alcohol, 1 c.c. HC1). BACTERIOLOGICAL METHODS. 163 Blot dry. Stain in dilute methylene blue 2 minutes. Wash in water. Dry. Mount. The spores are red and the bacilli blue. The difficulty with the process lies in the differentiation with acid alcohol. With the majority of spores it is difficult to get the fuchsin to penetrate them sufficiently to allow more than the most rapid dipping in the spirit. Successful preparations have quite a striking appearance, but the student should be able to recognise with certainty spores in ordinary carbol- thionin preparations and to distinguish them from beading of the bacillus by their regular shape and clean-cut outline. Flagella. — Film preparations are made from a growth on a solid medium. In making the films care is taken to avoid picking up any of the medium and to spread the bacteria as thinly and evenly as possible. Prepare the following mordant : — Tannin, 2 grammes. Water, 20 c.c. Ferrous sulphate solution of 1 : 2 strength, 4 c.c. Saturated alcoholic solution of fuchsin, 1 c.c. Pour mordant over film and heat without boiling 1 minute. Wash in water. Stain with carbol-fuchsin. Wash in water. Dry. Mount. This method is described as the most simple of the flagella staining processes. There are so many cultural and other methods of differentiating the flagellated bacteria that the demonstration of the flagella is rarely necessary. 11—2 CHAPTEE XII. vaccines anti-sera. Vaccines. A vaccine is a sterile standardised suspension of dead organisms in a neutral fluid. It is given hypodermically in doses graduated according to the actual number of bacteria present in each dose. The object of vaccine treatment is to raise the resistance of the individual to the organism with which he is infected by carefully graded and spaced doses of dead organisms. The toxins contained in the dead bacteria are liberated in the tissues, passed into the circulation, and thus lead to an over-production of antibodies which can be used to combat the living organisms of the disease. Less frequently vaccines are given to a healthy individual with the view of rendering him immune to a possible infection. The immunity induced by vaccines is an artificial acquired immunity. It is established comparatively slowly, over a period of weeks, and lasts a comparatively long time. The treatment of a patient with vaccines necessitates a co-operation between the pathologist and the clinician, since it is insufficient in the great majority of cases to raise the immunity of a patient to a given organism. The local and general treatment of the disease must also be undertaken on the usual lines. The cure of a case of gonorrhceal arthritis, for example, may be greatly expedited by means of a vaccine, but the vaccine may be almost useless in the presence of an untreated stricture or a posterior urethritis. If a local source of infection is left unrelieved the organisms present in it continue to multiply and to aggravate the general condition. A patient with puerperal septicaemia may die if only the uterus is drained, or if a vaccine is given without cleansing the uterus. A combination of both procedures has undoubtedly saved many lives. In such a condition as infective endocarditis, in which the source of infection cannot be dealt with, vaccine treatment is practically useless. VACCINES— ANTI-SERA. 165 There is no doubt that vaccines have been given in the past in a wild and extravagant manner, and the field of vaccine therapy is now becoming more restricted. The following are among the conditions for which vaccines may reasonably be given : — Chronic local infections, such as acne, recurrent boils and local abscesses, of which the causative organism is certainly known, and is usually a staphylococcus, or, in the case of uncomplicated acne, the acne bacillus. Acute streptococcal infections, particularly those in which the organisms have been isolated from the general circulation, and the local source of infection is accessible. Gonorrheal infections, particularly when chronic, less certainly in the stage of acute urethritis. In the following conditions the use of vaccines is more problematical, but worthy of trial in individual cases: — Pyorrhoea alveolaris and its probable complications. Chronic infection of the urinary tract with the B. coli. Local abscess, such as a bronchiectatic cavity, in which the organism isolated is the probable source of infection. Vaccines should not be given merely for the sake of doing- something. For example, a case of rheumatoid arthritis obtained no benefit from medical treatment. A catheter specimen of urine was taken and found sterile. A blood culture was sterile. Cultures from the faeces yielded a growth of the typical colon bacillus. Cultures were finally taken from the perfectly healthy gums, a coccus was grown, and a vaccine made from it. Such a proceeding is quite futile, if harmless. Stock vaccines should not be given on the chance of hitting off the correct species of organism, when it is possible to make a proper bacteriological investigation and to grow the causative organism. While it is probable that the majority of vaccines do no harm, it must be recognised that in some cases the injection of dead organisms is positively dangerous. Some cases of infective endocarditis are undoubtedly made worse by vaccine treat- ment, and the subjects of grave anaemia may succumb to large doses of an organism with which they are not actively infected. For example, a patient with pernicious anaemia of some years' standing who was in comparatively good condition was 166 CLINICAL PATHOLOGY. given a vaccine prepared from a streptococcus isolated from the mouth. He reacted strongly to the vaccine, which was repeated, and he was dead within a fortnight. The above are only the merest indications of when or when not to treat a patient with vaccines. Each case must be judged on its merits, but it can be stated broadly that the cases most benefited are some chronic septic conditions and general streptococcal infections. The dosage of a vaccine necessarily varies with the age, size, and general condition of the patient, as well as with the species of the organism injected. The variations as to age and condition are similar to those employed in any form of treatment. The dosage, according to species of organism, varies with the comparative virulence of the organism. In the cases of streptococci, pneumococci, and gono- cocci small initial doses of from 5 to 10 million cocci are given as a general rule. In the cases of staphylococci and colon bacilli doses of from 50 to 100 million organisms are given. Double these amounts may usually be injected in the second dose. The vaccine may be given as soon as it is prepared, but if a surgical operation has recently been performed, and there is reason to suppose that the patient has absorbed a material dose of toxin from the operation area, the giving of the initial dose should be postponed. If there is urgency in giving the vaccine, a probable idea of the causative organism justifies the use of a preliminary dose of a stock culture of that organism. Nor is there any grave objection to treatment by stock vaccines if the proper organism is known, although the balance of evidence seems to be that patients are more likely to improve on an autogenous vaccine — that is, a vaccine prejDared with the organisms obtained from the individual case — than on a vaccine derived from some other source. The time allowed to elapse between the doses is as a rule from 5 to 10 days. There is nothing to be gained by increasing the size of the dose to much more than double the initial dose, nor by markedly diminishing the intervals between the doses. The immediate effect of giving a vaccine is apparently to diminish the resistance of the individual to the infection. The diminished resistance is followed by a considerable rise in the immune bodies. The rise is succeeded by a fall to a point somewhat above the resistance before inoculation. The clinical signs vary with the rise in immunity, the temperature VACCINES— ANTI-SEEA. 167 falling and the condition improving. In most cases the patient experiences little or no discomfort after the vaccine is given, but in a certain percentage of cases a definite reaction follows. The reaction may be local, focal, or general, or all three. The local reaction consists of pain, redness and even oedema, at the site of injection. The general reaction com- prises a rise of temperature, with headache, malaise and sometimes sickness. A focal reaction consists in an aggrava- tion of the local condition. A marked reaction is evidence of an overdose for the individual. The control of vaccines by frequent estimations of the opsonic index has been already referred to (Chapter IV.). The method has been largely abandoned, and a sufficient guide to the effect of the treatment is afforded by the temperature chart, combined with clinical observation of the patient. Certain modifications of vaccine treatment may be men- tioned only, since they have yet to be put into practice on any large scale. Treatment by endotoxins has been attempted for several diseases, and in particular for typhoid fever. The endotoxins are obtained by grinding up the bacilli in such a way as to actually express from them the intracellular toxins. The sensitised vaccine of Besredka consists of a vaccine which has been brought into contact with the specific antiserum so that the organisms in the vaccine are combined with the anti- body in the serum. It is claimed that such a vaccine is non- toxic, leads to no preliminary lowering of the immunity, raises the immunity very rapidly, and produces an immunity which lasts a considerable time. Tuberculin is given in many different forms, and only one variety, tuberculin B E, is a genuine vaccine, in the sense that it consists of a known weight of tubercle bacilli emulsified with a mixture of glycerine and water. The old tuberculin consists of exotoxins mainly, and is obtained by evaporating down a 4 to 5 weeks' old culture of bacilli to one-tenth its bulk at a comparatively low temperature, filtering it and using the filtrate. Tuberculin T R is composed of endotoxins, and 1 c.c. of the T B contains the bacterial matter insoluble in water derived from 10 mg. of tubercle bacilli which have been repeatedly washed in water and centrifuged. The S B E con- sists of sensitised tubercle bacilli obtained by mixing the bacilli with antituberculous serum. 168 CLINICAL PATHOLOGY. The preparation of the tuberculins is largely left to the manufacturing chemist. The dosage of the tuberculins varies with the nature of the case, and is reckoned, so far as possible, from the weight of original solid substance employed to make the extract, or from the original solution. In the case of B E, for example, the preliminary dose is a dilution of the original solution, and is commonly "001 of a cubic millimetre. This may be given as the initial dose of any tuberculin. The dose is increased gradually and in such a way as the following :— '001, -002, '003, -004, -006, -008, •010, *015, "02, -03, etc. The maximum dose to be aimed at is 100 c.mm., or ^ 6 c.c. of the original solution. The intervals between the doses should be 3 days for the small doses, and from 1 to 3 weeks when the higher doses are reached. The rate of increase necessarily depends upon the reaction and progress of the patient. The value of tuberculin treatment cannot even yet be con- sidered as proved. There is no doubt that in careless hands much harm can be done with tuberculin. On the other hand, many careful observers have claimed satisfactory results from its use. The method of preparing vaccines. — The following are the various stages requisite for the preparation of an auto- genous vaccine : — Stage 1. The culture. — Cultures are taken from the patient in the usual way, and film preparations of the lesions are also examined if possible. A sub-culture on agar (or, if necessary for the growth of the organism, on serum agar) is made and incubated for 24 hours. A second sub-culture should be put up at the same time for the purpose of full cultural investigation. If the organism is in pure culture there is no difficulty at this stage, and the original culture on agar may be used if there is an}' urgency about the preparation of the vaccine. If more than one organism is present, it is advisable to make the vaccine from the more virulent bacterium, par- ticularly if it predominates in the film preparation and is most likely from the nature of the case to be the primary cause of the lesion. For example, if a Staphylococcus alius and a Streptococcus pyogenes are obtained from an acute cellulitis, VACCINES— ANTI-SERA. 169 the vaccine should be made from the streptococcus. In cases of doubt, or when there is reason to suppose that a combination of organisms is responsible for the condition, vaccines can be prepared from two or more organisms and subsequently mixed in whatever proportions are considered desirable. Stage 2. The suspension. — To the 24 hours' old sub-cul- ture on an agar slope add sterile 1 per cent, salt solution. The amount of saline to be added depends upon the thickness of the growth. A moderately turbid suspension of bacteria in saline is to be aimed at. Wash off the growth as far as possible into the saline by shaking the tube and pour the bacterial suspension into a sterile tube. Stage 3. The standardisation.— Make ready a Wright's opsonic pipette, 2 clean slides such as are used for blood work, clean swabs and ether, a surgical needle, and a bowl of 1 in 20 carbolic. Shake the saline suspension very thoroughly for several minutes and hand it to an assistant to continue the shaking. Make a pencil mark about half an inch from the end of the Wright's pipette. Cleanse the lobe of the assistant's ear care- fully with ether and prick it with the needle. Without apj)ly- ing pressure to the lobe draw up a volume of blood. Wipe the end of the pipette. Take the saline suspension from the assistant (the wool plug having been withdrawn) and draw up a volume of the suspension. Mix saline and blood thoroughly on a glass slide. Draw up mixture into tube. Blow out mixture on to a clean slide and make a thin film as in preparing a blood film. Place all materials which have been in contact with the organisms in the carbolic bowl. Stain the film with Leishman's stain. Fit a square aperture, such as is used for the enumeration of leucocytes, into the eye-piece of the microscope. Use the -j^-inch objective. Choose a thin and evenly spread part of the film and count 500 red cells together with all the organisms lying among them. Enumerate the red cells per cubic millimetre of the assistant's blood. Sufficient data have been obtained to arrive at the number of organisms per cubic centimetre of the saline suspension. The number of organisms counted among 500 red cells 170 CLINICAL PATHOLOGY. multiplied by twice the number of red cells per c.mm. gives the number of organisms per cubic centimetre of the suspension. The method given here is perhaps the simplest in common use. It is admittedly open to criticism, but is sufficiently accurate for all practical purposes. The preparation of the film from the mixture of blood and saline must be prepared before heating the suspension, since some organisms lose their staining properties on being sub- jected to heat. The actual enumeration of the film, however, is preferably postponed until after stage 5. Stage 4. The sterilisation. — Prepare a deep water bath or a saucepan capable of holding a considerable bulk of water. Fill almost to the brim with water at 60° C. Place over a Bunsen flame and lower the flame considerably. Leave a thermometer in the water. By adjusting the size of the flame the temperature can be maintained at 60° C. without appreciable variation. When the temperature has become constant, fit a rubber cap over the mouth and cotton-wool plug of the vaccine tube and immerse the tube in the water almost to the level of the rubber cap. The tube can be conveniently maintained in position by adjusting two long- handled test-tube holders. Keep the tube at 60° C. for 1 hour, observing the reading of the thermometer from time to time. At the end of 1 hour remove the tube. The actual temperature necessary to kill the organism varies with the species. The majority of organisms growing in pin-point colonies, such as gonococci, streptococci and pneumococci, are killed by exposure to 60° C. in from 30 to 45 minutes : organisms growing in coarser colonies, such as staphylococci and colon bacilli, require a temperature of at least 65° C. for an hour, and with some strains it may be necessary to raise the temperature higher still. Stage 5. The proof of sterilisation. — Incubate the vaccine tube for 24 hours. At the end of this time sub- culture from the vaccine on to an agar slope and incubate the sub-culture for a further 24 hours. If the sub-culture remains sterile, the vaccine may be considered sterile. The slide prepared in stage 3 may then be enumerated and stage 6 proceeded with. Stage 6. The dilution. — Prepare sterile measure glasses, VACCINES— ANTI-SERA.. 171 a small sterile bottle with a well-fitting ground glass stopper, sterile normal saline, and a bottle of lysol. Calculate the convenient dilution of the bacterial suspension. For example, if a streptococcal suspension has been found to contain 500 million cocci per cubic centimetre, and the initial dose wished for is 10 million, it is convenient to add 4 volumes of saline to 1 volume of the suspension, thus reducing the strength of the suspension to 100 million cocci per c.c. The initial dose will then be contained in one-tenth of a cubic centimetre. Sufficient lysol should be added to the saline to provide in the final mixture a lysol solution of a strength of 0*25 per cent. The addition of the lysol safeguards the vaccine from subsequent contamination. The mixture is finally placed in a sterile bottle and kept in a dark, cool place. The bottle should be marked with the name of the patient, the nature of the organism, the strength and the date. It is advisable to use no vaccines of a greater age than 3 months. The above steps are those preferably observed. It will be noticed, however, that 2 days are required for the sub-culture and 2 more for the proof of sterilisation, so that if every- thing goes well 4 days are required for the preparation of the vaccine. In cases of urgency a stock vaccine may be given at once. In acute streptococcal infections it is possible to prepare a vaccine in 24 hours. The original culture on agar is taken and, if pure, the suspension is made and heated to 65° C. for 1 hour and for 10 minutes at 70° C. It is then presumed sterile, and is diluted and injected. There is in practice no real risk in this procedure. To give the vaccine. — A 1 c.c. hypodermic glass syringe graduated in one-tenths of a cubic centimetre is required. The syringe is conveniently sterilised with boiling methylated spirit. A small quantity of the spirit is placed in a beaker and brought to the boil on a sand-bath over a Bunsen burner. The flame is turned out when the spirit is boiling briskly, and the syringe with needle attached is washed in and out several times with the boiling spirit. The vaccine bottle must be thoroughly shaken before drawing up into the empty syringe the required 172 CLINICAL PATHOLOGY. volume of the vaccine. The injection is preferably made subcutaneously into the forearm after cleansing the skin with ether. The puncture should subsequently be sealed with a drop of collodion. The prophylactic use of vaccines. — Vaccines may be given with the object of preventing disease. Almost the only preventive vaccination of this kind commonly performed in this country is that for typhoid fever. Vaccination against small-pox is of a somewhat different nature, since the individual is protected by an artificial attack of the modified disease. Preventive inoculation is also practised against plague, cholera, and other diseases. The effects of inoculation against typhoid are to markedly lessen the risk of infection and to modify the virulence of the attack if it does occur. There is sufficient statistical evidence that a considerably smaller percentage of inoculated persons become attacked after exposure to infection than of untreated persons, and that the incidence mortality rate among the inoculated is very low. The effect of the inoculation gradually wears off and the comparative immunity does not appear to last longer than from one to two years. Typhoid fever is not sufficiently prevalent in Britain to call for preventive inoculation , but inoculation should be advised for those proceeding to countries where the disease is rife. While abroad all the usual precautions against infection should be observed and re-inoculation should be performed, if practicable, within 18 months. The vaccine should be given a short time before the typhoid district is reached. Two doses are given with a 10-day interval between them. Comparatively large doses are commonly given, often from 500 to 800 million organisms, and it is reasonable to give a combined vaccine prepared from typhoid and paratyphoid bacilli. The inoculations in a con- siderable percentage of cases are followed by a definite local reaction and often by slight pyrexia and malaise. The patient's serum after inoculation should show well-marked agglutinating action upon the bacilli. The diagnostic use of vaccines. — Vaccines or bacterial extracts may be made use of for purposes of diagnosis. The reaction depends upon the fact that persons infected by a given organism are abnormally susceptible to the toxins of that organism; consequently the introduction of the toxins VACCINES— ANTI-SERA. 173 locally leads to an excessive local reaction and, if the dose is sufficient, to a general reaction also. Necessarily the dose given must be regulated, since an excessive dose might lead to severe reaction in normal people or to an excessive reaction in infected persons. The most widely used diagnostic extract of this nature is tuberculin, and it is given by several methods. The ophthalmic reaction of Calmette consists in dropping a tuberculin solution into one eye. Tuberculous patients develop a conjunctivitis, while normal persons are unaffected. A grave objection to this method lies in the comparative frequency with which a severe and intractable conjunctivitis follows, and for this reason alone the practice is not to be recommended in human medicine. The tuberculin may be given hypodermically in strong- doses, and this method again is not free from danger. The cuti-reaction of Von Pirquet is the most reliable and the most widely used method. The tuberculin employed is Koch's "old" tuberculin in 25 per cent, strength. A small skin area on both arms of the patient is cleansed with ether and scarified, as if for ordinary "vaccination," by means of a sterile surgical needle. The drawing of blood should be avoided. On one area the tuberculin is smeared, on the other normal saline as a control. The lesions are covered with dry sterile gauze and examined after 24 hours and again after 48 hours. There is no reaction in normal people, while tuberculous patients develop a definite red, raised papule, with the occasional addition of small vesicles. Von Pirquet's reaction is only reliable within certain definite limits. Patients dying of tuberculosis may not react at all and patients with healed tuberculous lesions may react strongly. Owing to the large number of adults with quiescent tuberculous foci a positive reaction in an adult is little evidence of active tuberculosis. A negative reaction in an adult is evidence against tuberculosis. In children the reaction, whether negative or positive, is of considerable value. Other diagnostic reactions are occasionally performed on similar lines. A cutaneous and an ophthalmic reaction has been made use of as a test for typhoid fever, and the subcu- taneous injection of mallein is largely practised in veterinary pathology as a means of detecting glandered horses. 174 CLINICAL PATHOLOGY. Anti-sera. Anti-sera consist of the blood sera of animals — in the majority of cases horses — which have been highly immunised against bacteria or their toxins. The injection of such sera into human beings is the method of artificially producing a passive immunity. The immunity thus conferred is very different from that aimed at in vaccine therapy, since the anti- bodies are, in the case of sera, manufactured by the animal and injected into the human body. The resulting immunity is in consequence very rapidly produced and of short duration. The antitoxic sera are in most instances standardised on the basis of the amount of toxin which they can neutralise. In the case of diphtheria antitoxin the smallest amount of diph- theria toxin capable of killing a guinea-pig of 250 grammes weight within 4 days is first determined. This is known as the minimum lethal dose. The amount of antitoxic serum which will neutralise 100 times the minimum lethal dose is found by mixing serum and anti-serum and then injecting the mixture into a guinea-pig without causing death. This amount is reckoned as 1 immunity unit of antitoxin. The anti-sera are obtained by the repeated inoculation of horses with bacteria or their toxins until a high degree of immunity is attained in the horse's serum. The animal is then bled from the jugular vein into a sterile receptacle. The clear serum which results after standing or centrifuging is pipetted off and stored in sterile glass capsules in measured doses. The anti-sera are given to human beings by subcu- taneous injections into the loose tissues of the axilla or abdominal wall. The dose may be repeated in 24 or 48 hours, but it is rarely advisable to give more than 3 doses. In favourable cases the temperature drops to normal within a few hours and the local and general condition rapidly improves. The injection of serum is not infrequently followed by the appearance of a diffuse and irritating rash, usually of the nature of urticaria and less frequently by painful effusions into the joints. The severe anaphylactic phenomena consisting of rapid collapse and death, which are readily induced in animals by repeating the injection of the serum after an interval of 2 to 3 weeks, are very rarely observed in man. The risk of VACCINES— ANTI-SEEA. 175 giving repeated doses at small intervals is almost nil, but the procedure is more liable to be followed by rashes and joint affections than if a single immunising dose is given. The preparation of anti-sera is beyond the scope of ordinary clinical pathology, since the use of large animals is involved, and reliable sera can be obtained from some of the leading chemical firms. Only a brief indication of the mode of preparation, and of the dosage and use, of the various sera can be given here. Anti- diphtheritic serum. — The horse is injected with the toxin of the diphtheria bacillus obtained from the nitrate of a 3 to 6 weeks old culture of the organisms in broth. The injections are continued during 4 to 6 months, and then stopped when a serum of high potency is obtained. The dose of serum given varies from 2 to 10 thousand units. The longer the lapse of time from the onset of infection the higher is the dose given. There can be no doubt as to the value of this serum, which should be given in all cases of diphtheria. Anti- tetanic serum is obtained by the injection of a horse with tetanus toxin in increasing doses. With most pre- parations a minimum of 25 c.c. of the serum should be given as the initial dose. The therapeutic effect of this serum in man is most disappointing, owing to the fact that the combination between toxin and antitoxin is unable to take place in the body if the toxin has previously combined with the nerve tissues. Consequently when symptoms have developed it is in the majority of cases too late to give the antitoxin as a curative. It is advisable, however, to give the serum in all cases when practicable with the view of fixing any further toxin that may be elaborated from the local lesion, while the toxin already combined in the nervous system is combated on ordinary medical principles. Anti-streptococcal serum. — The horse is immunised by a series of injections of a single strain or a combination of varieties of streptococci of increasing virulence. The value of this serum is somewhat problematical, but it may be used in cases of undoubted streptococcal infection, and may be given at once in doses of 20 c.c. or more and be followed by injections of the appropriate vaccine. It is of use in puerperal infections, in acute inflammations due to the streptococcus, and possibly in erysipelas. 176 CLINICAL PATHOLOGY. Anti-pneumococcus serum is prepared in a similar way to the foregoing, and has been used in the treatment of pneu- monia. The induction in the horse of a high immunity to the pneumococcus appears to be a matter of considerable uncertainty, and many of the sera used have been practically functionless. Anti-meningococcus serum may be given in 10 c.c. doses subcutaneously. Flexner's serum is given intraspinally by lumbar puncture in 80 c.c. doses. Good results have been recorded from Flexner's serum in the American epidemics, but in the sporadic cases in London the effect has not been so striking. Anti-colon bacillus serum is obtained by the immunisa- tion of horses to mixed strains of colon bacilli. In acute coli infections of the kidney the effect of the serum is sometimes striking, but it must be remembered that the clinical fluctua- tions in this condition apart from serum treatment are often remarkable. The serum should be given in 20 c.c. doses on 3 consecutive days. Anti-plague serum. — The horse is immunised with saline suspensions of the bacilli. The first doses are sterilised by heat. In the later doses living bacilli are given. The serum is usually given in 20 c.c. doses. In addition to the methods indicated above anti-sera have been given by the mouth, per rectum and by local applications. Striking results have also been recorded from similar uses of normal horse serum. It cannot be said that, with the exception of anti-diphtheritic serum, the results obtained with any of these sera have been particularly encouraging. At the same time, in a number of individual cases improvement seems to definitely follow the injection, while in many fatal cases the sera have been given as a last resort in the most desperate conditions. CHAPTER XIII. pebparation of culture media staining reagents. The Preparation of Culture Media. The main constituents of the commoner culture media and the changes which take place in the media as the result of bacteriological growth have been already referred to. The present chapter deals with the practical modes of preparation of the media. Sterilisation. — It is essential that all the media and the Fig. 12. — Hot-air Steriliser. receptacles which contain them should be perfectly free from all living organisms before they are used for inoculation. The following apparatus is required : — A thermometer graduated to 200° C. A hot-air steriliser. A steam steriliser. An autoclave. An inspissator. The hot-air~steriliser consists of a double-walled chamber p. 12 178 CLINICAL PATHOLOGY. of copper or iron heated below by a gas flame and fitted with a thermometer passing into the inner chamber. It is used for the sterilisation of glass flasks, test tubes, Petri dishes, etc., which can thus be rendered both sterile and dry. A tempera- ture of 170° C. for 1 hour is sufficient to destroy any organisms that may be present. The steam steriliser consists of a metal cylinder on legs enclosed in felt or asbestos and provided with a perforated tray fixed about 8 inches from the bottom. Water to the depth of 3 inches is placed in the bottom. The space above the tray should be tall enough to accommodate a litre flask fitted with a funnel. An ordinary potato steamer can be adapted to the purpose. Media placed in the steamer are sur- rounded with steam at 100° C. The majority of media, after being exposed to contamination, are steamed for 30 minutes on 3 consecutive days. The autoclave is used for rapid and effective sterilisation by means of steam at high pressure. It is not absolutely essential, and requires careful supervision when in use. The autoclave consists of a gun-metal cylinder provided with a movable or hinged lid, fitted on with screws and nuts, and with a pressure gauge and safety valve. The water is usually boiled at a temperature of 120° C, which requires a pressure of 30 lbs. to the square inch, and 30 minutes at this temperature is usually sufficient. The chamber must be filled with steam before beginning to raise the pressure. The autoclave must be allowed to cool to 100° C. before opening it or blowing off steam. All the above sterilisers should be allowed to cool down considerably before opening, otherwise the glass receptacles are liable to crack. The inspissator is required mainly for the preparation of blood serum media. It consists of a shallow sloped chamber provided with a water jacket and a thermometer. The serum tubes are kept at 75° C. in the chamber until the serum Fig. 13.— Steam Steriliser. PEEPAEATION OF CULTURE MEDIA. 179 solidifies. The top of the apparatus is of glass covered with thick felt, which can be lifted for inspection of the tubes in the interior of the chamber. All the receptacles, such as flasks and test tubes, used in the preparation of media should be perfectly clean and free from Fig. 14.— Autoclave. acid or alkali. They should be sterilised preferably before being filled, and always after exposure to contamination. The mouths of flasks and test tubes are corked with plugs of non- absorbent wool. The plugs should fill the mouths of the tubes and should project beyond them, but should not be too tight nor too bulky. Ordinary cleanliness should be observed in all 12—2 180 CLINICAL PATHOLOGY. operations connected with media-making in order to avoid the needless introduction of spore-bearing organisms. The methods of making the following media are given in brief, and the smaller details are left to the management of the individual worker. Fig. 15. — Inspissator. Stock nutrient broth. — This medium is widely used and forms the basis of many other media. Composition : — . 55 grammes. • 55 . 55 5,500 c.c. " Lemco "..... Sodium chloride Peptone ..... Tap water ..... To make : — Boil the water in a saucepan. Add NaCl, peptone and Lemco. Boil for 45 minutes and pour into flasks. Standardise (see below). Steam flasks for 1 hour. Stand in cool place till next day. Filter when cold. Either place in flasks for stock, and autoclave for 30 minutes, Or, as required, tube. To tube, fill the number of test tubes wanted to about one- third of their capacity and cork with wool plugs. PREPARATION OF CULTURE MEDIA. 181 Autoclave 30 minutes (or steam for 30 minutes on 3 con- secutive days). The standardisation is carried out as follows : — Take 10 c.c. of the stock broth. Add a few drops of phenol-phthalein. Carefully run in deci-normal caustic soda solution until a faint permanent pink colour is produced. Read off the amount of soda used and calculate the amount of normal soda required to neutralise 1 litre of the broth to phenol-phthalein. The amount required is commonly about 15 c.c. The broth is as a rule not exactly neutralised, but is made up to a definite known acidity. The acidity usually chosen is that known as + 5 on Eyre's scale, and is a favourable one for the growth of the majority of organisms, By an acidity of + 5 is meant that the normal soda added per litre is 5 c.c. less than that required to exactly neutralise to the phenol-phthalein end point. For example, if 10 c.c. of the broth required N 1*5 c.c. of ryr soda to neutralise, 1 litre would require 150 c.c. of r^ soda, or 15 c.c. of N soda. In such a case 10 c.c. of N soda per litre are added, leaving unneutralised an acidity of — 5 c.c. N soda, or + 5 on Eyre's scale. If an acidity of — 10 is required (an acidity recommended for the growth of some streptococci), 25 c.c. of N soda are added per litre. A broth solution with an acidity of + 5 is alkaline to litmus. Glycerine broth is made by the addition of 6 per cent, of glycerine to the stock nutrient broth. Glucose broth is made by the addition of 2 per cent, of glucose to the stock nutrient broth. Neutral red broth is made by adding 2 c.c. of a 2 per cent, aqueous solution of neutral red to 100 c.c. of nutrient broth. Litmus carbohydrate broths are made by adding 1 gramme of the carbohydrate to 90 c.c. of nutrient broth, which has been neutralised to phenol-phthalein, and 10 c.c. of litmus solution. The carbohydrates most commonly required are dextrose, lactose, mannite, ramnose, and salicin. Others which may be employed are arabinose, saccharoso, inulin, coniferin, sorbit, etc. 182 CLINICAL PATHOLOGY. The litmus solution required to give the necessary colour may be bought ready made or prepared as follows : — Materials required : — Litmus powder 20 grammes. 90 per cent, alcohol .... 200 c.c. Distilled water 200 „ To make : — Boil the litmus with 80 c.c. of the alcohol for 1 hour on a water bath. Pour off the clear liquid. Eepeat with 60 c.c. of the alcohol. Eepeat with the remainder of the alcohol. Digest the washed litmus in the distilled water. Filter. When tubing the carbohydrate media it is convenient to place in each test tube a small inverted tube filled with the medium in order to collect the gas which may be evolved as the result of bacterial growth. Agar-agar. — Agar medium consists of a solution of agar- agar in stock nutrient broth. It is best prepared from the powdered substance, and has the following composition : — Agar ....... 30 grammes. Nutrient broth, .... 1,000 c.c. To make : — Dissolve the agar in the broth by heating in the auto- clave for 15 minutes at 110° C. Standardise while hot (+ 5 on Eyre's scale). Cool to 60° C. Add the beaten white of 1 egg. (This clears the mixture, but is usually unnecessary if agar powder is used instead of the fibre.) Autoclave for 20 minutes at 115° C. Filter through a Chardin filter paper while hot. Tube if required. Steam for 1 hour on 3 consecutive days. The tubes should be prepared in two forms — for " stab " cultures, in which case the tubes are filled to about two-thirds their capacity and allowed to cool in the vertical position, and for " slope" cultures, in which case they are filled about one- sixth full and allowed to cool in a slanting position. MacConkey's neutral red agar. — This medium is made PREPARATION OF CULTURE MEDIA. 183 by adding the appropriate substances to the stock agar. It has the following composition : — Agar 30 grammes. Lactose ...... 10 „ Sodium taurocholate . . .5 „ 2 per cent, aqueous neutral red solution 20 c.c. Nutrient broth to 1 litre. Nasgar. — This medium consists of nutrose, ascitic fluid (or blood serum), and agar. It is made as follows : — Take Ascitic fluid . . . .15 c.c. Nutrose ..... 1 gramme. Agar powder .... 0*5 ,, Distilled water . . . . 35 c.c. Bring gradually to the boil and filter. Add 1 part of this mixture to 2 parts of agar. Mix at 60° C. Standardise (+ 5 on Eyre's scale). Autoclave 20 minutes at 115° C. Filter. Tube (in slope form) as required and steam. Oleic acid agar. — This medium has the following com- position : — Glycerine 2 per cent. Oleic acid ...... 0"1 „ Added to neutral agar medium. Gelatin. — Gelatin media consist of broth with sufficient gelatin dissolved in it to produce a solid medium at tenrpera- tures from 18° to 22° C. The necessary proportion of gelatin to broth is 25 per cent, of the former during the summer and 20 per cent, during the winter. It is important to obtain the best gelatin, otherwise a soft medium will result. Coignet's gold label gelatin is very satisfactory. To make : — Add the gelatin to warm nutrient broth. Dissolve while warm. Standardise while warm (-f- 5 on Eyre's scale). Add beaten white of 1 egg. Filter. Tube as required. (Put up both slope and stab media.) Litmus milk. — The milk must be quite fresh, and i84 CLINICAL PATHOLOGY. preferably the majority of the cream should have been removed. Steam for 30 minutes. Filter. Add 10 to 15 per cent, of the stock litmus solution. Add the minimum quantity of normal soda necessary to give a definite blue colour to the litmus (i.e., 0'5 to l'O per cent.). Tube. Steam the tubes for 1 hour on 3 consecutive days. Potato medium. — This medium is much less commonly used than formerly. Special test tubes are required, which resemble Buchner's anaerobic tubes in miniature. A few drops of glycerine are placed in the bottoms of the tubes, and sticks of potato well washed are cut and passed into the tubes, which are plugged with wool and steamed for 1 hour on 3 consecutive days. Unless used at once, it is preferable to cover the wool plugs with rubber caps. Dorset's egg medium. — This medium is used for the growth of the tubercle bacillus, and is prepared as follows : — Four eggs are well beaten up, 25 c.c. of water are added, and the whole is thoroughly mixed. The mixture is passed through muslin and tubed. The tubes in the form of slope cultures are placed in the inspissator and steamed for 4 hours at 70° C. They are then sterilised in the usual way. Blood serum media. — Blood, preferably that of a bullock, is obtained at the slaughter-house, and is taken from the wound, after a little blood has been allowed to flow, direct into a large sterile glass cylinder. The cylinder is placed on ice until the serum has fully separated, that is, as a rule, until the next day. With a sterile glass pipette the serum is sucked up from the cylinder and blown out into sterile test tubes. The serum is often tinged red from haemolysis of the cells, but this is of no importance and the colour goes in the subsequent processes. The tubes are then placed in a sloped position in the inspissa- tor and kept at a temperature of 75° C. for about 4 hours, or until they have become firmly set. The heating is repeated for 1 hour only on each of the 2 consecutive days. The media thus prepared are almost invariably sterile, but it is customary to incubate them for 24 hours in order to prove their sterility. PEEPAEATION OF CULTUEE MEDIA. 185 Instead of ox blood that of the sheep can be used, or human blood obtained at venesection. Hydrocele fluid or ascitic fluid can be made use of in the same manner. Loffler's blood serum has the following composition : — Four parts of serum to 1 part of nutrient broth with 1 per cent, of glucose added. The mixture is made before the tubes are filled, and a rather higher temperature may be required to obtain a satisfactory solidification of the medium. Ox-bile medium. — This medium is used for the growth of the typhoid bacillus, and is particularly useful if blood in the early days of typhoid fever has to be obtained from the finger. The typhoid bacilli grow readily in this medium and the common skin cocci do not. The gall-bladder of an ox is tied off and removed at the slaughter-house ; 100 c.c. of bile are mixed with 10 grammes of peptone and 2 grammes of sodium chloride. The mixture is autoclaved for 15 minutes at 120° C, filtered and tubed. The sterilisation of inoculated media. — It is necessary to destroy the organisms which have grown in the discarded culture media and to render the test tubes and plates fit for further use. Test tubes. — Place culture tubes without removing the wool plugs into a large saucepan with a well-fitting lid. Bring to boiling point and boil for 3 hours. Eepeat boiling on the following day. When cold remove plugs. Empty contents down sink and put tubes in a basin with hot water running over them. Wash with test-tube brush, using 25 per cent. HC1. Wash with cold water several times to remove acid. Place upside down in crates in a warm oven to dry and leave for several hours. Petri dishes. — Sterilise by boiling in the same way as the tubes. Place in a solution of hot strong Hudson's soap water for half an hour, after separating the plates. Wash thoroughly in the water. Wash with clear cold water. Wipe dry. Sterilise in hot air steriliser for 60 minutes at 170° C. (Washing soda must not be used.) 186 CLINICAL PATHOLOGY. To clean used microscopic slides. — Soak the slides in cold water to remove the lysol. Boil for 1 hour in Hudson's soap water. Take out and remove cover-glasses. Boil again for 15 minutes. Wash in hot water. The Preparation op Staining Reagents. The number of staining reagents required for the ordinary routine methods of clinical pathology is very small. Only the f orrnulse of the essential stains are given here, and it is remark- able how rarely one requires any stain which is not to be found in the scanty array of drop bottles disposable on one small shelf. The stains given here include those required for ordinary histological purposes as well as for bacteriological work. The special blood stains are described in the section on the blood, and a few other special stains are given in their appropriate places in the text. Carbol thionin. — This is the most useful general stain in routine bacteriological work. It has the following formula : — Saturated solution of thionin in 50 per cent, alcohol ..... 10 cc. Carbolic acid (crystal) .... 1 gramme. Distilled water 90 cc. The saturated alcoholic solution of thionin requires about 5 per cent, of thionin, and should be kept in the incubator at 37° C. for one week. It is then filtered, kept in stock, and made up as required with the carbolic acid and water. Methylene blue. — A saturated watery solution has the following composition : — Methylene blue (Griibler) . . about 20 grammes. Distilled water 400 cc. The stain is added to the distilled water in a bottle with a well-fitted stopper, and the bottle is kept in a hot oven (or in the incubator at 37° C); for several days. If all the stain is dissolved more is added until the mixture is saturated. A saturated alcoholic solution is made in the same way by PEEPAEATION OP CULTURE MEDIA. 187 saturating about 5 grammes of methylene blue in 100 c.c. of absolute alcohol. The saturated watery and alcoholic solutions are kept as stock, and a 1 per cent, dilution of the watery solution is used for ordinary staining purposes. The alcoholic solution is required for the next stain. Lb'ffler's methylene blue. — Formula : — Saturated solution of methylene blue in alcohol ....... 30 c.c. Caustic potash 1 per cent 1 „ Distilled water ...... 100 „ Carbol fuchsin. — Formula : — Basic fuchsin (Griibler) ... 1 gramme. Absolute alcohol . . . .10 c.c. Carbolic acid 1 in 20 . . . . 100 c.c. The stain is shaken with the alcohol and the carbolic added to it. This solution is kept as stock and is filtered into drop bottles as required. Gentian violet (alcoholic). — Formula : — Gentian violet . . . about 10 grammes. Absolute alcohol .... 300 cc. The mixture is kept on an oven or in the incubator for one week, and if not saturated at the end of this time more gentian violet is added. This solution is kept as stock and filtered before use. Gram's iodine. — Formula : — Iodine ...... 1 gramme. Potassium iodide .... 2 grammes. Distilled water 300 c.c. A clear solution results, which does not require filtering. Giemsa's stain. — Formula : — Azur ii. eosin ..... 3 parts. Azur ii. . . . . . . . 0*8 ,, Glycerin (Merck pure) .... 250 ,, Methyl alcohol (Kahlbaum) . . . 250 ,, Safranin. — This is a useful counter-stain to Gram's method for organisms in sections. Formula : — Safranin ..... 0'5 gramme. Distilled water .... 100 c.c. The stain is filtered on the section when used. 188 CLINICAL PATHOLOGY. Hsemalum. — This is the most universal nuclear stain for tissues. It is also convenient for the staining of leucocyte drops by Strong's method. Formula : — 1. Pure hffiinatein .... 2\5 grammes. Alcohol 95 per cent. ... 95 c.c. Dissolve 2. Ammonium alum . . . 125 grammes. Distilled water .... 2,000 c.c. Dissolve. Add 1 to 2. Add to the mixture Glacial acetic acid ..... 325 c.c. Shake well and allow to ripen on a shelf in the light, if possible for from 1 to 3 months. Filter before use. Eosin. — This is an excellent tissue stain for sections. Alcoholic solution. A saturated solution is made in methy- lated spirit. About 2 grammes are required for 100 c.c. of spirit. Five per cent, of this solution in distilled water or spirit is used for staining purposes. Watery solution. The saturated solution is made in distilled water. About 10 grammes are required for 100 c.c. of water. Two per cent, of this solution in distilled water is used. Van Gieson's stain. — This stain is a very widely used differential stain for histological purposes. It has the follow- ing composition : — Saturated watery solution of picric acid . 50 c.c. Watery solution of acid fuchsin 1 per cent. . 15 ,, Distilled water . . . . . . 50 ,, Scharlach R. — This stain is the most satisfactory for the demonstration of fat in tissues or in film preparations. Fat globules are stained red by it, and all other tissues are left unstained. It is prepared as follows : — Put 100 c.c. of 75 per cent, absolute alcohol in distilled water into a bottle. Add Scharlach E. Shake vigorously and allow to stand for several days in the cold. When the fluid is saturated filter it. SECTION III. PUNCTUKE FLUIDS. CHAPTER XIV. General Procedure— Pleural Fluids — Pericardial Fluids. CHAPTER XV. Peritoneal Fluids — Cerebro-spinal Fluids — Synovial Fluids —Cysts, etc. CHAPTER XIV. GENERAL PROCEDURE PLEURAL FLUIDS — PERICARDIAL FLUIDS. By puncture fluids are meant such body fluids, whether exudates, transudates, or the contents of cysts, as are commonly removed for examination by means of a needle and syringe. The fluids most frequently taken for investigation are those derived from the pleural and peritoneal cavities and from the cerebro- spinal canal. No attempt is made here to give a detailed account of the complete examination of these fluids. We are concerned only with such investigations as reveal the nature of the patho- logical processes producing the effusions and such as are of practical value in clinical medicine. The complete chemical analysis of any exudate has a valuable scientific interest, but requires very many hours of laborious work, which is at the present of the nature of experimental research rather than of practical clinical value. With the majority of fluids we depend mainly upon determining the nature of the cells and bacteria present in them, and can by these means in almost every case give to the clinician an accurate diagnosis of the nature of the disease. Purely chemical tests are in this connection of little value to us. General Procedure. The procedure varies somewhat according as the fluid to be examined is obviously purulent or not. In the case of puru- lent exudates the examination is conducted in a precisely similar manner to that employed for the investigation of pus from any other part of the body. In the case of fluids not obviously purulent the procedure is as follows : — (1) Inspection. — The naked-eye appearance of these fluids is often of considerable help. It should be noted whether they GENERAL PROCEDURE— FLUIDS. 191 are clear, turbid, or flocculent ; if they contain blood, and, if so, whether the blood is in great or small amount, and intimately mixed or not ; if a clot forms on standing, and the appear- ance of the clot. (2) Chemical examination. — This can be mainly omitted in the majority of cases so far as clinical diagnosis is concerned. The only tests that need be employed are for the reaction, the specific gravity, the amount of coagulable proteid, and the amount of globulin. The globulin or proteid content of cerebro-spinal fluid in particular is of diagnostic significance. The other tests are rarely necessary for diagnosis. (3) Cytology. — By cyto-diagnosis is meant an investiga- tion of the number and nature of the cells present in a fluid. Such investigation should be made as soon as possible after the fluid has been withdrawn. The cells which may be present in these fluids are mostly of three varieties, namely, small lymphocytes, polynuclear neutrophils, and endothelial cells. A relative predominance of one of these cells, when present in excess, is among the most reliable diagnostic signs in clinical pathology. Small lymphocytes in excess are diagnostic of the chronic infective granulomata, of which syphilis and tuber- culosis are by far the most important examples. The clinical distinction between syphilis and tuberculosis in the case of the central nervous system is in the great majority of cases clear ; syphilis of the pleural and peritoneal sacs is an extremely rare pathological condition ; consequently the demonstration of an excess of small lymphocytes in any of these fluids commonly suffices to definitely substantiate a diagnosis. These small lymphocytes or, as they are called by some, lymphoid cells, cannot be distinguished on histological grounds from the small lymphocyte of the blood, the cell which is relatively increased in the circulating blood in tuberculosis and syphilis. The cell is also probably identical with the lymphoid cells found in the tissues in large numbers in the neighbourhood of tuberculous, and to a less obvious extent of syphilitic, lesions. The part played by the lymphoid cells is obscure, but it is evident that this is the type of cell which is actively attracted into the blood, and from the blood into 192 CLINICAL PATHOLOGY. the exudates and tissues, by the toxins of the tubercle bacillus and the spirocheeta pallida. Polynuclear neutrophils. — The presence of these cells is definite evidence of an acute inflammatory process. Acute inflammation of the serous sacs or of the cerebro-spinal canal may be induced by bacteria or by an aseptic irritant acting as a foreign body. In human pathology the exciting cause is in the vast majority of cases a pyogenic organism. The demon- stration of polynuclear neutrophils therefore calls for a further examination by microscopical and cultural methods in order to identify the causative organism. The distinction between clear, turbid, and obviously purulent fluids all containing the polynuclear neutrophil as the predominant cell is only one of degree. The process has in each case the same pathological basis. For example, a clear or faintly turbid fluid with- drawn from the chest after an attack of lobar pneumonia may be shown on careful examination to contain both polynuclears and pneumococci, and thus to be of the same nature as the thick pus of an ordinary empyema. When blood is present the demonstration of an occasional polynuclear neutrophil is of no significance, and one has to judge in such cases whether the leucocytic content of the deposit is in excess of what is natural to the number of red cells, remembering that in normal blood the proportion of white cells to red is only about 1 to 1,000. Endothelial cells. — These cells are derived from the lining membranes of the serous cavities and their presence is evidence of a passive transudate. They are rarely found, except in very small numbers, in cerebro-spinal fluid. They may be very numerous in pleural or peritoneal fluids. The cells are of much the same character in all situations in the body, and consist of large, more or less rounded cells, the cytoplasm of which is basophilic and often vacuolated. The edges of the cells are frequently frayed and irregular. The nucleus contains as a rule two or three well-marked nucleoli. The endothelial cells are phagocytic and may contain red cells, and, in the case of mixed cellular deposits, lymphocytes, polynuclear cells, or bacteria. They often occur in plaques of considerable size. In a good preparation these cells are quite unmistakable, but if the film has been made too thick the cells may be so shrunken as to be almost indistinguishable from. PLATE X. Small Lymphocytes. Endothelial Cells. (Tuberculous Pleural Exudate.) (Peritoneal Fluid : Cirrhosis of Liver.) (Leishman's Stain.) (Leishman's Stain.) Polynuclear Neutrophils and Meningococci. Polynuclear Neutrophils and Pneumococei. (Cerebro-spinal Fluid.) (Pleural Exudate.) (Leishman's Stain.) (Carbol-thionin.) The Cells of Puncture Fluids. PLATE X. GENEKAL PROCEDURE— FLUIDS. 193 small lymphocytes. In such cases the nature of the cells is commonly revealed by examining the edge of the film where the fluid is thinner and the cells have dried more quickly and have consequently shrunk less. An excess of endothelial cells is to be found in a variety of conditions, including renal disease, general cardiac failure, and any local obstruction to the circulation, as in malignant disease or cirrhosis of the liver. Other cells. — Almost any variety of cell may on rare occasions be found in these fluids. The eosinophil cell is exceptionally predominant, and is of doubtful significance. It may be found in parasitic effusions, as in the content of a hydatid cyst, or after the rupture of a cyst into the pleural or peritoneal cavities. It may form a considerable percentage of the cells present in the clear exudates which occasionally follow lobar pneumonia, and is then of good prognostic significance, since such cases apparently do not proceed to suppuration. The eosinophil has also been found in some numbers in tuberculous exudates. The large hyaline is never the predominant cell, but is not infrequently found in small numbers, particularly in associa- tion with the polynuclear neutrophil. Malignant cells, whether sarcomatous or carcinomatous, are frequently described and practically never identified. The cells which are sometimes figured as sarcoma cells are either small lymphocytes or quite indistinguishable from them. The cells often described as carcinoma cells are in every way similar to endothelials. It is true that fragments of growth may be washed off into a serous fluid and may very rarely be identified microscopically, but in the great majority of malignant cases the predominant cell in an exudate is not to be distinguished from either an endothelial cell, or much less commonly, a small lymphocyte. In the routine examination of a very large number of serous effusions I have only once recognised with any certainty the cells of a malignant growth. It has not been sufficiently insisted upon that a carcinoma or sarcoma cell floating free has no distinguishing marks by which it can certainly be identified. (4) Bacteriology. — The method of bacteriological investi- gation necessarily varies with the type of cell present in the exudate, and consequently a portion of the fluid should be p. 13 194 CLINICAL PATHOLOGY. examined cytologically before an attempt is made to discover the presence of a causative organism. If the predominant cell is an endothelial no bacteriological investigation is necessary, since there is no known organism which leads to an over-production of this type of cell, at any rate in preponderating numbers. The presence of bacteria in such fluids is due to a contamination either from the patient's skin or from the needle and syringe. When the predominant cell is a polynuclear neutrophil the bacteriological investigation must be conducted on exactly the same lines as that for acute inflammatory exudates in any part of the body. The causative organisms themselves may often be seen in the film preparations made primarily for the identification of the cells, and cultures are then made upon the appropriate media. If no organisms are seen a growth may still be obtained in the cultures, which should be made on media suitable to the organism suspected from the nature of the case. In a small proportion of acute inflammatory exudates organisms may be seen in the films, but, owing probably to the previous exposure of the bacteria in the body to bacteriolytic substances, the attempt to grow them in culture media fails. In such cases a fairly accurate bacteriological diagnosis can usually be made from the nature of the case and the morphological appearance of the organisms. The pneumococcus in pleural or other exudates, and the meningococcus in cerebro-spinal fluids, not infrequently stain very faintly, and sometimes fail to grow upon the ordinary media. In another small percentage of cases polynuclear neutrophils are the predominant cell, but no organisms are seen in the film preparations and none grown in culture. Some of these fluids are in reality produced by pyogenic organisms which have escaped recognition. Others are not the product of organisms but of some mechanical irritation, such as an injury or the presence of new growth, or an effusion of blood acting as a foreign body. Others, again, may turn out to be tuberculous effusions in which the predominant lymphoid cell has degenerated and the clearly staining polynuclear neutrophil, which is acting as a phagocyte of the cellular debris, appears to predominate. Primary tuberculous effusions may further become secondarily infected by pyogenic organisms, and in such cases the underlying pathological process may be entirely GENERAL PROCEDURE— FLUIDS. 195 missed, unless a knowledge of the clinical condition of the patient suggests that the tubercle bacillus also should be specially sought for. When the small lymphocyte is the predominant cell there is no object in putting up the ordinary culture media and search- ing for the presence of pyogenic organisms. Any bacteria which may be seen in the ordinary film preparations or grown on the stock media are evidence of failure in the aseptic technique. When an excess of small lymphocytes with a relative predominance of 80 per cent, or more over the other cells is found, a diagnosis of either a syphilitic or a tuberculous infection can be made with a considerable degree of confidence. Since on clinical grounds the differential diagnosis between these two pathological processes can in the great majority of cases be readily made, the laboratory diagnosis of a lympho- cytic effusion is usually sufficient. Such a diagnosis, however, is an indirect one, and thus liable to a small but definite percentage of errors ; consequently further investigation is preferable whenever practicable. In syphilitic cases it is useless to look for the spirochete in the serous fluids, but a Wassermann reaction should be performed with the patient's serum, or with the fluid, or with both. In the case of tuber- culous effusions an attempt should be made to identify the bacillus, but unfortunately in the majority of these fluids the bacilli are extremely scanty and require special methods to display them, such as will be described subsequently. A small percentage of lymphocytic effusions are neither tuberculous nor syphilitic. Very occasionally miliary growths of carcinoma or sarcoma on the pleural membranes, or less commonly on the peritoneum, may act as chronic irritants and produce an exudate rich in lymphoid cells. Such exudates are liable to be mistaken, both on clinical and pathological grounds, for tuberculous exudates. Similar exudates may accompany an aneurysm of the thoracic or abdominal aorta, or an enlarge- ment of the mediastinal glands by new growth, or Hodgkin's disease. All these rare pathological states are more commonly associated with an excess of endothelial cells, and the percentage of error in ly mphocy tic effusions is very small ; it is sufficient, however, to remind one that no single physical sign in medical diagnosis is pathognomonic. An instance may be given of a pleural effusion rich in small lymphocytes, a portion 13—2 196 CLINICAL PATHOLOGY. of which was injected into a guinea-pig. The animal subsequently died of general tuberculosis. The patient died shortly afterwards, and no evidence of tuberculous disease could anywhere be found. The probable explanation is that a guinea-pig already tuberculous was inoculated with the sterile fluid. The incident is merely quoted as an example of ordinary human fallibility when dealing with circumstantial evidence. Methods. — The methods to be adopted in the examination of the various fluids do not materially differ, but it is convenient for the sake of clearness to describe the procedure for each fluid. Pleural Fluids. All puncture fluids must be obtained with strict aseptic precautions. The patient's skin should be painted with iodine solution, and the needle and syringe must have been boiled. The needle should be a fairly long and stout one, and the syringe should be capable of holding at least 10 c.c. The place chosen for puncture necessarily varies with the physical signs present, but in the majority of cases the puncture is best made midway between the posterior axillary and scapular lines in the ninth or tenth space. If much fluid is present the syringe when full is detached from the needle and emptied into a sterile test tube and the process repeated for a second tube. The fluid withdrawn may be obviously purulent or more or less clear. Purulent pleural exudates are examined as follows : — (1) Observe whether they are offensive or non-offensive. The majority of these effusions have little odour ; a minority stink most evilly. Stinking pus from the chest is of diagnostic significance, since it indicates pretty certainly that one is not dealing with a simple empyema, that is with an abscess confined to the pleura. The smell is produced in the great majority of cases by a long, thin saprophytic bacillus whose normal habitat is the respiratory or alimentary tract. In a very small percentage of cases the smell may be due to, or augmented by, the presence of the bacillus coli or other members of that group of organisms. The significance of these bacilli is that a communication has taken place between the pleural cavity and the lung or between the pleura and an GENERAL PROCEDURE— FLUIDS. 197 abdominal viscus. The presence of a stinking fluid, therefore, combined with the information acquired by further examina- tion, suggests one of the following alternatives : — A primary abscess of the pleura (or empyema) has ruptured into the lung ; a primary abscess of the lung has ruptured into the pleura ; an abdominal abscess has ruptured through the diaphragm into the pleura ; a bronchiectatic, and not a pleural, abscess has been punctured. The prognosis for any of these alternatives is evidently less favourable than for an uncomplicated empyema. (2) Make film preparations. Stain with carbol-thionin and examine. The cells present will be found to consist almost entirely of polynuclears. Organisms are usually to be seen in addition. The organism most commonly met with is an extracellular diplococcus with pointed ends, the pneumococcus. The great majority of empyemata follow lobar pneumonia, and in most cases the pneumococcus is present in large numbers and in pure culture. The diagnosis of the causative organism can be made with considerable certainty from film preparations in many cases, but it is advisable always to confirm by cultural tests. Numerous other organisms may appear in pairs in pus films, and the pneumococcus often forms quite short chains of 4 to 8 members. The organisms found next in order of frequency are streptococci. These may appear in chains of considerable length, are often intracellular, and usually prove on culture to be of the ordinary S. pyogenes type. Streptococcal purulent effusions are not infrequently met with in young children as a sequel of broncho-pneumonia, and may also occur as part of a general septicaemia or as a terminal infection in chronic general diseases. Streptococcal effusions are of much less favourable prognosis than pneumococcal. Staphylococci are occasionally found in these fluids, but they are practically never the primary cause of a local empyema. The presence of a staphylococcus is suggestive either of a general pyaemia or of an infection secondary to some other process, most commonly a tuberculous one. Tubercle bacilli may be present in apparently purulent exudates. They will, of course, not appear in the carbol- thionin preparation, but are to be suspected on the following grounds : — If the cells in the film preparation are extremely 198 CLINICAL PATHOLOGY. degenerate and no bacteria are to be seen ; if the cultures on ordinary media are subsequently sterile ; if only staphylococci or the long, thin bacilli of a stinking exudate, or both, are found ; if the condition was a pyo-pneurno-thorax and not a simple empyema; if there is other evidence of tuberculosis in the patient. Saprophytic bacilli. — These are practically confined to the stinking exudates, and are usually associated with other organisms. They may be very numerous in the film pre- parations, and appear mainly as long, thin curved, and often beaded, extracellular bacilli. They are sometimes so numerous as to form a regular background to the film. Their diagnostic import has been already referred to. They are in all probability non-pathogenic, and they do not grow upon the ordinary media in aerobic culture. Influenza bacilli are occasionally met with in purulent pleural exudates. They appear as minute intracellular bacilli, and should be proved to be negative to Gram's stain. Other bacteria, such as are rarely found in the chest, include colon bacilli, typhoid bacilli, bacillus proteus, bacillus pyocyaneus, the pneumobacillus. (3) Identify the causative organisms by cultural or other methods. The bacteria seen in the film preparations should suggest the further procedure necessary for their exact identification. When pneumococci are suspected, subculture from the pus into litmus milk and on to agar. In the case of streptococci or staphylococci take the primary cultures on to agar and into broth. In suspected tuberculous fluids proceed as follows : — Shake up about 2 c.c. of the pus with about 10 c.c. of 20 per cent, antiformin (page 156). Stand, preferably in a warm place, until the pus is mainly dissolved. Centrifuge at a high speed. Pour off the supernatant fluid, and stain films of the deposit by the Ziehl-Neelsen method. The bacilli may be present in such films in considerable numbers ; more often they are very scanty, and they may not be found at all. If no bacilli are found a portion of the antiformin residue, after being washed, should be injected into a guinea-pig and another portion cultivated upon Dorset's egg medium. Influenza bacilli should be subcultured on to nasgar, or preferably blood-agar. GENERAL PROCEDURE -FLUIDS. 199 " The majority of other bacteria are rarely met with. In the case of bacilli of the colon group a primary broth culture should be made and subsequently plated out on Petri dishes. More or less clear pleural exudates.— These are examined as follows : — (1) Naked-eye appearance. — The great majority of these fluids are of a pale straw colour and form an almost complete jelly-like clot on standing. The exudates as a rule clot more firmly than the transudates. The presence of blood in any quantity should be noted. (2) The chemical examination. — This need be by no means exhaustive, and unless a considerable quantity of fluid is avail- able can be omitted altogether. The reaction of these fluids is generally alkaline. The specific gravity is commonly above 1,020 in the case of exudates and below 1,020 for transudates. The coagulable proteids can be roughly estimated by acidifying the fluid in a test tube, boiling, and allowing the precipitate to settle. The exudates often become almost solid on boiling. (3) The cy tological examination. — This is of great importance and should never be omitted. It is convenient to have two test tubes of the fluid in order that one portion may be left uncontaminated for a subsequent bacteriological investigation. If only one tube is available a portion should be poured off into a second tube for cyto-diagnosis. The clot must first be broken up, and this can be done either by shaking the tube or stirring up the contents with a glass rod. The fluid is then centrifuged at a moderate speed, preferably on a water or electrically driven centrifuge, but a hand centrifuge will serve the purpose. All centrifuge tubes should be carefully rinsed out with distilled water before use. After centrifuging empty out the supernatant fluid by simply turning the tube upside down. The last drop, con- taining the majority of the cells, will remain in the bottom of the tube. (It is a wise precaution to preserve the supernatant fluid, if there is not a large quantity available.) Wrap a very small piece of absorbent cotton wool around the tips of a small pair of fine-pointed forceps. Soak the wool in the centrifuged deposit and make moderately thick films in the centres of 2 slides. Stain one slide with carbol- thionin, the other with Leishman's stain. 200 CLINICAL PATHOLOGY. The Leishrnan-stained film should be treated in the same manner as a blood film, but given the following times : — Leishman alone, ^ minute. Leishman -4- 2 volumes of distilled water, 5 minutes. Distilled water only, 2 minutes. Examine the films with an oil immersion lens. In the majority of these pleural fluids the cells are small lymphocytes ; in the minority either polynuclear neutrophils or endothelial cells predominate. The lymphocytes are often very numerous, and 20 or 30 are commonly seen in one field of a j^th inch objective. They often form at least 80 per cent, of the cells present, the remainder consisting of endothelials, and large lymphocytes, with occasional polynuclear or other cells. Such a lymphocytic effusion is almost certain evidence of a tuberculous affection, syphilitic disease of the pleura being practically unknown. Very rarely a lymphocytic exudate may occur with pleural or mediastinal neoplasms. A deposit of endothelial cells occurs most frequently in cardiac or renal dropsy. They may also be found with malignant growths. Polynuclear neutrophils may predominate in an apparently clear or in a slightly turbid fluid. They have the same significance as in an obviously purulent fluid. Eosinophil cells are occasionally found in the clear fluid which exceptionally follows a lobar pneumonia, and still more rarely in a tuberculous exudate. When present in excess a Irydatid cyst should always be suspected and the fluid examined accordingly (page 209). (4) The bacteriological examination. — This varies with the type of cell present. In the case of a lymphocytic exudate proceed as follows : — Fill 2 centrifuge tubes (of about 10 c.c. capacity each) two- thirds full of the fluid. Add one- third of absolute alcohol. Invert several times and stand for 5 minutes. A copious precipitate forms. Centrifuge at a high speed for 10 minutes. Pour off the supernatant fluid. If the deposit is comparatively small in amount make thick films from it and stain for tubercle bacilli in the ordinary way. If the deposit is very bulky (as is usually the case) fill the tubes with 20 per cent, antiformin. Shake thoroughly and stand in a warm place GENERAL PROCEDURE— FLUIDS. 201 until the precipitate is almost dissolved. Centrifuge again at a high speed for 10 minutes. Make films of the deposit and stain for tubercle bacilli. A prolonged search is necessary to demonstrate the bacilli, and in the case of the majority of pleural fluids of tuberculous origin no bacilli can be found. In cases of exceptional importance the antiformin deposit should be washed two or three times with sterile saline and one portion rubbed over the surface of Dorset's egg medium, the other portion injected into the leg of a guinea-pig. Prefer- ably the centrifuged deposit of the original fluid is injected, without other treatment, into a guinea-pig. Where endothelial cells are in excess no bacteriological examination is necessary. With an excess of polynuclear neutrophils the procedure is the same as for purulent exudates, except that it is advisable to add the fluid in greater bulk to the media. As an alter- native the fluid may be centrifuged in sterile tubes and the deposit of pus used for inoculation of the media. Lung puncture. — It occasionally happens that a chest deemed to contain fluid proves on exploratory puncture to be filled with solid lung. On such occasions a small quantity of blood-stained fluid is removed in the needle and should be reserved for film and culture preparations. The pneumococcus or other organisms can often be identified by these means, and a diagnosis of the condition made. Lung puncture may be performed deliberately as a means of diagnosis, but the procedure can hardly be said to be entirely free from risk, and should be avoided as a routine method. Pericardial Fluids. Puncture of the pericardium is rarely performed. It is advised to make the puncture with a fine needle in the fourth left intercostal space half an inch from the edge of the sternum. The fluid withdrawn should be examined in exactly the same way as a pleural fluid both by cytological and bacteriological methods. Those fluids for which pericardial puncture is performed are usually purulent and frequently associated with a purulent pleurisy. CHAPTEE XV. peritoneal fluids — cerebrospinal fluids — synovial fluids cysts, etc. Peritoneal Fluids. Before puncturing the peritoneal cavity the bladder must be emptied. The puncture is best made in the middle line and midway between the umbilicus and the pubes. The fluid is examined in much the same way as a pleural fluid. Purulent peritoneal fluids are rarely withdrawn by puncture, being more commonly obtained during a laparotomy for general peritonitis. The organisms present in such fluids usually include the bacillus coli. Cocci are often seen in addition. Streptococci in pure culture are of the worst pos- sible prognosis. Pure pneumococcal exudates are more fre- quent in young children, are as a rule associated with comparatively slight constitutional disturbance, and are of fairly good prognosis. B. pyocyaneus is occasionally met with, and is usually associated with a particularly virulent peritonitis. Purulent fluids are sometimes obtained in associa- tion with the tubercle bacillus, a secondary infection with the B. coli, or other intestinal organisms having taken place. Almost all of the pyogenic organisms are found from time to time in the peritoneal cavity, and the examination of purulent peritoneal fluids is conducted on exactly the same lines as those already laid down for the examination of " pus r ' generally. Clear peritoneal fluids are examined in the same way as clear pleural fluids. Lymphocytic fluids indicate a tuberculous lesion ; endothelial fluids are associated with cardiac or renal dropsy, with cirrhosis of the liver, with malignant disease, or with any abdominal lesion which tends to obstruct the portal circulation. Polynuclear neutrophils may be present in apparently clear peritoneal fluids and in association with the ordinary pyogenic PEEITONEAL FLUIDS— CYSTS. 203 organisms. With such fluids ifc is always wise to examine films of the deposit for tubercle bacilli, otherwise the underlying cause of the condition may be missed. The tubercle bacilli may be present in considerable numbers. The cytology of peritoneal fluids is for some cause not quite so satisfactory as that of pleural fluids. The cellular deposit is very frequently a mixed one, and the diagnostic value of the findings is not very great unless the predominant cell is present in overwhelming numbers. Lymphocytic fluids should be examined for tubercle bacilli in the same way as pleural fluids. Cultures should be made of the acute inflammatory or polynuclear-containing fluids. Opalescent fluids are occasionally withdrawn from the peritoneal cavity, and still more rarely from the pleural sacs. The opalescence is not removed by filtration, nor by centri- fuging, nor by extraction with ether, and is apparently due to the presence of a lipoid body combined with a proteid. The significance of these striking-looking fluids is not known, but they seem to be most frequently associated with the dropsy of parenchymatous nephritis. Extremely rare are genuine chylous fluids, in which the opalescence is due to actual fat and is removed by extraction with ether. Such fluids are associated with lesions of the thoracic duct, and occur in filariasis. They are to be distin- tinguished from the pseudochylous fluids referred to above. Cbrebro-spinal Fluid. The fluid is obtained by puncture through the fourth lumbar space, a special hollow needle provided with a stilette being required for the operation. Needle and stilette should be made of flexible drawn nickel and of a length of 3J inches. Do not use an ordinary steel exploring needle, since it is liable to break in the tissues. An all-glass syringe should be fitted to the needle, but it is advisable so far as possible to avoid strong suction with it. The patient is placed in the left lateral position with the back well bent and the knees drawn up so as to approximate to the chin. A line is drawn across the back to join the highest points of the iliac crests, and the interspinous space immediately below this line is defined with the tip of the 204 CLINICAL PATHOLOGY. finger. An area of skin is painted with iodine, and may be anaesthetised with the ethyl-chloride spray. The needle is plunged firmly into the interspinous space one-fourth of an inch to one side of the middle line and pushed steadily onwards and directly forwards until the canal is reached. In the majority of adult patients the needle has to be passed almost up to the hilt, and the correct position of the point is recognised by the sudden and easy passage of the needle just as the canal is reached. The patient may also complain of a sensation of pain or tingling in the leg. On withdrawing the stilette the fluid may gush out, or, as is more frequent, escape drop by drop. The flow can sometimes be started by cautiously applying suction with the syringe. The fluid is collected in clean sterile test tubes, and if the first sample is tinged with blood a second tube should be ready as soon as the fluid runs clear. It is important to obtain a sample free from blood. About 10 c.c. of fluid can be removed with impunity. After withdrawing the trocar seal the puncture with collodion, and direct that the patient be kept in bed with the head low for a period of 24 hours. The fluid is examined as follows : — (1) Naked-eye examination. — The normal cerebro-spinal fluid is a clear, limpid fluid resembling water. In disease small flocculent threads may be seen floating in the fluid, an appearance particularly common in syphilitic and parasyphilitic conditions. Sometimes on standing a delicate clot, having the appearance of a fine cobweb, slowly forms in the tube. This formation of a clot is certain evidence of disease, and suggests either tuberculous or meningococcal meningitis. In suppura- tive meningitis, whether due to the meningococcus or to other pyogenic organisms such as the pneumococcus, every grade of turbid fluid is found, from a mere cloudiness to actual pus. The presence of blood, if bright in colour, is usually due to the puncture of a vessel ; if dark, intimately mixed with the fluid and persisting during the whole time the fluid is running, it is suggestive of intradural haemorrhage, such as occurs in fracture of the base of the skull. (2) Cytological and bacteriological examinations. — If normal spinal fluid is centrifuged for 10 minutes at a moderate speed, if the tube is emptied by inversion and a PEEITONEAL FLUIDS— CYSTS, ETC. 205 fairly thick film the size of a sixpence is made with a cotton-wool swab in the manner described for pleural fluids, practically no cells are found in the film. The presence of more than 2 or 3 cells to a film in preparations made by this method is evidence of disease. It often happens in disease that no obvious sediment is found after centrifuging ; the last drop may nevertheless be rich in cells. The cells present in these fluids are practically of only two kinds — small lymphocytes and polynuclear neutrophils ; endothelial cells in excess are never seen. If small lymphocytes are present the fluid is either syphilitic or tuberculous. If a syphilitic lesion is suspected a Wassermann reaction must be performed with the fluid and with the patient's serum. In syphilis of the central nervous system the reaction is typically negative in the fluid and positive in the serum. In tabes and general paralysis the reaction is usually positive in both serum and fluid. If a tuberculous lesion is suspected proceed as follows : — Half-fill two centrifuge tubes with the fluid, and add to each an equal volume of absolute alcohol. Mix thoroughly and stand for a few minutes. In the case of normal fluids practically no turbidity results ; in meningeal disease, whether tuberculous, syphilitic, or suppurative, a more or less marked opalescence forms in the fluid, owing to the precipitation of proteids. This stage of the proceedings therefore yields confirmatory evidence of the cytological findings. The tubes are then centrifuged at as high a speed as possible for about 10 minutes. In the majority of cases a small but obvious sediment is found at the bottom of the tubes. As thick films as possible are made from the sediment and stained for tubercle bacilli in the ordinary way. Bacilli may be present in considerable numbers ; more commonly they are very scanty ; a careful search, however, should reveal the bacilli in at least 70 per cent, of the cases. If polynuclear cells are found some form of septic meningitis is present. The causative organism should be sought for in the films. The meningococci are seen as Gram-negative diplococci, the majority of which are within the polynuclear cells ; but a number of extracellular cocci are nearly always present as well. In meningococcal exudates the first cultures should be made on nasgar and in milk, and in a certain number of cases, even when the organisms are abundant in the 206 CLINICAL PATHOLOGY. films, the cultural results are negative. Once a growth has been obtained the sub-cultures grow readily, provided they are made at frequent intervals. Pneumococci are sometimes found in the spinal fluid, and are recognised as Gram-positive, lanceolate, extracellular diplococci. The cultures should be made in milk and on agar. The pneumococcal cases run a rapidly fatal course of 1 to 3 days as a rule ; consequently the prognosis is far less favourable than in the meningococcal infections, which run a more chronic course with a fairly high percentage of recoveries. Other organisms which may be found in the cerebro-spinal fluid include streptococci, influenza bacilli and colon bacilli. Diphtheroid bacilli and staphylococci are occasionally grown in culture, but are usually to be regarded as skin contaminations. To recapitulate the findings of a spinal fluid deposit. Absence of cells is very strong evidence against any variety of meningitis. The normal fluid examined in the manner described here yields no cells in the film preparation. Small lymphocytes mean syphilis or tuberculosis. Polynuclears mean septic meningitis, of which the most common variety is due to the meningococcus, the next common to the pneumo- coccus. The examination of the cellular deposit may be obscured by the presence of blood, and it must also be recognised that the preponderance of the cells, whether lymphocytes or polynuclears, is a matter of degree. A few polynuclears will be found in a tuberculous or syphilitic meningitis. Lymphocytes are fairly numerous in meningococcal infections, and particularly in the more chronic cases. A positive interpretation of the film requires that the percentage of the predominant cell should be at least 70. (3) The chemical examination. — If 10 c.c. of fluid have been withdrawn a few simple chemical tests should be per- formed, since these are confirmatory of previous findings. The globulin content of the fluid is a guide to the presence of meningeal inflammation, whether tuberculous, syphilitic, or septic. It can be investigated by the method of Nonne and Apelt. A saturated solution of ammonium sulphate is prepared as follows : — 85 grammes of the pure salt (Merck) are boiled in a flask with 100 c.c. of distilled water until no more salt is taken up. The solution is allowed to cool and is filtered. PEEITONEAL FLUIDS— CYSTS, ETC. 207 Equal parts of the solution and of the fluid are mixed and allowed to stand at room temperature for 3 minutes. After 3 minutes note — (1) Opacity; (2) Opalescence ; (3) Slight opalescence ; (4) Trace of opalescence. 1, 2, and 3 are positive ; 4 is negative. The spinal fluid must not be heated and must contain no blood. Very similar readings are obtained by the simple addition of absolute alcohol, as described under the method for demonstrating tubercle bacilli in tbe fluid. The spinal fluid should also be examined by the ordinary Fehling test for the presence of a reducing substance. A trace of sugar is present in the normal fluid ; it is usually absent in cases of meningitis. Synovial Fluids. The puncture of joints for diagnostic purposes should only be undertaken in ideal surroundings and with the strictest aseptic precautions. Provided there is a large excess of fluid in one of the large joints and every care is taken, there is no real risk in the procedure. The point at which to make the puncture necessarily varies for each joint, but no difficulty should be experienced if the needle is plunged directly through the skin into the most obviously distended portion of the joint cavity. The needle should be of sharp steel fitted with an all- glass syringe and with a stilette. After withdrawal of the needle the puncture wound must be sealed with collodion. The removal of the fluid from a joint has further advan- tages. The relief of tension when a moderate quantity of fluid is withdrawn is in many cases beneficial, and the subse- quent preparation of an autogenous vaccine may be made use of as an aid to treatment. In the puncture of a distended knee joint an unexpectedly small quantity of fluid is commonly withdrawn owing to the blocking of the needle with synovial fringes. By altering the position of the needle a further flow is often obtained. The normal synovial fluid is a sticky viscous material of unmistakable character. In disease it may be not obviously 208 CLINICAL PATHOLOGY. altered in appearance, or it may be turbid, or it may be obviously purulent. In all cases film preparations should be made without centrifuging. In practically every variety of arthritic disease the predominant cell is a polynuclear neutrophil, while lymphocytic and endothelial cells are usually present in addition. The polynuclear cell is predominant, not only in acute lesions due to gonococci or other pyogenic organisms, but also in the chronic distended joints of rheumatoid arthritis. Lymphocytes may be more numerous in acute tuberculous joints, but the cytology of synovial fluids is not of striking diagnostic value. The cells in carbol-thionin preparations are seen lying on a deeply-stained background of the synovial fluid. All film preparations should be carefully searched for bacteria, and these should be seen in all the acute septic cases due to streptococci, pneumococci, or staphylococci, and in many of the gonorrhceal cases if a very careful and prolonged search is made. In these affections the findings of the film preparations must always be confirmed by cultural methods. In tuberculous cases no organisms are seen in the carbol- thionin films unless a secondary infection has taken place. The material withdrawn in such cases is often suggestively caseous, and the cells are necrotic and many of them unrecognisable. A portion of the fluid should be treated with antiformin, centrifuged and examined for tubercle bacilli in the ordinary way. The bacilli may be fairly numerous. In the large group of arthritic affections known as " rheu- matoid " no organisms are seen in the films and none are grown in any culture media. Paracentesis of acute rheumatic joints is rarely performed, partly because the effusion is so often slight in amount and is mainly periarticular. A short- chained streptococcus has been recovered in these cases, and is by some considered to be the cause of rheumatic fever. A similar coccus has been found in almost every rheumatic lesion, including the angina, the nodules, the valvular vegetations, and, in cases of infective endocarditis, in the circulating blood. The etiology of rheumatic fever is, however, still sub judice. Hydrocele and spermatocele fluids. — These are obtained by puncture through the tensest portion of the cysts, care being taken to avoid wounding one of the scrotal vessels. Hydrocele fluid is commonly clear and straw-coloured. On PEKITONEAL FLUIDS— CYSTS. 209 standing a quantity of cholesterin crystals separates out. The cells present are mainly endothelial, and no organisms are found. In cases where secondary infection has occurred polynuclear neutrophils predominate, and the ordinary pyogenic organisms, most commonly staphylococci, are obtained. Spermatocele fluid is usually milky in appearance, and in almost all cases large numbers of spermatozoa are seen under the microscope. The spermatozoa are best examined unstained, a drop of the fluid being placed on a slide with a cover-slip over it and watched under the ^-inch objective. Stained preparations of dried films can be made with carbol- thionin or Leishman's stain. Cysts. For all cyst fluids a chemical and a microscopical exami- nation are required. Bacteriological investigations are rarely called for. In the majority of cases the nature of the cyst is clearly determined on clinical grounds or at operation, and the laboratory investigations are for purposes of confirmation. The examinations made should naturally be those most appro- priate to the type of cyst suspected. When the nature of the cyst is quite obscure the following routine procedure should be adopted. Note the amount and naked-eye appearance of the fluid. Test the reaction and the specific gravity. Esti- mate roughly the amount of coagulable proteid. Test for the presence and amount of urea. Test for the presence of a reducing substance. Estimate roughly the amount of chlorides present. Examine the centrifuged deposit in fresh and stained specimens, as to the nature of the cells, and the presence of crystals or of hydatid hooklets. If there is reason to suspect a pancreatic cyst, examine also for the presence of ferments. Prepare paraffin sections of the cyst wall, if available. The following is a brief description of the more important cyst fluids : — Hydatid cysts. Specific gravity about 1,010. Proteids very scanty or absent. Chlorides abundant. Sugar occasionally present. Urea present in very small amount. Hydatid hooklets in centrifuged deposit. 14 210 CLINICAL PATHOLOGY. The really diagnostic feature is the presence of hooklets. The hooklets are sharply pointed at one end and barbed. Occasionally a well-formed scolex with a circle of hooklets may be found (page 334). Pancreatic cysts. Specific gravity, 1,010—1,020. Keaction alkaline. Proteids in varying amounts. Chlorides usually scanty. Urea often a trace. Cholesterin usually present. Trypsin, lipase, and diastase present. The characteristic constituents of these cysts are the ferments, which can be found in the majority of cases. They are tested for as follows : — Trypsin. — Put in one test tube about 5 c.c. of the fluid ; in a second test tube 5 c.c. of the fluid and bring to the boiling N point ; in a third test tube 4 c.c. of the fluid, and 1 c.c. of — N NaOH ; in a fourth tube 5 c.c. of ^ NaOH. Label each tube. Add to each tube veiy thin strips of hard-boiled white of egg. Cork the tubes with wool plugs, place in the incubator at 37° C. and examine at the end of 1 hour, 2 hours, and 12 hours. Definite digestion of the proteid should have occurred in tubes 1 and 3 at the end of 2 hours, and digestion should be complete the next morning. Lipase. — Exactly neutralise the fluid with dilute HC1. Add a trace of calcium chloride. Add a few drops of ethyl butyrate in a test tube and a few drops of litmus solution. Put up a control tube of the boiled fluid. Incubate for 12 hours at 37° C. The litmus is turned red if lipase is present. Diastase. — Add a small amount of starch emulsion to the fluid. Incubate and examine at the end of 1, 2, and 12 hours. Put up control tubes. A drop of the starch and fluid mixture is taken out and mixed with a drop of iodine solution. When diastase is present the blue coloration is lost and replaced, first by a red colour, finally by an absence of colour. Renal cysts. --Renal cysts proper may arise from a hypernephroma, from a congenital cystic kidney, or from a contracted granular or cardiac kidney. Rare single cysts of PERITONEAL FLUIDS— CYSTS. 211 considerable size may be found, arising, as a rule, from the lower pole of an apparently healthy kidney. The composition of such cysts is very variable, and both urea and uric acid may be absent. The contents of a hydronephrosis have more obviously the composition of urine, but the specific gravity is usually below 1,010 ; albumin is frequently small in amount. The reaction may be acid, a point which immediately suggests a renal origin. The most important constituent is urea, which may amount to from 0"4 to l'O per cent. It must be remembered, however, that many cystic fluids contain a trace of urea, though a percentage of more than 0*2 is very suggestive of a renal origin and more than 0*5 is practically diagnostic. Ovarian cysts. — The source of the fluid may sometimes be recognised from the character of the cellular deposit. The cells in some cases may be indistinguishable from the endothelial cells of a peritoneal fluid ; in other cases columnar epithelial, or even ciliated, cells may be found. Cholesterin crystals are common in these fluids, but may occur in almost any variety of cyst. Other abdominal cysts. — These include mesenteric, retroperitoneal, and omental cysts. All of these are rare, but they may be of considerable size. The fluid contents present no particularly characteristic feature. Dermoid cysts may be recognised by their contents and by the nature of the cyst wall. The contents may include any of the skin appendages, such as bones, teeth, or hair, the last being the most frequent. The cyst wall consists of true skin, and consequently in a microscopic section all the layers of the true skin are found, including the stratum lucidum and the stratum granulosum. These cysts have to be distinguished from sebaceous cysts, which contain only a pultaceous material* consisting of fatty bodies, cholesterin, and debris. Sebaceous cysts, in common with thyro-glossal and other cysts of epiblastic origin, have an epidermal lining. Some thyro-glossal cysts are lined with a columnar ciliated epithelium. 14—2 SECTION IV. THE URINE. CHAPTEE XVI. Routine Examination — Variations in Amount —Variations in Appearance. CHAPTER XVII. Variations in Acidity, and Acidosis — Variations in Specific Gravity — Urea — Proteids — Carbohydrates. CHAPTER XVIII. Urinary Deposits — Urinary Calculi. CHAPTER XIX. Special Investigations of the Urine — Bacteriology of the Urino-genital Tract. CHAPTER XVI. ROUTINE EXAMINATION VARIATIONS IN AMOUNT — VARIATIONS IN APPEARANCE. An examination of the urine forms a part of the routine investigation of every patient. For the purposes of an ordinary clinical examination a specimen of urine passed at the time can be made use of, but in the case of patients con- fined to bed a sample taken from the total urine passed in the 24 hours is to be preferred. For special purposes the urine must be withdrawn by catheter ; and a catheter specimen is necessary for bacteriological investigation other than that for the presence of tubercle bacilli : the presence of pyogenic cocci or bacilli in an ordinary specimen of urine has no significance whatever. For the detection of small traces of albumin or of pus in the urine of women a catheter specimen is similarly essential, since a sample of urine passed in the natural manner is very frequently contaminated by leucocytes and the albuminous secretion of the vagina. Further, the comparatively recent advances of skilled surgery permit, in special cases, of the examination of the secretions of each kidney by means of ureteric catheterisation. The routine examination of an ordinary specimen of urine should always be conducted according to a settled scheme of investigation, otherwise in an important minority of cases some point essential to the appropriate treatment will be missed. The simple tests described below must be applied in every case, whether there is any reason to suspect any abnormal sub- stance in the urine or not. The urine may be found loaded with albumin or sugar in the case of apparently healthy persons who are seeking advice as to the necessity of some operation that may well be postponed, but which would be advisable for normal persons. The unexpected detection of pus in the urine may turn the diagnosis of a supposed splenic enlargement into that of a left pyonephrosis, and numerous similar instances of the importance of routine examination could readily be adduced. 214 CLINICAL PATHOLOGY. The Boutine Examination of the Urine. (1) The amount passed. — The measurement of the urine passed in the 24 hours may be omitted if no alteration in the urinary secretion is to be expected and nothing abnormal is detected by the ordinary tests. In any case the amount of urine necessarily varies considerably according to the quantity of fluid taken and the amount lost by the skin. The average output of urine by the healthy adult male in the 24 hours is from 40 to 50 ounces, the majority of which is excreted during the daytime. (2) The naked-eye appearance. — The urine directly after being passed should be perfectly clear and of an amber colour. The colour is mainly due to the pigment urochrome. While the naked-eye inspection of a sample of urine is of value in leading to the detection of many gross pathological changes, confirmatory tests should always be employed, since it is often impossible to distinguish by the eye numerous sub- stances of similar appearance and widely different nature. The certainty of an observer's naked-eye diagnosis usually varies inversely with the carefulness of his routine examination. (3) The reaction. — The reaction of the freshly-voided normal urine to litmus paper is definitely acid from the presence of acid salts and in particular of acid sodium phos- phate. An alkaline reaction obtained in the sample of a 24 hours specimen which has been standing is of no significance. An alkaline reaction in a fresh specimen calls for further investigation. (4) The specific gravity. — The normal specific gravity of the urine is about 1,020 and is taken by means of the urino- meter. No simple examination is more frequently bungled than is this taking of the specific gravity, which in some pathological states is of considerable clinical importance. The following points should always be observed. The sample examined must be one from the 24 hours' output, since variations in a single specimen are too dependent upon external circum- stances. It must be placed in a glass sufficiently deep to contain the urinometer in almost its whole length, and sufficiently wide to prevent the instrument clinging to the sides. The reading must be taken with the eye on a level with the surface of the urine and the surface must be free ROUTINE EXAMINATION— VARIATIONS. 215 from bubbles. The urinometer itself must be periodically inspected, since in the cheap instrument in common use the little scale is merely attached by a minute piece of sealing- wax and may become considerably dislodged from its true position. (5) The presence of albumin. — Albumin in quantity detectable by the ordinary tests is normally absent from the urine, and its presence in even small amount is of important clinical significance. The albumin present in disease consists in the great majority of cases mainly of serum albumin, with varying amounts of serum globulin. One of the two following tests should be employed in every examination, and either test if properly performed is perfectly reliable. (a) The heat test. — The urine, if cloudy, must be filtered, and if alkaline must be rendered acid to litmus with dilute (33 per cent.) acetic acid. Fill a clean test tube two-thirds full with the clear acid urine. Slowly heat to boiling point over a small Bunsen flame the top half-inch of the fluid, rotating the tube, but not shaking it. Look at the column of fluid with the light coming from behind and holding the tube against a dark background. If a cloud, however faint, appears in the heated portion, add a few drops of the acetic acid, whether the urine was previously acid or not. If the cloud persists albumin is present ; if the turbidity vanishes again it was due to phosphates. If the urine remains clear after heat-" ing, add a little acetic acid; a slight turbidity due to albumin may rarely be found. If the urine remains clear, albumin is absent. The failure to examine the tube against a dark background is responsible for innumerable cases in which a small, but often important, trace of albumin has been altogether missed. Unless a considerable cloud of coagulable proteid is present the turbidity becomes transparent when held against a bright or comparatively bright ground. Even should the urine be slightly turbid with bacteria (gross turbidity from this cause can be modified by centrifuging at a high speed), the additional cloud of albumin can be detected in this way. If the urine is turbid from the presence of phosphates the turbidity will disappear on adding the acetic acid.. If the turbidity is due to the presence of urates and is not completely removed by filtration, gently warm the upper half 216 CLINICAL PATHOLOGY. of the tube, which will become clear, before proceeding to heat the top half-inch of the fluid. If such a urine is heated rapidly the turbidity of the albumin may merely replace that of the urates and escape observation. (6) The nitric acid test (Heller's test).— This test is readily performed, but not so readily interpreted. It is further subject to certain difficulties not met with in the previous test. To perform the reaction place 1 inch of nitric acid in a clean test tube. Slowly allow to run down the side of the sloped tube about an equal quantity of the filtered urine. Allow to stand for a minute or two. In the presence of albumin an opaque white ring forms at the junction of the two layers of fluids. A similar ring appears if proteoses are present, but dis- appears on heating, to return on cooling. A diffuse turbidity may appear if mucin is present, but no ring. With very con- centrated urines a whitish " fluffy " opacity with undefined margins may form at the junction of acid and urine. In such cases the urine should be diluted with an equal volume of water and the test repeated, when if no ring forms albumin is absent. A coloured transparent ring is merely due to the presence of urinary pigments. Numerous other tests are employed for the detection of albumin. Some are sufficiently delicate to react with normal urine, others are comparatively complicated or require special and little used reagents. It is advisable to make use of one of the two tests given above and preferably the former. Which- ever test is used care is essential and some experience valuable ; consequently the repeated change from one routine test to another for the same substance is to be avoided. (6) The presence of glucose. — The reagents most com- monly employed in clinical pathology for the detection of glucose in the urine are those of Fehling and Nylander. The tests are performed as follows : — (a) Fehling' S test.— The urine, if strongly ammoniacal, should first be rendered acid with acetic acid. In one test tube bring to the boil about 2 inches of Fehling's solution. (If the copper sulphate and soda solutions are kept in separate bottles, equal quantities of each solution should be taken.) Boil the same amount of urine in another test tube. Add the boiling urine gradually to the boiling Fehling's solution. Allow the mixture to stand till cool before deciding that the ROUTINE EXAMINATION— VARIATIONS. 217 test is negative. In the presence of glucose a red or yellow precipitate of cuprous oxide is formed. A well-marked precipi- tate occurring on the addition of only a small proportion of urine is almost certain evidence of glucose, but may be due to lactose in the urine of nursing women. A definite precipitate on cooling after the addition of the entire equal volume of urine is probably due to sugar. A precipitate which forms only after prolonged boiling of the mixture of urine and reagents, or a greenish discoloration and turbidity without a true coloured precipitate, may be due to traces of sugar or to other substances. The substances which may react with Fehling's solution, and which are comparatively often met with in the urine, are uric acid or creatinine in excess, that is in highly concentrated urines, glycuronic acid and the products of certain drugs, of which the most important are chloral, chloroform, carbolic acid and salicylates. To prepare Fehling's solution: — (1) Powder and press dry between filter papers 34'64 grammes of pure copper sulphate. Dissolve in '200 c.c. of warm distilled water. Cool and make up to 500 c.c. (2) Dissolve 180 grammes of sodium potassium tartrate (Rochelle salt) in 300 c.c. of hot water. Filter and add 70 grammes of pure caustic soda. Cool and make up to 500 c.c. (3) Mix equal volumes of 1 and 2. 10 c.c. of the mixture is exactly reduced by '05 gramme of glucose. (b) Nylander's test. — This test is preferred by some because it is delicate and is not affected by an excess of uric acid or creatinine. The reagent is reduced by glycuronic acid, lactose, and the products of certain drugs. Nylander's reagent is prepared as follows : — 10 grammes of caustic soda are dis- solved in 100 c.c. of water ; 4 grammes of sodio-potassium tartrate and 2 grammes of bismuth subnitrate are added. After shaking thoroughly, the mixture is filtered and kept in the dark. To perform the test add 1 c.c. of the reagent to 10 c.c. of urine. Boil the mixture ; allow to cool. In the presence of much sugar a black precipitate of metallic bismuth separates out ; in the presence of small quantities the urine becomes dark brown, and a fine cloud of bismuth slowly settles down. 218 CLINICAL PATHOLOGY. In cases of doubt, and when it is particularly desirable to know that the reducing substance is glucose, the following scheme of procedure should be followed. A reduction of Fehling's solution having been obtained, Nylander' test may be applied, and if this is negative the reduction of the Fehling's solution may be taken as due to an excess of uric acid or creatinine. If Nylander's solution is not readily available this part of the procedure may be omitted. Next one portion of the urine is preserved and another portion is rendered acid if necessary and mixed with powdered German yeast, such as may be obtained from any baker. The mixture is placed in aDoremus ureometer tube so as to completely fill the long limb of the tube and about half the bulb, and to leave no air bubbles at the top of the tube. The tube is placed in a warm place, preferably an incubator at 37° C, and left for from 12 to 24 hours. A second tube containing yeast and water only should be put up as a control, since a small quantity of gas may be given off from the yeast. The top of the tube is then examined for bubbles of gas, proving that fermentation has taken place. The contents of the tube are then filtered, and Fehling's test is again performed both with the filtrate and, for the sake of comparison, with the reserved and unfermented portion of urine. If fermentation has taken place and if the reducing substance has been removed by the process, glucose was present in the urine. Lactose, uric acid, creatinine, glycuronic acid, and the products of drugs are not appreciably affected by yeast. The Doremus tube may be replaced by a test tube com- pletely filled and carefully inverted, with its mouth immersed in a beaker of water. (7) The nature of the deposit. — If the sample examined is that from a 24 hours specimen a deposit of some kind will probably have settled down. If, however, the urine is perfectly clear, and no abnormal substances have so far been detected in it, further examination is unnecessary as a routine. If any abnormal substance, particularly albumin, has been detected, or there is any reason on clinical grounds to expect renal disturbance, a deposit should be examined for. The deposit should be examined both by the naked eye and with the help of the microscope. The naked-eye examination of a deposit is useful, because it allows the description of the ROUTINE EXAMINATION— VARIATIONS. 219 amount of any abnormal substance. For example, the centri- fuged deposit of a clear urine may contain leucocytes, and in the case of urine with a bulky deposit in the specimen glass the microscope may similarly show leucocytes. The difference is one of degree only, but is a guide to the severity of the inflammatory process. The diagnosis of a urinary deposit by naked-eye examination alone is utterly fallacious, and can only be relied upon by those who have never troubled to prove the frequency of error by exact investigation. It is true that the nature of a deposit can be correctly guessed in a propor- tion of cases from the appearance alone, and that an abnormal deposit will often give an obvious clue to the examination required, but further investigation by chemical means, or more particularly by the aid of the microscope, should never be omitted. The microscopical examination of a urinary deposit is readily and easily made. If the deposit is bulky a small portion should be removed from the bottom of the specimen glass by means of a pipette and transferred to the centre of a slide. A cover-glass is carefully let down on the drop of deposit, which should be sufficient in amount to spread across the cover-slip without the inclusion of air bubbles, and not so large that the cover-slip floats about on the surface of the slide. No staining process is required. The slide is examined with a f-inch and with a ^-inch objective. If the deposit is slight in amount or has not settled to the bottom of the glass the supernatant fluid should be poured off and the lower portion of the urine shaken up and then centrifuged at a moderate speed. Centrifuge tubes should always be washed out with water before use. After centrifuging pour off the supernatant urine by simply inverting the tube, and then shake out the last drop upon the slide. Even if no naked-eye deposit appears after centrifuging, the last drop should be examined under the microscope, since casts, pus cells, red cells, or other bodies may still be present. The above description applies simply to the ordinary routine examination in all classes of disease. In cases where the urinary excretion is normal, that is to say in the great majority of cases, the entire examination takes less than 10 minutes. An account follows of the variations from the normal which may be detected by the routine examination, 220 CLINICAL PATHOLOGY. their meaning and an account of such further investigation as may be required. Variations in the Amount of Urine passed. As already mentioned, considerable variations in the amount of urine take place under purely physiological conditions. Such variations are largely eliminated in the case of patients at rest in bed on a fixed diet, and they will not be considered here. The urine is largely increased in diabetes and very largely in the condition known as diabetes insipidus, a disease associated with thirst and the passage of large quantities of pale sugar- free urine of a very low specific gravity. A considerable increase in the urine is present in chronic interstitial nephritis with cardiac hypertrophy. An increase is also commonly found in amyloid disease, and may occur as a purely functional condition in hysterical subjects. An increase may further be artificially produced by diuretic drugs. A decrease in the amount of urine passed occurs in acute nephritis, when the output may be reduced to a few ounces or less in the 24 hours. A similar but as a rule less pronounced decrease takes place in cardiac failure with dilatation of the right ventricle. Such a decreased output, due to cardiac failure, often occurs in the last stages of the chronic interstitial form of nephritis. Some decrease in the urine is practically always present in all febrile states as well as in conditions associated with an increased loss of water by other channels, as in diarrhoea or vomiting. Partial suppression of urine may also be induced by certain poisons, such as turpentine or cantha- rides. Decrease in the urinary output has to be distinguished from retention of the urine, such as commonly takes place in stricture of the urethra or in obstruction due to an enlarge- ment of the prostate. Pietention of the urine may also occur in certain cerebral conditions, such as meningitis. Complete blocking of both ureters by calculi leads to entire suppression of urine or " calculous anuria." Variations in the Appearance. The more obvious variations may be those of colour or those of transparency. (a) Variations of colour.— The urine may be paler than KOUTINE EXAMINATION— VARIATIONS. 221 normal, and specimens of this kind are usually those of a low specific gravity associated with polyuria. Diabetic urine, however, is nearly always pale and of a high specitic gravity. It has a somewhat characteristic limpid appearance. Concentrated urines are darker than normal, and such urines, if acid, frequently deposit a quantity of amorphous urates. The most important urines of dark colour are those associated with the presence of blood or bile. Blood in the urine may be in such amount as to colour the urine bright red, and specimens are not infrequently met with which appear to contain rather more blood than urine. Blood in lesser amount darkens the urine and gives it a peculiar " smoky" " appearance. Blood in small traces may be present without any alteration in the appearance of the urine. During menstruation the examination of the urine should be avoided owing to its frequent contamination by blood. Blood may be found in the urine in numerous conditions. The blood may come from any part of the urinary tract — from a chancre of the penis, from the urethra after injury, from the prostatic plexus of veins in enlargement of the prostate, from the bladder in cases of vesical calculus, tuber- culosis or new growths, from the ureter during the passage of a stone or from the kidneys in cases of nephritis both in its acute form and less commonly in its chronic form, in eclampsia, in calculus, tuberculosis, neoplasm, or in paroxysmal hematuria. Among less common causes of hematuria are hydronephrosis, congenital cystic kidneys, kinking of the kidney over an abnormal renal artery, injuries to the kidney, and acute coli infections of the urinary tract. The exact site of the haemorrhage can usually be determined by an exami- nation of the history and physical signs of the patient together with a further examination of the urine, and in cases of diffi- culty by a cystoscopic examination. A further clue to the origin of the blood may be obtained by observing the relation of the blood to the urine in a sample as it is passed. If the majority of the blood is passed with the first portion of urine the haemorrhage is probably from the urethra or prostate. If the majority is passed at the end of micturi- tion, it probably comes from the bladder. If blood and urine 222 CLINICAL PATHOLOGY. are intimately mixed throughout, the bleeding may be of renal origin. In addition to the presence of actual blood in the urine, the same coloration may be due to haemoglobin only. The haemoglobin may be either oxyhaenioglobin or methaeruoglobin. Haemoglobinuria occurs in black-water fever, and recurrent paroxysmal attacks of haemoglobinuria may take place in apparently healthy individuals in this country, particularly after exposure to cold. The following tests for blood and haemoglobin should be performed : — The guiac test. — Boil 1 inch of urine in a test tube. Allow to cool. Add 2 or 3 drops of tincture of guiacum. A white pre- cipitate forms. Pour gently down the side of the tube 1 inch of ozonic ether. Allow to stand. A blue ring appears at the junction of urine and ether if blood is present. This test is a fairly delicate one for the presence of blood or haemoglobin. It is given also by the urine of a patient who is taking iodides. In the presence of pus in any quantity a greenish blue ring is given. Since the test is not infallible, and may be given by either blood or haemoglobin, and may not be given in the presence of minute quantities of blood, one or both of the following tests should always be performed in addition. The microscopic test. — Centrifuge the urine if necessary. Place a drop of the deposit on a slide and cover with a cover-glass. Examine under a ^-inch objective with the diaphragm partly closed. Eed blood corpuscles should be recognised with certainty, even if very few are present. Should the student be in any doubt as to whether the corpuscles seen are red cells or not he should make a dried film preparation and stain with Leishman's stain. The red cells in such a preparation are commonly distorted, but retain their characteristic staining reaction. The microscopic test is the only infallible one for the presence of blood, and is very easily performed. In cases of pure hemoglobinuria no red cells may be found, but the guiac KOUTINE EXAMINATION— VARIATIONS. 223 test will be positive. An occasional red cell may be found in a centrifuged deposit when the guiac test is negative. The spectroscopic test.— With a positive guiac reaction and a proportional number of red cells in the urinary deposit it is unnecessary to confirm the result by the spectroscope. In cases of supposed hemoglobinuria the spectrum of the urine must always be examined. A small direct-vision spectroscope can be used, and the urine if very deeply tinged should be diluted with water. If the substance present is methemoglobin a band is seen in the red between C and D in addition to the two bands of oxyhemoglobin (see page 93). Bile in the urine is another common cause of alteration of colour. Bile is present in the urine in every variety of jaundice with the exception of congenital family cholemia. Bile salts are usually but not always present as well as the pigment. Bile in the urine is, as a rule, fairly obvious to the naked eye, and if a test tube containing the urine is shaken a greenish-yellow froth appears at the top. The following tests for bile should be performed : — Gmelin's test. — Filter several cubic centimetres of urine through a clean filter paper. When all the urine has passed through dip a glass rod in yellow nitric acid and then on the filter paper. A spreading ring of colours appears round the area touched by the acid. The colours are from within outwards yellow, red, violet and green. The test is not positive unless the green colour is certainly seen. Should the bile be present in small amount a positiva reaction may be obtained if a considerable bulk of urine is filtered two or three times through the paper. Iodine test. — Take 2 c.c. of urine in a test tube. Carefully pour on the top of the urine 2 c.c. of tincture of iodine. A green ring appears at the junction of the liquids. Both the above tests are for the presence of bile pigments. A simple and fairly delicate test for the presence of bile acids is the following : — Hay's test. — Pour a few cubic centimetres of urine into a watch glass. Gently sprinkle on to the surface of the urine a little flowers of sulphur. 224 CLINICAL PATHOLOGY. In the case of normal urine the sulphur floats. "When bile salts are present the surface tension of the urine is lowered and the sulphur sinks. For clinical purposes Hay's test is sufficiently accurate. Pettenkofer's test, using cane sugar and sulphuric acid as the reagents, is not very satisfactory with urine as the test fluid. Urobilin may cause a darkening of the colour of the urine. It is not present in any quantity in normal freshly-voided urine. It may be present in excess with fever, in pernicious anaemia, in congenital family cholaernia, and in other diseases. If urobilin is present in considerable excess the absorption band of the spectrum, which appears at the junction of the green and blue, can be seen in direct examination of the fresh urine with the direct-vision spectroscope. The normal urine shows no bands other than a general darkening of the violet end of the spectrum. Urobilin present in lesser amount can be demonstrated as follows : — Saturate the urine with ammonium chloride to precipitate the urates. Filter. Saturate the filtrate with ammonium sulphate. Add a drop of sulphuric acid. Shake with a mixture of 2 parts ether and 1 part chloroform. Pipette off the ether-chloroform layer and examine for the urobilin spectrum. Among other and rarer causes of dark urines are alkapton, hsematoporphyrin, melanin and carbolic acid. Alkaptonuria is a rare congenital abnormality which persists for life and is attended by no symptoms. The urine is of normal colour when passed and becomes of a dark, almost black, colour on standing. The urine readily reduces Fehling's solution and darkens Nylander's solution, but without the production of a precipitate. The urine is not fermented by yeast. Ferric chloride added to the urine drop by drop produces a striking and momentary deep blue colour. Haematoporphyrinuria. — Haematoporphyrin is present in the normal urine, but only in very small amount. In the condi- tion known as haematoporphyrinuria the urine is obviously dark and may be of a deep port wine colour. The condition arises in EOUTINE EXAMINATION— VAEIATIONS. 225 patients who have been taking sulphonal in excess, and from this cause has occurred in almost epidemic form in lunatic asylums. Unless the condition is recognised at once and the sulphonal stopped death is likely to result. A urine contain- ing haematoporphyrin in excess does not reduce Fehling's solution and does not give the guiac reaction. If the pigment is present in great excess direct examination of the urine shows the spectrum of alkaline haBmatoporphyrin. The spectrum has 4 bands — one between C and D, one at D, one just on the red side of E, and a broad band between the green and the blue. If the spectrum is not clearly obtained, extract the pigment in the following manner : — To 100 c.c. of urine add 20 c.c. of dilute soda. Allow the precipitate of pigment and earthy phosphates to settle. Decant the supernatant fluid. Transfer precipitate to a filter paper and wash with water. Extract precipitate with alcohol acidified with hydrochloric acid. Examine extract for spectrum of acid haBmatoporphyrin. The spectrum of acid haBmatoporphyrin has 2 bands — a narrow band in the orange near the D line, and a broad band in the yellow. Melaninuria is a rare condition which may be found in patients with melanotic sarcomata, and occasionally with non- malignant pigmented papillomata of the skin. The urine as a rule is dark when passed, but becomes considerably darker on exposure to the air. The addition of a few drops of nitric acid causes immediate blackening of the urine. No reduction of Fehling's solution is produced and no reaction with the guiac test. There is no spectrum. A dark urine which fulfils these conditions in all probability contains melanin, but there is no simple and satisfactory direct test for this substance. Carboluria may occur in any variety of carbolic acid poisoning. With susceptible patients the absorption of a very small quantity of carbolic acid, such as takes place after the application of a " carbolic cap " to the head, may lead to the production of carboluria. The patient may show little or no symptoms. The urine becomes considerably darker on standing or after the addition of nitric acid, and in well- marked cases is of a distinctive greenish-blue colour. This p. 15 226 CLINICAL PATHOLOGY. colour and the evidence of exposure to carbolic acid helps to distinguish the urine from that of melaninuria. The pigments produced in the urine by carbolic poisoning are chemically similar to those of alkaptonuria. Fehling's solution may be reduced. Certain urines of striking appearance sometimes met with follow the absorption of harmless or comparatively harmless substances. Methylene blue taken by the mouth, or given hypo- dermically, leads to the production of a remarkable bright green (not blue) urine. There is at the present a considerable epidemic of these urines owing to the activity of the quack medicine-monger who sells methylene blue " kidney pills " to purge the urine of the credulous. The coloration of the urine may persist for several days after taking the drug and is quite harmless. The mental shock to a neurotic patient on passing bright green water may, however, be considerable. Eosin has been extensively used as a coloring matter for cheap sweets, and a curiously dichroic urine may result. The tint of the urine is exactly that of a dilute solution of eosin and is easily recognised. Among other substances santonin may produce a greenish urine, and rhubarb or senna a reddish-brown coloration. Resorcin used as an ointment may lead to a striking greenish coloration of the urine. (b) Variations of transparency. — Among the causes of turbidity of the urine are the following. Urates are a frequent cause of turbidity of the urine, particularly on standing, the urates separating out as the urine cools. The separation of urates is the rule in acid, concentrated urines, whatever may be the cause of the con- centration. Turbidity due to urates is readily recognised, since on warming the urine it becomes clear. Phosphates may produce a turbidity in neutral or alkaline urines. On acidifying the urine with dilute acetic acid the turbidity goes. Bacteria commonly produce a turbidity in urines which have been standing exposed to the air ; their presence in such urines is of no significance. In pathological conditions the urine may be turbid from the presence of bacteria at the time of passage The turbidity has the exact appearance of that ROUTINE EXAMINATION— VARIATIONS. 227 produced in a broth culture tube by the growth of organisms. It does not alter on warming or on the addition of an acid, nor is it removed by nitration. It is little affected by centrifuging, except at a very high speed. A drop of the urine, or pre- ferably of the centrifuged deposit, examined under the microscope, is seen to be swarming with bacilli or cocci. The examination of urines for bacteria will be described subsequently. Fat in the urine or lipuria is an extremely rare condition. The fat may be present in such amount that the urine looks like milk, and on standing a creamy layer rises to the top. Lipuria of this degree is usually due to the presence of filarise, the embryos of which should be looked for in the urine and in the blood. In this variety of lipuria, which is commonly known as chyluria, because the fat escapes from a ruptured lymph vessel, the fat may appear as amorphous granules under the microscope. Lipuria, in appreciable degree, may very rarely accompany diabetes, pregnancy, and phosphorus poisoning. It has been described in other conditions. The fat in such cases may be in the form of oil droplets. (A few oil drops in the urine are almost the rule in catheter specimens and come from the lubricating fluid used for the catheter.) In the case of milky urines it is always advisable to make sure that no fat-containing substance, such as milk, has been added to the urine by the patient. In cases of doubt a catheter must be passed. Turbidity of the urine due to fat can be removed by extraction with ether, and the presence of fat in the ether extract should be verified. 15—2 CHAPTEE XVII. variations in acidity, and acidosis variations in specific gravity — urea — proteids carbohydrates. Variations in Acidity. Variations in the ammonia nitrogen (acidosis). — The normal acid reaction to litmus paper may be converted into an alkaline one by the growth of staphylococci in the urine on standing. The change in reaction results from the conversion of urea into ammonium carbonate. The normal urine also may be faintly alkaline at the height of digestion, and may be rendered alkaline by certain drugs such as potassium citrate. The habitual passage of an alkaline urine is abnormal, and in the majority of cases is due to the growth of organisms in the urinary tract. The organisms found in an alkaline urine are usually staphylococci, or the bacillus proteus, and the site of infection is more commonly the bladder than the kidney. Infections with the bacillus coli are almost always accompanied by an acid urine. In order to measure the acidity of the urine phenol-phthalein may be used as an indicator, and sufficient deci-normal soda run into a measured quantity of urine until neutralisation is effected. The estimation of the acidity is the first stage in the estimation of the ammonia nitrogen by an accurate and simple method. The procedure is as follows : — Measure out 25 c.c. of urine into a beaker and dilute with about double that volume of distilled water. Add 2 or 3 drops of phenol-phthalein. N Run in r-pr NaOH from a burette until a faint permanent pink colour is produced. Note the number of cubic centimetres of NaOH used. Measure about 10 c.c. of commercial (40 per cent.) formalin into a second beaker. Add phenol-phthalein. N Neutralise exactly with — NaOH. VAKIATIONS IN ACIDITY AND ACIDOSIS, ETC. 229 Add the neutral formalin to the neutral urine. The pink colour disappears. . N Run m r^r NaOH until the pink colour returns. Note the number of cubic centimetres of NaOH used. N The result is calculated in terms of j^. NaOH for the acidity. That is to say, the acidity of the urine is given as the number of N cubic centimetres of r-x NaOH required to neutralise 100 c.c. of urine to phenol-phthalein. Thus if 10 c.c. of soda were used in the first titration to neutralise 25 c.c. of urine, the acidity of the urine is — X 100 = 40. The ammonia result should be expressed in grammes of ammonia per 24 hours. The number of cubic centimetres of soda used in the second titration of the urine is the equivalent of the number of cubic centimetres of ammonia present in the 25 c.c. of urine. Supposing the number of cubic centimetres of soda used in the second titration to have been 10, then 10 c.c. ^NaOH = 10 c.c. ^ NH 8 = 10 X -0017 gramme NH 3 . Therefore the ammonia passed in the 24 hours = 10 X # 0017 24 hours' urine in cubic centimetres X " ~^5~~ The reaction depends upon the combination of the ammonium salts with formaldehyde to form urotropin, and the consequent liberation of the acids previously combined with ammonia. The method is an extremely accurate one, but yields results higher than those obtained by the older and more complicated method of Folin, since the amino groups of the amino-acids react with the formaldehyde and are included in the ammonia total. Variations in the acidity and ammonia nitrogen of the urine are of considerable clinical significance, and may be very marked in any of the conditions which may be associated with " acidosis." The most important pathological states of which acidosis may be a feature are starvation, eclampsia, diabetes, and chloroform poisoning. A relative increase in the ammonia nitrogen is the rule in eclampsia and in the condition known as delayed chloroform poisoning. The onset of coma in diabetes is similarly accompanied by the appearance of 230 CLINICAL PATHOLOGY. abnormal acids in the urine. The more important acids which may be found in the urine in such cases are diacetic and B-oxybutyric acids. The excess of ammonia nitrogen in the urine is commonly explained as following the excessive production of ammonia needed to combine with the abnormal acids. The presence of acidosis can be proved by an examination of the urine, and its onset can frequently be detected before the clinical state is manifest. The acidity of the urine as estimated by a simple titration with soda and phenol-phthalein is too variable to be of much clinical assistance. The estimation of the ammonia output alone has much less significance than a comparison between the percentage of ammonia nitrogen and the percentage of the total nitrogen in the urine. Under normal conditions the ammonia nitrogen forms about 3 per cent, of the total nitrogen, and in acidosis considerably more. In order to estimate the total nitrogen it is necessary to use Kjeldahl's method. Kjeldahl's method is not given here because it is too difficult for ordinary clinical use in that it requires a somewhat cumbrous apparatus with which the operator must be thoroughly conversant. A reliable guide to the extent of the acidosis can be obtained from a comparison of the ammonia and the urea nitrogen. The urea is readily estimated by one of the methods to be described shortly. The calculation is made as follows : — The molecular weight of ammonia or NH 3 being 17, the nitrogen fraction of ammonia is ^. The molecular weight of urea or CO/,. 2 being 60, the 28 7 nitrogen fraction is -ttc or ^. The amount of ammonia in grammes in a given sample is estimated by the formalin method, and fourteen-seventeenths of this represents the ammonia nitrogen. The urea is similarly calculated in grammes in the same sample of urine, and seven- fifteenths of this weight is nitrogen. Under normal conditions the ammonia nitrogen is about one-twentieth of the urea nitrogen, and in conditions asso- ciated with acidosis it may rise to one-fourth. A rise in the VARIATIONS IN ACIDITY AND ACIDOSIS, ETC. 231 ammonia nitrogen to one-tenth of the urea nitrogen is definite evidence of acidosis. Vaeiations in Specific Geavity. Variations in the density of the urine observed in catheter specimens or single samples are of little significance. The specific gravity must be taken from the sample of a 24 hours specimen of urine. The specific gravity of the urine is unaffected by substances in suspension, and very little affected by any albumin that may be present. It is dependent upon the salts in solution, and varies directly with the amount of urea present. The specific gravity is raised in practically all conditions associated with a diminished urinary output, such as occurs during violent exercise, with pyrexia, in acute nephritis and in cardiac failure. An increased urinary output with a urine of high specific gravity is characteristic of diabetes. In this disease the specific gravity is commonly from 1,035 to 1,045, and may rise higher. The specific gravity is generally lowered when the urinary output is increased, as in chronic interstitial nephritis and amyloid disease. The passage of a large quantity of urine of a specific gravity constantly at or below 1,010, and containing a trace of albumin, is characteristic of chronic interstitial nephritis. Variations in the specific gravity must always be considered in conjunction with the amount of urine passed and the percentage of urea present, and repeated observations must be made before deductions of any value can be drawn. The Urea. The amount of urea excreted by the kidneys varies within fairly wide limits, and the variations naturally depend to a large extent upon the amount of nitrogen in the food and upon the activity of the individual. A patient at rest in bed on a milk diet will excrete less urea than a similar patient at work on an ordinary mixed diet. The majority of urea estimations performed on the urine of patients in hospital wards in the usual perfunctory manner are scarcely worth the 232 CLINICAL PATHOLOGY. sodium hypobrornite expended on them, yet if due allowance be made for the condition of the patient valuable clinical information can be obtained from urea estimations in suitable cases. The exact estimation of the urea in a sample of urine is a matter of some difficulty, but the results obtained by one of the following methods are sufficiently accurate for all clinical purposes. Under normal conditions the urine contains about 2 per cent, of urea, and the daily excretion is from 30 to 40 grammes. Estimations of urea should always be made if possible from a measured 24 hours specimen of urine. The deductions to be drawn from these estimations will be considered later. The methods described depend upon the decomposition of urea by alkaline hypobroniite into nitrogen and carbon dioxide. The carbon dioxide is absorbed by the alkali, and the nitrogen is collected and measured. The whole of the nitrogen, however, is not evolved by this method, and whereas 1 gramme of urea should yield 373 c.c. of nitrogen, only 354 c.c. are actually evolved, or about 92 per cent. When sugar is present in the urine a greater yield of the nitrogen, or about 99 per cent., is obtained. The scale of the ureometers in common use is corrected for the yield of normal urine, consequently in cases of diabetes the results are too high, and should be multiplied , 92 by 99' Before making the estimation the urine should be tested for albumin, and if more than a trace is present a measured quantity of urine is taken, acidified with acetic acid, raised to the boiling point, cooled, filtered, and made up to the original volume with distilled water. If much albumin is present the urine must be diluted before boiling. The estimation should be carried out by one of the two forms of apparatus described below : — (1) Gerrard's ureometer. — The apparatus consists of a graduated glass cylinder, the open top of which is fitted with a rubber cork, through which passes a glass T-piece. One limb of the T-piece has a short piece of rubber tubing attached, which can be closed by a clip. The other limb is connected with a long rubber tube, which passes by means of a glass junction through a rubber cork, filling the mouth of a VARIATIONS IN ACIDITY AND ACIDOSIS, ETC. 233 glass bottle (a). The glass bottle contains a small tube marked to contain 5 c.c. At the lower end of the glass cylinder is an opening which connects by a piece of rubber tubing to the bottom of a wide glass tube (c). The glass tube is attached to the cylinder by a metal fitting, and can be pushed up and down upon the cylinder. The hypobromite solution used must be freshly made, other- wise it will decom- pose and give off gas. The bromine and the caustic soda should therefore be kept separate and mixed as required. Bromine is supplied in glass cap- sules holding 2'5 c.c. To mix, place 25 c.c. of 40 per cent, caustic soda in a strong glass bottle fitted with a glass stopper. Place one of the bromine capsules in the bottle. Fit the stopper tightly. Shake the bottle smartly so as to crack the bromine capsule. Do not use weak caustic soda, otherwise the irritating bromine vapour will escape. To make the estimation : — Place the 25 c.c. of hypobromite solution in the glass bottle. Measure 5 c.c. of the urine into the small tube. Wipe the outside of the tube and stand it carefully in the bottle so that the urine does not mix with the hypobromite. Do not cork the bottle. Pour water into the wide tube. Fig. 16. — Gerrard's Ureometer. 284 CLINICAL PATHOLOGY. Now cork the bottle tightly. Open the clip on the T-piece. Eaise or depress the wide tube until the water in the graduated cylinder is at the zero mark, and level with the water in the tube. Close the clip on the T-piece. Tilt the bottle so that the urine mixes with the hypobromite. Fig. 17. — Mayhew's Ureoraeter. Wait for 15 minutes, giving the bottle an occasional shake after the first effervescence has subsided. Lower the tube until the water in tube and cylinder are again level. Bead off the amount of nitrogen in the cylinder. The scale is graduated in percentages of urea. The apparatus must be tested from time to time to make sure that there is no leakage in any of the rubber connections or their attachments. The same hypobromite may be used for a second estimation if required. (2) Mayhew's ureometer. — This simple form of apparatus VABIATIONS IN ACIDITY AND ACIDOSIS, ETC. 235 is very convenient for clinical purposes. The apparatus con- sists of a glass cylinder composed of one long straight central part, which is graduated, and two shorter terminal parts bent at an angle to the graduated part. The short limb nearest to the zero mark has a wide mouth provided with a well-fitting rubber cork. A small glass tube graduated to contain 1 c.c. of urine completes the apparatus. To make the estimation : — Prepare hypobromite solution as for Gerrard's apparatus. Pour hypobromite into the wide-mouthed opening in such amount that when the long limb is horizontal the fluid rises about halfway in each of the short limbs. Fit in the cork and hold the long limb vertical with the stoppered end uppermost. The hypobromite level should be at the zero mark in the long limb, and there should be several cubic centimetres in the upper shorter limb, with a consider- able space between the two volumes of fluid. Place the apparatus against a rest with the long limb horizontal and remove the cork. Fill the small glass tube with urine to the 1 c.c. mark. Wipe the outside of the tube. Holding the apparatus horizontal in the left hand, slip the glass tube into the wide-mouthed limb. The foot of the tube should be in the hypobromite, but the mouth of the tube above the level of the solution, so that urine and solution do not mix. Fit in the rubber cork. Alter the long limb in one movement from the horizontal to the vertical position. Hang up the apparatus and leave for 15 minutes. The urine mixes with the hypobromite. The nitrogen is liberated into the upper bend of the apparatus and forces the solution down the graduated limb. Head the level of the fluid in the graduated limb. The scale is graduated in percentages of urea as well as (in most forms of the apparatus) in grains per ounce. The calculation should always be worked out for the urea output of the 24 hours. The statement that a certain sample of urine contains a certain percentage of urea conveys very little information. The variations in urea content of different samples of urine from the same patient over a period of 24 236 CLINICAL PATHOLOGY. hours are considerable. Consequently a mixed sample of the urine must be taken, and the amount of the urine must be measured. Also the diet of the patient must so far as practic- able be taken into consideration. Among the conditions in which the urea output is increased are fevers and diabetes. More important are the conditions in which the urea output is diminished. These include both medical and surgical affec- tions of the kidney. The factors of the diet and the general circumstances of the patient having been taken into account, the urea output may be taken as a rough but useful clinical guide to the activity of the kidneys. Slight variations in the amount of urea are of little moment, and in all cases a series of observations should be made without alteration of diet. In both acute and chronic nephritis the urea output is diminished. In acute nephritis the diminution is a well-marked feature, and a rise in the urea excretion is evidence of improvement. In surgical affections of the urinary tract urea estimations are frequently of value. In cases of prostatic enlarge- ments with back pressure some estimation should always be made of the amount of normal renal tissue available to the patient. The general clinical condition gives a fair idea of the state of the kidneys, but the clinical estimate should always be aided by examination of the urine. It may be stated roughly that it is dangerous to operate upon such patients when the specific gravity is constantly less than 1,008 and the urea less than 0*6 per cent, of an average measurement. When the specific gravity is above 1,010 and the urea percentage about l'O or over, operation should be successful. It is occasionally necessary to estimate the urea in a ureteric catheter specimen, and particularly when informa- tion is required as to which kidney is diseased, or as to whether the diseased kidney is functionating. The urea percentages in such specimens should be interpreted with caution, since slight differences on the two sides are found in health, but gross differences may be of great assistance in diagnosis. Attempts have been made to provide a more exact clinical method by which the excretory activity of the kidneys can be estimated. A function of the kidneys is to remove waste products from the body, and if they fail to do so these VARIATIONS IN ACIDITY AND ACIDOSIS, ETC. 237 products accumulate in the blood. The most reliable method therefore of investigation involves a comparison of the blood and the urine. Under normal conditions the salts in solution in the urine are double those in solution in the blood. Under abnormal conditions the ratio may be actually reversed. The ratio between the inorganic salts in solution in the urine and those in the blood is known as the haemorenal index. The estimation of the salts in the blood and urine can be performed in one of two ways. A direct analysis is not practi- cable, since the amount of blood available is very small. One method consists in taking the freezing points of the serum and the urine and comparing them. The method is known as cryoscopy, and depends upon the fact that the lowering of the freezing point of a liquid varies directly with the amount of salts in solution. The method is a long and tiresome one to perform. In the other method the electrical conductivity of a volume of the serum is measured by a modification of the Wheatstone bridge. The serum is replaced by an equal volume of a sample of urine, passed at a period corresponding to the time at which the blood was drawn, and the conductivity of the urine is measured. The ratio of the conductivity of the serum to that of the urine is the ratio of the inorganic salts in solution in these fluids. The actual measurement of the conductivity is simple and takes a few 7 minutes only. A special and somewhat expensive apparatus is required, and a fairly extensive trial has not convinced me that the test provides any real clinical information such as cannot be obtained by a careful examination of the patient and his urine by ordinary methods. Peoteids in the Urine. The routine tests for the presence of albumin in the urine have already been given. When albumin is present the amount should always be stated, and for all practical purposes it is sufficient in nearly all cases to measure the amount by the following rough scheme. If a cloud only appears, such as would cause no appreciable bulk of precipitate at the bottom of the test tube, note that a faint trace, a trace, or a heavy cloud of albumin is present according to the opacity which is produced after boiling and acidifying. 238 CLINICAL PATHOLOGY. If a precipitate is formed in bulk, boil the whole contents of the test-tube and allow it to stand. When the precipitate has all collected at the bottom, hold an ordinary tape measure against the test tube, and read the level of the precipitate and of the urine. Express the depth of the precipi- tate as a fraction of the depth of the column of urine as one- sixth, one-fourth, one-third, etc. Should a slightly more accurate method be required, Esbach's albuminometer can be used. This instrument does not measure traces of albumin, and if very much albumin is present it is necessary to dilute the urine and to allow for the dilution after taking the reading. To use the apparatus : — Fill the tube with the urine (which should be acidified if necessary) up to the mark U. Pour in the reagent up to the mark E. Cork the tube and mix by inverting several times. Allow to stand for 24 hours. Bead the level of the precipitate. The scale gives the grammes of proteid present in a litre of urine. The percentage is consequently obtained by dividing by 10. The reagent has the following composition : — Picric acid .... 10 grammes. Citric acid . . . . 20 ,, Water 1 litre. The method is not remarkably accurate, since the space occupied by the precipitate continues to vary with the time the tube is allowed to stand, and all albuminous precipitates do not appear to take the same time to settle. The percentage of proteids, however, is given with sufficient accuracy for clinical purposes, and the exact estimation of proteids in the urine is a tedious process and conveys no further information of practical value. The nature of coagulable proteids in the urine is of no particular clinical significance, and they consist as a rule mainly of serum albumin with a lesser amount of serum globulin. The amounts of serum albumin and serum globulin can be estimated as follows : — Bender about 200 c.c. of urine slightly alkaline with ammonia. Filter. VARIATIONS IN ACIDITY AND ACIDOSIS, ETC. 239 To 100 c.c. of the filtrate add solid ammonium sulphate to saturation. Allow to stand overnight. The total proteids are precipitated. Filter off the precipitate through a weighed ash-free paper. Dry and weigh. Incinerate and weigh the ash. Deduct the weight of the ash ; the difference gives the total proteids. To another 100 c.c. of the filtered urine add 100 c.c. of a saturated solution of ammonium sulphate. Proceed as before. The precipitate contains the globulins, and the difference between the two precipitates may be reckoned as albumin. The following are among the more important conditions in which albumin occurs in the urine : — Albumin may occur after violent exercise, severe exposure, and after excessive eating or drinking. In the previously normal individual the albuminuria which may be induced by such means rapidly disappears. Fever, particularly if considerable or prolonged, is commonly associated with a trace of albumin. In chronic interstitial nephritis albumin is present only as a trace so long as cardiac compensa- tion lasts. In diabetes there is commonly a trace of albumin. Albumin in large amount is present in acute nephritis of all varieties, including the nephritis of eclampsia and chronic parenchymatous nephritis, also in amyloid disease of the kidney and in cardiac failure with partial suppression of urine. In cases of albuminuria due to any form of renal disease casts are nearly always present in addition. Albuminuria may also occur and in considerable degree without any known structural alteration in the kidneys or heart. Such cases are known as functional or postural albu- minuria, and are not uncommon in young people. Such albuminuria tends to disappear when the patient is at rest and to reappear on taking exercise or even on assuming the erect posture. Casts are absent and the arteries are not affected, nor do these cases commonly appear to progress to 240 CLINICAL PATHOLOGY. any of the recognised forms of nephritis. Albuminuria with- out renal or vascular degeneration may likewise follow one of the infectious fevers. The occurrence of blood or pus in the urine is always associated with the presence of albumin, and it is frequently of importance to determine whether the amount of albumin is such as could be accounted for by the quantity of pus or blood or is in excess of it. Albumin present in greater amount than can be accounted for by the amount of pus coming from a probable focus in the bladder, for example, would point to involvement of the kidneys as well. It may be taken as a rough guide that a very considerable amount of pus in the urine produces little more than a trace of albumin. The presence of blood leads to a higher degree of albuminuria than the same amount of pus, and hematuria occurring with a contracted granular kidney obscures the albumin due to the nephritis. In such cases the presence of granular casts would indicate renal involvement. The hEematuria which accom- panies acute nephritis is usually associated with a degree of albuminuria obviously much in excess of the amount of blood present. The exact significance to be attached to the presence of albumin in the urine is of great importance in life insurance, and every case in which even a trace is discovered calls for a very complete examination, not only of the urine but of the patient also. There is no doubt that transient albuminuria may occur without any serious involvement of the kidney, and in exceptional cases a considerable percentage of albumin may be passed over long periods without any apparent detriment to the individual. Proteoses may be present in fevers, in long-standing cases of suppuration, and rarely, if ever, in nephritis ; they may be tested for as follows : — Saturate the urine with ammonium sulphate. Heat to coagulate the proteins which may be present. Filter and extract the precipitate with alcohol to remove the urobilin which gives the biuret reaction. Extract precipitate with boiling water, which dissolves the proteoses. Test the solution by the biuret reaction and with Millon's reagent. VARIATIONS IN ACIDITY AND ACIDOSIS, ETC. 241 The biuret reaction is performed as follows : — To the aqueous solution add caustic soda and 2 drops of a 1 per cent, solution of copper sulphate. A pink colour is produced. Millon's reagent consists of mercury dissolved in concen- trated nitric acid. Add the reagent to the solution and heat. A deep red coloration is produced. The examination of the urine for proteoses is only occasion- ally called for. There is, however, an extremely rare disease of the bone marrow in which almost the entire marrow is transformed into a sarcoma-like substance, and in these cases an abundant proteosuria is present and is pathognomonic of the disease. The exact nature of the protein, which is called the Bence-Jones protein, is not known, but it is readily recognised in the urine by the following simple test : — Filter the urine into a test tube. Place the test tube in a water bath at about 45° C. Slowly raise the temperature of the water. When the temperature rises to between 50° and 60° C. the urine becomes turbid from the coagulated proteid. On raising the temperature still further the turbidity lessens and finally disappears entirely. On cooling the urine again the precipitate returns. Mucin is not infrequently present in the urine in associa- tion with pus, and in connection with numerous crystalline deposits. The presence of mucin out of solution is of no particular significance except that it may be mistaken on naked-eye examination for pus. When the mucin is in solution it may be detected by dilut- ing the urine with an equal volume of water and adding a few drops of acetic acid, when the mucin is precipitated as a white cloud insoluble in excess of acid. Carbohydrates in the Urine. Glucose is the most important carbohydrate which may be present in the urine, and tests for its detection form part of the routine examination of all specimens. The qualitative tests for glucose have already been given. Should glucose be present its amount must always be estimated. There are p. 16 242 CLINICAL PATHOLOGY. numerous quantitative methods in common use, and the following can be recommended as simple and accurate. It is known as Gerrard's method. Gerrard's method depends upon the fact that the colourless (or almost colourless) double cyanide of potash and copper dissolves the coloured cuprous oxide precipitate as it is formed in Fehling's solution. The estimation is performed as follows : — Dilute the urine according to the intensity of the Fehling's reaction obtained. Thus, if an abundant precipitate resulted on the addition of a few drops of boiling urine, dilute the urine 10 times ; in cases of moderate glycosuria a dilution of 5 times is sufficient. The dilution to be aimed at is one in which from 10 to 15 c.c. of the diluted urine reduce 10 c.c. of Fehling's solution. If the first dilution is unsuitable another can be made. All dilutions must be measured accurately and can be made with tap water. Einse out a burette with a few cubic centimetres of the diluted urine before filling. Measure out carefully with a delivery pipette 10 c.c. of Fehling's solution and expel into a porcelain evaporating dish. Add about BO c.c. of Gerrard's solution. Bring to the boil over the flame. Eun in the diluted urine from the burette. See that the mixture is boiling all the time and keep it stirred with a glass rod. The reaction is complete when the last trace of blue colour has gone from the mixture. The colour partially returns on cooling. The titration should be performed as rapidly as possible. The calculation is made as follows : — 10 c.c. of Fehling's solution = '05 gramme glucose. If 20 c.c. of urine diluted 1 in 10 have been used, then the actual amount of urine needed was 2 c.c, and 2 c.c. of urine contain "05 gramme glucose, therefore 100 c.c. of urine contain •05 X 100 K or 2*5 grammes. Knowing the total measurement of the urine, the output of glucose for the 24 hours can be calculated from the percentage. VARIATIONS IN ACIDITY AND ACIDOSIS, ETC. 243 The Gerrard's solution used is not concerned in the calcula- tion, since the cyanide undergoes no reduction. Gerrard's solution is prepared as follows : — Dilute 100 c.c. of Fehling's solution with 300 c.c. of water. Boil. Run in a 5 per cent, solution of potassium cyanide until the blue colour has gone. Make up the mixture to 500 c.c. with water. The solution may remain a very faint blue, but it should be as free from colour as possible. It will keep for several weeks. Glucose is present in the urine under the following con- ditions : — The urine of untreated diabetics usually contains glucose in considerable amount, and is of a high specific gravity and large quantity. The "alimentary" glycosuria of middle- aged or older persons is largely dependent upon the intake of carbohydrates. On a strict carbohydrate-free diet the glucose commonly disappears from the urine, and such persons on a comparatively mixed diet may continue to excrete a moderate amount of glucose for years and live to an old age. The diabetes of younger people, that is of an age less than 25, almost invariably runs a rapidly fatal course ending in coma. The glucose in the urine of such cases is much less affected by diet than is the glycosuria of old persons. The rapidly fatal form of diabetes may, however, occur at any age. In addition to diabetes proper, glucose may occur in the urine in a considerable variety of affections, but in almost every instance the glucose is in small or comparatively small amount, is transitory, and is not associated w 7 ith the symptoms of diabetes. In diabetes the glycosuria is the most striking- sign of the disease, w T hile in other affections the presence of glucose in the urine is an interesting but casual phenomenon only. Glycosuria of this nature may arise under the following conditions : — Alimentary disturbances, such as starvation or the inges- tion of excessive carbohydrate meals. Toxic causes, in particular after the taking of morphine, strychnine, phloridzin, or phosphorus, and during acute fevers. Nervous diseases such as haemorrhage into the pons. 16—2 244 CLINICAL PATHOLOGY. cerebral tumour or cyst, meningitis, syphilitic and para- syphilitic diseases of the central nervous system. Pregnancy may be associated with a glycosuria as well as a lactosuria. Diseases of the secreting glands, as of the thyroid in exophthalmic goitre and myxoedema, of the pituitary gland in acromegaly, of the pancreas in acute or chronic pancreatitis or in tumours of the pancreas. Renal affections, of which phloridzin diabetes is the best known type. Hepatic diseases, as in cirrhosis of the liver and in gall stones. In all cases in which a reducing substance is present in the urine its nature should be determined, and if the substance proves to be glucose its amount should be estimated. The investigation does not, however stop at this point. It is necessary to discover if the patient has the symptoms and signs of diabetes or if he is suffering from some other obvious affection known to be occasionally associated with glycosuria. In all diabetic cases the effect of diet on the output of glucose has to be d^ termined over a series of observations. A further and important examination of the urine has also to be made in addition to the ordinary routine processes. The urine has to be examined for evidence of acidosis. The abnormal substances which may be present in the urine are B-hydroxybutyric acid, aceto-acetic acid and acetone. The B-hydroxybutyric acid is the mother substance, which on oxidation yields aceto-acetic acid and water. . CU 3 • CH (OH) CH 2 . COOH + = CH 3 . CO. CH 2 . COOH + H 2 0. The aceto-acetic acid is readily decomposed into acetone and C0 2 . CH 3 . CO. CH 2 . COOH = CH 3 . COCH 3 . + C0 2 . The substances to be tested for in the urine are acetone and aceto-acetic acid. If these are present B-hydroxybutyric acid may be presumed. The following test for aceto-acetic acid should be employed : — Gerhardt's reaction. — Filter fresh unboiled urine. Add dilute ferric chloride drop by drop until no more precipitate forms. VARIATIONS IN ACIDITY AND ACIDOSIS, ETC. 245 Filter and add a few more drops of the ferric chloride. A claret-red colour is produced, which disappears on prolonged heating. Certain drugs, such as antipyrin or salicylates, give a similar colour, which, however, persists on heating. If the acid is present in considerable amount it is sufficient to add 4 or 5 drops of the ferric chloride to the urine, an obvious deep red colour resulting. The colour does not always disappear completely on boiling. Acetone in the urine and in the patient's breath may be recognised by its characteristic " fruity " odour. The odour of acetone is more readily recognised by some individuals than by others, and many persons are quite unable to detect it by the smell unless it be present in exceptional amount. The peculiar odour of typhoid patients is in a similar way very characteristic to some observers. The test for acetone which depends upon its conversion into iodoform is not very satisfactory, and the following test is preferable: — Rothera's test.— To 5 c.c. of urine add 3 grammes of ammonium sulphate. Add 3 drops of a freshly-prepared solution of sodium nitro-prusside. Add 2 c.c. of ammonia. Acetone gives a slowly developing permanganate colour. The presence of these bodies in the urine of diabetic patients is of considerable importance, since the onset of coma is associated with their occurrence. In cases of glycosuria, which can be cured by appropriate diet, acetone and diacetic acid are almost invariably absent : their presence is therefore indicative of the graver form of diabetes and of the onset of coma. Exceptional cases are, however, met with in which these bodies may be present at intervals for many years without grave symptoms. Diacetic acid is also found in the other conditions which have previously been mentioned as being associated with acidosis. The " acidity " of the urine also, as previously de- scribed, should be periodically investigated in cases of true diabetes. An abnormal acidity is evidence of an abnormal metabolism of the body tissues, whether of proteids or fats, and the ratio of the ammonia nitrogen is in reality of considerably 246 CLINICAL PATHOLOGY. more clinical significance than the amount of glucose in the urine. Lactose is not infrequently found in the urine of pregnant or nursing women. Its occurrence is of no particular import- ance except that it is apt to be mistaken for glucose. Lactose has a similar reducing action upon Fehling's solution to glucose. Both lactose and glucose yield osazone crystals. The form of the crystals in the two cases is, however, different. Glucosazone crystals are long feathery sheaves ; the lactosazone form more circular bunches of spidery crystals (see plate). Lactose is not fermented by yeast, and by this test is readily differentiated from glucose. The osazone reaction in the urine is obtained as follows : — Place about 50 c.c. of urine in a beaker. Add 1 gramme of sodium acetate. Add *5 gramme of phenyl hydrazin hydrochlorate. Stir well and place on a water bath. Leave for one hour. Allow to cool slowly at room temperature. Examine a drop of the deposit (after centrifuging at a low speed if necessary) on a slide beneath a cover-slip and with the low power of the microscope. The test is rarely successful when only very small quan- tities of carbohydrate are present. Pentose may occasionally be met with in the urine. Urines which contain pentose reduce Fehling's solution and yield an osazone crystal. Pentose does not ferment with yeast. Pentoses give the following tests with phloroglucinol and orcinol : — Phloroglucinol reaction. — Mix equal parts of con- centrated hydrochloric acid and water in a test tube. Add a little phloroglucinol. Add 5 to 10 drops of urine. Warm in the water bath. The solution gradually becomes cherry red and a precipitate forms. Allow to cool. Shake with amyl alcohol. PLATE XL Glucosazone Crystals. Lactosazone Crystals. Pancreatic Test C " Crystals. Uric Acid Crystals. PLATE XI. O o VARIATIONS IN ACIDITY AND ACIDOSIS, ETC. 247 Examine the red amyl alcohol solution with the spectroscope. The spectrum shows an absorption band between D and E. Orcinol reaction. — This reaction is performed in exactly the same manner. The solution in the water bath becomes first red, then violet, and finally blue. The amyl alcohol extract is bluish green and shows an absorption band between C and D. The same reactions are given with glycuronic acid, which may appear in the urine in considerable amount after the administration of certain drugs, such as chloral, chloroform and morphia. Occasionally it occurs spontaneously in the urine. Urines containing glycuronic acid give much the same reactions as those containing pentose. Glycuronic acid is optically active ; the free acid is dextro-rotatory, and the combined acid, which alone occurs in the urine, is Iebvo- rotatory. The presence of these substances in the urine is of importance, since they may be mistaken for glucose. The occurrence of pentose in the urine is of considerable scientific interest in that it may occur in apparently healthy persons as a notable example of one of the rare inborn errors of metabolism. CHAPTER XVIII. urinary deposits — urinary calculi. Organised Urinary Deposits. The examination of urinary deposits with the microscope is more important and more neglected than almost any other laboratory investigation. The student working in the wards is more concerned, and rightly, with the physical signs and symptoms of the patient ; consequently he prefers to content himself with a note of the naked-eye appearance of a deposit, or at the most with a rough chemical test. The recognition of the various formed elements which may be present in the urine requires some practice, and this is more conveniently acquired in a laboratory than in the wards. The time available in the wards is limited, and the ward microscope, however excellent an instrument it may have been in its youth, is apt to reflect more light from its brass work than it admits through its lenses. Fortunately for those students and practitioners who have been unable to avail themselves of a laboratory course, an acquaintance with urinary deposits is fairly readily acquired without personal instruction. The deposits particu- larly lend themselves to accurate reproduction by diagram, and are among the minority of pathological objects which can be recognised by reference to a plate. An attempt is made here to describe, and so far as possible illustrate, such deposits as are likely to be met with in the urine. Those who require a wider knowledge of the subject are referred to the excellent text-book of Rieder and Delepine. The methods of examining a urinary deposit may be again recapitulated. If a considerable deposit is present in the specimen glass do not centrifuge. Draw up a portion of the deposit in a pipette. If the deposit is scanty or absent, wash out thoroughly two centrifuge tubes. Shake up the bottom portion of the specimen after carefully pouring off the super- natant fluid. Fill the centrifuge tubes with urine and centri- UKINARY DEPOSITS— URINARY CALCULI. 249 fuge for a few minutes at a moderate speed. Do not stop the centrifuge after turning off the power, but allow it to run down. Remove the tubes and make use of the one that contains the more deposit. Turn the tube upside down : the bulk of the deposit will remain in the last drop, which will not fall out, Prepare a clean slide and cover-glass. Shake out a drop of the urine containing the deposit on the centre of the slide. Let down a cover -glass on the slide. If the right amount of deposit is taken there will be no air bubbles and the urine will not spread over the slide beyond the cover-glass. Examine with the microscope vertical and the diaphragm partly closed. Use both a f-inch and a ^-inch objective. Blood. — Red blood corpuscles are recognised under the higher objective by their shape, size and colour. The size and shape are not infrequently altered by crenation '; the colour is very distinctive. With a little practice a single red cell can be recognised with certainty. The objects most frequently con- founded with red blood corpuscles are oil globules and uric acid crystals. Oil globules are often seen in the deposits of catheter specimens, and are derived from the lubricant used for the catheter. They are more circular than red cells and of very varying sizes from circles smaller than red cells to obvious large globules. They are yellowish in colour, but of a different tint from the corpuscles. Uric acid crystals mislead only by their colour ; they can be recognised by their regular outlines and crystalline shape. Should there be any doubt as to the nature of the corpuscles, a film preparation should be made and stained with Leishman's stain. Blood may also appear in the urine as a contamination from some other part of the body, and in cases of doubt, particularly in specimens obtained from women, a catheter specimen should be examined. The presence of blood in the urine will probably have been confirmed by the guiac test or by the spectroscope. The amount of blood which can be detected by the microscope may be so small as to escape observation by the other tests. In all cases of hematuria an estimate should be made of the relative proportions of red cells and haemoglobin present. There is no difficulty in this. If sufficient blood is present to obviously colour the urine a very large number of red 250 CLINICAL PATHOLOGY. cells will be present in the deposit. In cases of marked hemoglobinuria red cells will be almost entirely absent or very scanty and very distorted. Pus (Plate XII.). — A curious convention has grown up about the meaning of the phrase " pus corpuscles." The student has been led to look for some new and strange pathological entity. Pus corpuscles are phagocytic cells, and as such consist of polynuclear neutrophils with occasional large hyaline cells and epithelial cells. It is true that the corpuscles may be more or less degenerated, but the majority of cells in most samples of pus are perfectly well formed and normal in appearance. There is no actual dividing line between a polynuclear cell and a pus corpuscle. If polynuclear cells are sufficiently numerous to produce a naked-eye deposit, pus is present. If few cells are found the process is the same, and the difference is one of degree only. Urines containing pus cells in large numbers give a greenish colour with the guiac test, but the microscope is the only reasonably accurate method of detecting pus. The perform- ance of the potash test for pus is a mere waste of time and is not described here. Pus corpuscles are readily recognised in unstained specimens by their size, their round shape, the commonly bilobed or poly- nuclear form of their nuclei, and by their refractile granular appearance. "When a considerable degree of pyuria is present the pus may be partially obscured by the presence of " mucus " in addition. The pus in such cases is very viscid and difficult to control when preparing a microscopic specimen. When viewed under the microscope the cells are partially hidden under the " mucus" and can only be seen by careful focussing. Such specimens floated in water may resemble slimy membranes in appearance and may have the shape of the bladder or urethra. Prostatic casts are of similar composition. In cases of doubt a thin film should be spread on a slide with a platinum wire, dried, stained for 3 minutes with carbol- thionin, washed in water, blotted dry, and examined with an immersion lens or even with a ^t-inch objective after mounting in Canada balsam. The polynuclear cells are often somewhat shrunken in such specimens, but are perfectly recognisable, and in addition any bacteria which may be present can be noted. PLATE XII. ®m@>^^ © ® ® # ® © © ® M &_ Pus Corpuscles. (Urinary Deposit.) Epithelial Cells. (Urinary Deposit.) -". . ® (A o O ® . O Sfe © ^^^ ' *i°». »*•'■ ® o §J«fe. O Casts, etc. (From the Deposit of a Case of Acute Nephritis.) f \ ^■f * •*^ Tyrosine Crystals. (From Urine, after Separation.) Spermatozoa. (From a Spermatocele.) (Stained Specimen.) URINARY DEPOSITS— URINARY CALCULI. 251 It is occasionally necessary to determine if a urine which contains blood has pus present in addition, since polynuclear cells are necessarily present when the bleeding has been free. The question is fairly easily answered by the micro- scopic examination of the deposit. In the case of blood the average field of the ^-inch objective shows large numbers of red cells and 1 or at most 2 or 3 leucocytes. If pus is present in addition the field will show a dozen or more leucocytes among the red cells, and fields will be found in which the leucocytes are gathered in small clusters. Conversely in cases of con- siderable pyuria occasional red cells will usually be detected among the leucocytes. The presence of polynuclear leucocytes in a urinary deposit is pathological, provided that they came from some part of the urinary tract. It is essential for the detection of small traces of pus in the urine of women that a catheter specimen should be examined. In men the pus may come from the urethra in cases of gonorrhoea, and it is not infrequent to find a few leucocytes, probably derived from the prostatic urethra, in the urine of patients who have had gonorrhoea many years previously. The pus in such cases may or may not be due to the actual presence of gonococci. The pus cells are frequently united by "mucus" into long thread-like processes visible to the naked eye and known as prostatic threads. These threads are most often present in the first specimen of urine passed in the morning, and may be seen best after massage of the prostate. Epithelium (Plate XII.). — Epithelial cells are com- monly present in the urine, and in deposits taken from the urine of female patients are as a rule very abundant. Epithelial cells in the urine have no pathological significance, but must be recognised, since they are apt to be mistaken for pus corpuscles, or even portions of new growth. Epithelial cells may be recognised by their size, being com- monly much larger than the polynuclear cells, by their shape, which is rarely round, by the absence of granularity, and by their single central nucleus. They cannot be distinguished from the single cells of a neoplasm. Epithelial cells differ considerably in shape and size, and the student is commonly asked to recognise from the nature of the cell that portion of the urinary tract from which it is 252 CLINICAL PATHOLOGY. derived. I am by no means convinced that this is possible. The commonest form of epithelial cell is a very large angular cell of the squamous type. It is the predominant cell in the urine of women and may be entirely derived from the vagina. Smaller pear-shaped and tailed cells are often predominant in ureteric specimens, and less commonly small round cells which closely resemble polynuclears, but differ from them in having a round nucleus. Such cells are presumably derived from the ureter or pelvis of the kidney. The ureteric lining is very friable and readily damaged, and specimens obtained by ureteric catheterisation are frequently grossly contaminated by blood. It is only in very skilled hands that epithelial cells only are rubbed off in normal, cases, and these are commonly present in abundance and have to be carefully distinguished from pus corpuscles. New growths. — In descriptions of the methods of diagnosing new growths of the bladder or kidney the detec- tion of particles of growth in the urine or in the eye of the catheter holds a time-honoured place. It is perfectly reasonable to look for such particles, but it is extremely rarely that one is able to identify them. The recognition of a single cell, or even a small cluster of cells, from a neoplasm is quite impossible. The more solid fragments in such urines nearly always turn out to be blood clots. Fragments of growth may, if present, be recognised after teasing out if necessary, and flattening in a drop of salt solution between a slide and cover-slip. A drop of dilute methylene blue may be allowed to run under the cover-slip if desired for staining purposes. Definite villus-like papillary processes lined with epithelium can only come from a neoplasm. Such processes have to be distinguished from particles of pus and epithelial cells bound together by " mucus." Pus of this description presents an undulating outline, but no regular processes. Actual naked-eye fragments of a papilloma or carcinoma are in cases of doubt preferably fixed and sectioned. The distinc- tion between a simple and a malignant growth in such chance particles may be impossible. Faecal elements. — Faecal elements may be present in the urine, particularly of female patients as a chance contamina- tion. The student on examining the urinary deposit of a catheter specimen, and being confronted with striated muscle URINABY DEPOSITS -URINARY CALCULI. 253 fibres, may be puzzled to account for them. Striated muscle fibres are yellow in colour, and show more or less clearly the transverse striping. They are quite unmistakable, and in a catheter specimen are definite evidence of abnormal com- munication between bladder and intestine. The most common cause of such communication is a recto-vesical fistula, which may first attract attention by the production of a cystitis. If muscle fibres are absent, vegetable fibres may be recognised by their shape and spiral appearance, and in the absence of these elements faecal contamination can be inferred only from the smell and the general heterogeneous appearance of the deposit. Casts (Plate XII.).— Casts are among the most important of the formed elements in the urine. If casts are present in any numbers and of certain varieties^ we can be satisfied that some form of nephritis is present, and can sometimes deter- mine which form. Tube casts are products of the cells lining the renal tubules, and their permanent form and outline are probably due to the fact that they have been formed in the tubules and have remained some time in situ. If the tubular epithelium is normal and the renal tubules are properly patent, no deposition of casts can take place. The presence of tubular casts in the urine is consequently evidence of renal disease. Casts vary greatly in size, and to a less extent in shape. A cast of average size is just recognisable under a §-inch objec- tive, and when only a few casts are present they are best searched for with this power. A J-inch objective is necessary for their verification, and should always be used in addition. Casts are recognised by the nature of their contents, which will be subsequently described, but particularly by their shape and their definite outline. In shape casts are long and more or less narrow cylinders with rounded ends. Occasionally one end is rounded, the other ragged as if fractured. A cast has a clear-cut and sharply defined outline. The limiting edge of the cast distinguishes it from the only objects that could reasonably be mistaken for it. Amorphous urates, and less commonly amorphous phosphates, may be found in little masses which exactly resemble casts in shape, and for the reason in some cases that they are probably formed in the renal tubules. Such urtitic casts are distinguished in a care- 254 CLINICAL PATHOLOGY. ful examination by the comparative irregularity of their edges and the absence of a definite outline. Casts on the whole are remarkably like the representations of them in many text- books, and if the beginner sees a well-formed cast he almost always recognises it. If he is in doubt as to whether the object he sees is a cast or not, it may be anything from an epithelial cell to a scratch on the glass, but it is very rarely a cast. Casts are of several varieties, each of which has a some- what different significance. The casts are divided according to their structure into cellular, granular, and amorphous varieties. Cellular casts are furthur subdivided according to the nature of the cells present into epithelial, erythrocytic, and leucocytic casts. Epithelial casts contain the mononuclear cells of the shed renal epithelium. The epithelial cells may be more or less degenerated, and their nuclei may be absent. Some cells may appear vacuolated, others granular. Some may contain globules of fat. The cast may be entirely filled with cells, or may be partly hyaline. The presence in casts of epithelial cells, not greatly degenerated, is practically confined to the acute stage of acute nephritis. Erythrocytic casts are readily identified, and may consist of casts packed with obvious and undegenerated red cells. Some casts may contain both red cells and epithelial cells, and the red cells may be more or less broken up. These casts are similarly found in acute nephritis, and particularly in the acute stages. Free red cells are always present in the urine in addition. Leucocytic casts, that is casts which contain polynuclear neutrophils, are not commonly met with. They may be found in septic conditions of the kidney, such as " surgical " or " consecutive " nephritis. A few polynuclear cells are nearly always present in addition to the casts in a case of acute nephritis, and a few degenerated epithelial cells are also seen lying free. Granular casts are casts which contain no formed elements other than fine, or more rarely coarse, granules. Granular casts are perhaps the most common variety seen, and usually predominate in chronic parenchymatous nephritis as well as in acute nephritis after the first few days of the disease. The majority of granular casts are probably derived in the same way as the epithelial casts, and differ only in URINARY DEPOSITS— URINARY CALCULI. 255 representing a later stage in the cells which have now undergone complete granular degeneration. Granular casts may less frequently arise from a granular change in the amorphous forms. Amorphous casts. — The commonest variety of amorphous cast is the hyaline cast. This varies considerably in size, and may be very large and spirally twisted in its long axis. The content of the cast is as a rule quite structureless, and almost entirely transparent. These casts may escape observa- tion unless the light reflected through the preparation is reduced to a minimum. Hyaline casts may be found in every form of nephritis, and may be the only variety present in cases of contracted granular kidney. Very occasionally a cast of this nature may be found in the urine of an apparently healthy person. Amyloid casts are rarely found, and are more refractile than hyaline casts. They are frequently fissured, and some- times give the amyloid reaction with one of the appropriate staining mixtures such as methyl violet, or iodine and sulphuric acid. Amyloid casts are not particularly associated with lardaceous disease of the kidneys. Spermatozoa (Plate XII.) are occasionally found in the urine, and should be readily recognised by their shape. The long, thin and tapering tail, with the prominent oval head, are quite unmistakable. Spermatozoa in the urine may show active movement even in a specimen which has been standing for some time. Spermatozoa are readily stained by the ordinary dyes, such as carbol-thionin. Animal parasites are not commonly met with in the urine of patients in this country. Occasionally the common thread worm (oxyuris vermicularis) or its ova may be found in the urine of little girls. Filariee are very rarely detected in the urine, even in those countries in which filariasis is common. The only parasitic infection of the urinary tract at all commonly met with is that of bilharzia haematobia. In cases of hsematuria coming from tropical or sub-tropical climates, such as Egypt or parts of South Africa, where this parasite is numerous, its presence should always be suspected. Infection of the bladder by bilharzia is recognised with certainty by the detection of the characteristic ova in the urine. When haenia- turia is present the ova are generally numerous, and they may 256 CLINICAL PATHOLOGY. persist for many months after residence in an infected district. A specimen of the urinary deposit should be put up in the ordinary way, and the ova can readily be seen with the low power of the microscope, and the details of the contained embryo can often be made out on using the ^-inch objective. If the ova are very scanty the patient should be told to empty his bladder, and then to pass the last few drops of urine, evacuated by active straining, into another receptacle. The ova are large oval bodies with a definite capsule terminating in a single sharp, short spine which is quite characteristic. Within the ovum can often be seen a coiled, ciliated embryo which may show active movement. If fresh water is added to the urine the embryo may burst its way out of the ovum and swim about freely. The ova are occasionally found in the faeces, and in this situation are usually provided with lateral and not terminal spines. Unorganised Urinary Deposits. While the greater number of the organised urinary deposits are only present in the urine as the result of disease, the majority of the unorganised deposits are not necessarily of any pathological import. The detection of a crystalline or non-crystalline substance in the urine is no evidence that it is being excreted in excess. The only conclusion that can be drawn is, that the sample of urine on cooling and standing is unable to contain the substance in solution. The majority of normal urines and almost all concentrated urines form some deposit on standing. Those urines from which no deposit can be obtained, even after standing and centrifuging, are nearly always derived from cases of polyuria. The detection of uric acid or calcium oxalate crystals in the urine of a patient with renal symptoms is very little evidence that a calculus of similar composition will be found in the kidney. The following are the more important unorganised deposits : — Amorphous urates (Plate XIII.). — The deposition of amor- phous urates from an acid urine on cooling is an extremely common phenomenon. The urates have a marked tendency to absorb the urinary pigments, and a concentrated acid urine will frequently throw down several inches of a thick pink deposit of amorphous urates. Such deposits may be URINARY DEPOSITS— URINARY CALCULI. 257 found in almost any febrile urine, or in the urine of a perfectly normal individual who has recently taken an unusual amount of exercise. The amorphous urates are recognised by their occurrence in an acid, or less commonly, in a neutral urine, by their pinkish colour, by the manner in which they re-dissolve on warming the urine, and by their appearance under the micro- scope. Examined under the microscope, these urates appear as fine, loose, amorphous granules of a yellowish-brown tint. Almost the only substance they can be mistaken for is amor- phous phosphate. The phosphates usually form coarser granules, which are colourless, which dissolve on the addition of an acid but not on warming, and which usually occur in an alkaline urine. The amorphous urates consist mainly of acid sodium urate. Acid ammonium urate (Plate XIII.) is much less commonly met with, and practically only occurs in alkaline urine. It may be found in normal urine which has been standing for a long time, and which has undergone ammo- niacal fermentation. The customary crystalline form of ammonium urate is a very striking one. The crystals are deeply pigmented, of a considerable size, and of very irregular shape. One or more elongated spinous processes proceed from the crystals, giving them the appearance of a radish with its roots. Uric acid (Plate XL). — Well-formed crystals of uric acid are found only in urines of acid reaction. They occur under much the same conditions as do deposits of amorphous urates. Deposits of uric acid in concentrated urines are con- sequently of no significance, but a continued excretion of urine rich in such deposits is associated with certain patho- logical states of which leukaemia and acute gout are the most noteworthy. Uric acid crystallises in a variety of forms which differ widely in size and shape. Nearly all forms are coloured by the urinary pigments, but colourless uric acid crystals occur. A common variety of uric acid deposit is readily visible to the naked eye as bright red crystals, which have a tendency to adhere to the sides of the specimen glass. These crystals, if passed in considerable amount, are referred to by the patient as "gravel." Under the microscope the commonest shape of the uric acid crystal is that of the " whetstone," and a number of such crystals united together p. 17 258 CLINICAL PATHOLOGY. at one end and radiating from each other at their free ends, constitute the macroscopic gritty red granule. Other forms of uric acid which may occur are crystals somewhat similar to those described as " whetstones," but flatter, more pointed, and " lozenge "-shaped; also smaller crystals, more quadrilateral in shape and with a tendency to cling together in clusters. Another variety of crystal, which may be colourless, is almost circular, and presents more or less regular striations radiating from the centre to the periphery. Cones, needles, and rod- like forms are less commonly met with, and various of these abnormal forms are doubtless impure. Amorphous phosphates are found as a deposit in alkaline or less frequently in neutral urine. The colourless deposit at the bottom of a test tube is frequently mistaken for pus, and cannot certainly be distinguished from it by naked-eye examination. Under the microscope the deposit is seen to consist of white amorphous granules, not unlike those of urates. The distinction between phosphates and urates is given under the description of the latter. The amorphous phosphates consist of calcium and magnesium phosphate. Triple phosphate (Plate XIIL), or ammonium magnesium phosphate, forms the crystalline deposit which is the one most commonly met with in ammoniacal urine, and very rarely in slightly acid urine. Deposits containing triple phos- phates are frequently mixed with amorphous phosphates, and less commonly with acid ammonium urate. Triple phosphate crystals are of no significance in a stale specimen of urine, and in comparatively fresh specimens they have the same meaning as the alkaline reaction with which they are associated. Consequently the majority of freshly-voided alkaline urines which contain triple phosphate crystals con- tain also pus and micro-organisms. The crystals are usually of considerable size and of somewhat variable shape. In almost all samples some of the more characteristic forms will be present. The common form is that known as the " coffin lid " crystal, and is a straight prism with obliquely-cut terminal facets. Less common are feathery and fern-shaped crystals. Stellar phosphates (Plate XIII.) or crystals of calcium hydrogen phosphate maybe found in neutral or nearly neutral urine, occasionally in health, but more commonly in blood diseases and joint affections. These crystals are, like other PLATE XIII. Triple Phosphate Crystals Calcium Hydrogen Phosphate Crystals. o'gHj #»• . , y_t •• " 'tf 1 *!* 'fpfitf Vw'. * z^ &£«* '*:,:■<•'- - '" >• Q O □ <^\ o D D O Amorphous Urates Calcium Oxalate Crystals. Ammonium Urate Crystals. Cystine Crystals. URINARY DEPOSI TS— URINARY CALCULI. 259 phosphates, readily soluble in acetic acid. The most common form assumed by the crystals is that of a long, narrow and flat prism, usually pointed at one end. The prisms may be collected together in bunches or rosettes. Less commonly the crystals may be very fine and feathery and collected into tufts. Calcium oxalate (Plate XIII.) crystals may be found in acid or alkaline urine, but more frequently in the former. They often occur in excess after the ingestion of certain fruits and vegetables, such as apples, tomatoes and rhubarb. It is stated that a continual passage of calcium oxalate crystals in excess is characteristic of chronic pancreatitis. The crystals are com- monly found in perfectly normal urine, particularly if it has been allowed to stand for some hours, and they may form a considerable naked-eye deposit not unlike pus in appearance. The type of crystal most commonly found, and the one which accompanies any other varieties which may be present, is a small colourless octahedron. It is among the smallest of the urinary crystals, and is only just visible under the §-inch objective, the ^-inch being required for identification. When seen from above with the pointed end uppermost the lines of the facets show as intersecting diagonals on a square, giving the crystal the appearance of an envelope. In addition to the " envelope " forms, oval, spherical and dumb-bell forms are met with. The crystals are practically insoluble in acids. Calcium carbonate, commonly found in the urine of herbivors, is rarely met with in human urine. It may occur in alkaline and stale urine, and usually in association with phosphates. The deposit may be crystalline, the ciystals having the shape of small dumb-bells, or amorphous. The nature of the deposit is made clear by the addition of a little dilute hydrochloric acid, when the deposit rapidly dissolves with effervescence. Cystine (Plate XIII.) is among the rarest of urinary deposits, and at a large London hospital perhaps one case of cysti- nuria will come under observation in five years. Nevertheless the student is, for examination purposes, expected to be very familiar with the cystine crystal. The condition is an interest- ing one, since it forms one of the rare congenital abnormali- ties of metabolism, and its occurrence appears to be confined to the children of first cousins. Although traces of cystine 17—2 260 CLINICAL PATHOLOGY. can be obtained from the normal urine, the occurrence of cystine crystals in the urine is clear evidence of this rare condition. Cystinuria may persist for years without producing any symptoms ; it is, however, frequently associated with the formation of renal calculi composed of cystine. The deposition of the crystals takes place in acid or faintly alkaline urine. The appearance of the crystals under the microscope is very characteristic, since they consist of flat, colourless, regular, hexagonal plates. All the crystals in any one deposit will not be regular, but a certain number of perfectly formed ones are nearly always found. Cystine is readily soluble in ammonia, but is insoluble in water, ether, or acetic acid. Leucine and tyrosine. — These substances, which result from the decomposition of proteids, are not found in normal urine. The conditions in which they are most frequently met with are acute yellow atrophy of the liver and phosphorus poisoning. The two crystals almost always occur together in the urine, and they may form a considerable naked-eye deposit in the specimen glass. Tyrosine is less soluble than leucine, and separates out from the urine more readily and in greater abundance. Tyrosine (Plate XII.) crystals take the form of brush-like tufts of very fine needles, and are either colourless or greenish- yellow. To obtain the crystals in a pure form from the urine : — Eemove coagulable proteids, if present, by boiling and filtering. Precipitate the pigments and extractives by basic lead acetate. Shake well. Allow to stand. Filter. Pass H 2 S through the filtrate to remove the lead. Filter and evaporate down the clear filtrate. Colourless crystals of tyrosine separate out on cooling. The material obtained on evaporation can be further tested for tyrosine by Hofmann's reaction. In this reaction the material is heated with water in a test tube, and a few 7 drops of Millon's reagent are added to the hot solution, which becomes rose-coloured in the presence of tyrosine. Leucine crystals take the form of yellow spheroids, which show both radial and concentric striation. Leucine is readily soluble in acids and alkalies. URINARY DEPOSITS— URINARY CALCULI. 261 Foreign bodies in urinary deposits.— A complete list of the adventitious material which may be found in the urine would rival the well-known list of foreign bodies met with in the vagina, though there is no record of a bust of Napoleon in a specimen glass. Patients may, however, deposit almost any available object in the urine. The follow- ing include some of the more common adventitious objects which may be puzzling to the student. Marks on the cover-glass have been frequently and earnestly scrutinised with a view to interpretation, and the mistake is easily made. If the cover-glass is not scrupulously clean and it is difficult to make it so, or if it is scratched, the dust and scratches on its surface are the objects first seen on focussing the objective down. The scratches may assume fantastic shapes and may even remotely resemble casts, while the surrounding dust is mistaken for the urinary debris. The mistake is at once rectified by continuing to focus down until the actual urinary deposit comes into view. Scratches on the slide itself may of course be similarly mis- taken for urinary contents, and have to be differentiated by their shape and appearance. They present no real difficulty. Air bubbles should not be present in a carefully put up sample of deposit ; still they sometimes occur. They are circular in form unless compressed. They are recognised by their broad, dark and often double outlines, and their clear, shining centres. Fat globules may occur in disease, but are more frequently present as a foreign body in catheter specimens. They are recognised by their circular form, their variability in size, their sharp margins and refractile centres. They turn black on adding osmic acid. Starch granules are fairly frequently found in the urine, particularly of children, their source being the dusting powder used. They are recognised by their oval shape and concentric lamellation. They turn blue on the addition of iodine. Sulphur granules are occasionally found in the deposit of urine which has been subjected to the sulphur test for bile salts. The granules appear as dark irregular clumps of crystalline bodies. Hairs are commonly present in the specimen glass, and are usually pretty obvious to the naked eye. They are as 262 CLINICAL PATHOLOGY. a rule pigmented, and their structure becomes more obvious on the addition of caustic soda. Fibres of cotton or linen are very commonly met with, and are usually derived from the cloth used in cleaning the speci- men glass. They are necessarily of variable size and shape, but are all more or less cylindrical and twisted, and usually have frayed ends. The only pathological objects for which they can be mistaken are casts, and the resemblance is not particularly striking. Urinary Calculi. The calculi which may be found in an}- part of the urinary tract can be divided into those which are comparatively common and those which are extremely rare. We are only concerned here with the composition of the various calculi, and this is readily determined by a short and simple scheme of analysis. The exact percentage composition of mixed calculi is naturally a more laborious proceeding, but does not form a part of ordinary clinical pathology. Speaking generally, calculi may vary in size from a small concretion, such as ma} 7 be passed by the urethra, to a mass the size of a clenched fist. They are conmionry pigmented. They may be smooth or rugged. They tend to take the shape of the viscus in which they have been formed, as, for example, the pelvis of the kidney. If multiple they may be facetted. The fractured surface frequently shows concentric rings. Calculi are commonly of mixed composition, and the nucleus may be formed of a different material to the cortical portion. The comparatively common stones are calcium oxalate, uric acid, ammonium urate and phosphatic calculi. Calcium oxalate stones mixed with a certain amount of calcium phosphate would appear to be the most common variety. Calcium oxalate calculi are as a rule hard, of a reddish- brown colour, and with a granular surface which has given them the name of " mulberry " calculi. They may be branched. Calculi of triple phosphate are white and crumbling, are practically only found in the bladder, and may be formed around a foreign bod} 7 . Uric acid calculi are less common and, contrary to the statements of many text -books, are extremely rarely found in the kidney, though the} 7 are not very infrequently met with in the bladder. The rarity of uric acid calculi in the kidney is URINARY DEPOSITS— URINARY CALCULI. 263 fortunate, since the permeability of uric acid to the X-rays is the same as that of the belly- wall ; consequently they can- not be recognised by X-ray examination. The calculi are usually small, chocolate-coloured, fairly hard, and have a smooth surface. While pure uric acid calculi are rare, uric acid is fairly commonly found in association with some other substance such as calcium phosphate or oxalate. The mixed calculus can be recognised by X-ray photography. Urate stones consisting of ammonium, with a lesser amount of sodium urate, are hard stones of a brown colour. Calcium phosphate is not a common variety of calculus, but a certain amount of this substance is not infrequently present in association with calcium oxalate, triple phosphate and, occasionally, calcium carbonate. The composition of the commoner calculi should be sought according to the following scheme of examination. If the reactions for those substances are not given, or only traces of them are found, some of the rarer substances must be tested for, as subsequently described. The chemical examination of urinary calculi. If the stone is of considerable size, cut it in half. Examine the cut surface, and if the nucleus differs in appearance from the cortical portion, scrape out the nucleus first and examine it separately. Powder up the calculus in a clean mortar. Then apply each of the following tests : — (1) Place a little powder in the bottom of a clean test tube and add dilute HC1. If effervescence occurs carbonates are present. If effervescence is doubtful, place some powder on a slide with a cover -glass over it. Run the dilute acid between slide and cover-glass and watch under the microscope for the evolution of gas bubbles from the granules of powder. Carbonates are rarely obtained except as traces. (2) Place a little powder on a platinum foil. Heat over the Bunsen flame. When the powder is incinerated shake it out into a clean test tube. Add dilute HC1 as in 1. 264 CLINICAL PATHOLOGY. Oxalates were present if effervescence occurs. If in doubt repeat on a glass slide under the microscope. The oxalates were converted into carbonates by the heating on the platinum foil. If oxalates or carbonates are present the base may be presumed to be calcium. The presence of calcium may, if preferred, be confirmed by the usual tests in solution, for example : — + Ammonium carbonate = white precipitate. + Ammonium oxalate = white precipitate, insoluble in acetic acid ; soluble in dilute HC1. + Calcium sulphate = no precipitate. (3) Place a little powder in a clean porcelain crucible, and perform the murexide test as follows : — Add a few drops of nitric acid. Heat on a sand bath in the fume cupboard. If an effervescence of gas takes place and a bright red residue is left: — Allow to cool. With a glass rod add to a portion of the residue 1 drop of caustic soda. The residue turns bluish-violet. To another portion of the residue add 1 drop of ammonia. The residue turns purple. A positive murexide test is evidence of the presence of Uric Acid or Ammonium Urate. To determine which is present : — Place some fresh powder in a test tube. Add a little caustic soda and heat. Examine for the evolution of ammonia by the smell and by a piece of moist litmus paper held in the mouth of the test tube. If ammonia is detected the material contained ammonium urate ; if, as is more usual, no ammonia is present, the material was uric acid. (4) Dissolve some of the powder in dilute warm HC1 in a test tube. If necessary filter. Add a few drops of nitric acid. URINARY DEPOSITS- URINARY CALCULI, 265 Add ammonium molybdate solution in quantity equal to the amount of the original solution. The ammonium molybdate solution must be comparatively fresh, and there should be little or no precipitate in the bottom of the bottle. Heat and allow to stand. If a yellow precipitate forms, phosphates are present. The phosphates may be earthy phosphates or, less frequently, ammonio-magnesium phosphate. To distinguish between the phosphates test for the presence of ammonia as in (3). If any of the above tests are strongly positive it is not necessary to proceed further. But if the tests are negative or only traces of the above substances are found, the following rare constituents of urinary calculi must be sought for : — Cystine. — Burn the powder on a platinum foil over the flame. Cystine burns rapidly with a blue flame and gives off a sharp odour. Dissolve some powder in ammonia. Cystine is readily soluble, and on spontaneous evaporation of the ammonia the typical hexagonal plates separate out. Xanthine. — The powder on the platinum foil burns away, but without a flame. Try the murexide test. There is no effervescence on adding nitric acid, and the residue left after heating is yellow. Add a drop of caustic soda to the dried residue, after cooling ; in the presence of xanthine the residue becomes orange. Warm, and the colour changes to red. Fibrin. — The powder burns on the platinum foil with a yellow flame and an odour of burnt feathers. The powder is insoluble in alcohol or ether, but soluble in hot caustic potash. Add acetic acid to the caustic potash solution. The fibrin is precijDitated with an evolution of H 2 S. Urostealith. — The powder burns with a yellow flame and an odour of resin. It is soluble in alcohol and ether. CHAPTER XIX. special investigations of the trine — bacteriology of the urino- genital tract. Special Investigations of the Urine. By special investigations are meant those which are outside the ordinary routine examination of the urine and those which are not commonly called for even if any abnormal substance is detected. There exist a very large number of such investi- gations, but the majority of them are of more scientific than practical interest and cannot be described here. Only those methods are given which have some bearing upon the clinical diagnosis or treatment of disease, and which do not involve any very specialised knowledge of chemistry. The estimation of uric acid. — Uric acid belongs to the group of bodies known as purines, and is the chief nitrogenous excretory product of birds. In man the average daily output of uric acid varies from 0*5 to 1 gramme. The amount is increased with any abnormal destruction of nuclei, as in the leukaemias, when four or five times the normal quantity may be excreted. A lesser increase accompanies high fever. In cases of gout the uric acid output is markedly low in the intervals of freedom, but just before an attack and during the height of the attack a marked increase in the output takes place. Hopkins' method of estimating the uric acid in the urine as modified by Folin is that most commonly employed. The following solutions are required : — N (1) A standard ^ solution of potassium permanganate. 1 c.c. of this solution = 0*00375 gramme uric acid. (2) A 10 per cent, solution of ammonium sulphate. (3) A solution containing — Uranium acetate .... 5 grammes. Ammonium sulphate . , . 500 ,, 10 per cent, acetic acid ... 60 c.c. Water 650 „ SPECIAL INVESTIGATIONS OF THE URINE, ETC. 267 (4) A measured 24 hours' supply of urine, which must be well stirred before a sample is taken. Proceed as follows : — Stage 1. — Into a 500 c.c. flask measure 200 c.c. of urine and 50 c.c. of the uranium acetate solution. Mix and allow to stand for half an hour, or until the precipitate has settled. By this procedure a mucoid substance is precipitated from the urine, which would, if left, render subsequent filtration of the ammonium urate very slow and tedious. Stage 2. — Filter the bulk of the supernatant fluid through a dry filter paper into a dry vessel. Measure 125 c.c. of this (containing 100 c.c. of the original urine) into a beaker. Add 5 c.c. of concentrated ammonia. Mix and allow to stand for from 12 to 24 hours. In this stage the uric acid is precipitated as ammonium urate, which is insoluble in the presence of the ammonium sulphate. Stage 3.— Filter. Wash the precipitate on to the filter with the 10 per cent, ammonium sulphate solution. Wash two or three times with the same solution to remove the chlorides. Remove the filter paper. Open it. W T ash off the precipitate into a beaker with a stream of hot distilled water. Stage 4. — The ammonium urate precipitate is now suspended in about 100 c.c. of distilled water. Add 15 c.c. of concentrated sulphuric acid. Titrate with the permanganate solution while warm. The titration is completed when a faint permanent pink colour is diffused through the solution. In performing the titration a burette with a glass tap must be used, since the permanganate acts upon rubber. The calculation is made from the number of cubic centimetres of the permanganate solution used, and this number multiplied by 0*00375 gives the number of grammes of uric acid in 100 c.c. of urine. On account of the solubility of ammonium urate, however, a correction has to be made, and 0'003 gramme of uric acid should be added for every 100 c.c. of urine used. 268 CLINICAL PATHOLOGY. The estimation of purines. — The purine bases include purine, xanthine, and hypoxanthine, and are increased in the leukaemias and in febrile conditions. The "purine bodies" include the purine bases and uric acid, and they can be readily estimated by the "Walker Hall purinometer, -with sufficient accuracy for clinical purposes. The variations in such a disease as myeloid leukaemia are very considerable. The purinometer is provided with full instructions for use, which may be recapitulated here. The apparatus consists of a tall glass vessel divided by a stop- cock into a lower reservoir and an upper graduated cylinder. The following solutions are required : — (1) Magnesia mixture .... 100 c.c. 20 per cent, ammonia solution . . 100 ,, Pure talc (finely ground) ... 10 grammes. The magnesia mixture has the following composition : — Magnesium chloride crystals . . 100 grammes. Ammonium chloride Ammonia .... Water to .... (2) Silver nitrate .... Ammonia (strong) . Talc Distilled water The solutions may be made in bulk and kept. (3) A measured 21 hours sample of urine. To use the instrument close the stopcock and pour in urine (previously freed from albumin if necessaiy) to the 90 c.c. mark. Open the stopcock. Add 20 c.c. of the No. 1 solution. Shake well and allow to stand until all the precipitate of phosphates has settled into the lower chamber. Close the stopcock. Add No. 2 solution until the total fluid reaches the 100 c.c. mark. Continue to rotate the cylinder until the precipitate in it has become a yellowish white. Stand in a room of even temperature away from the light for 21 hours. Read the upper level of the precipitate in the cylinder. 110 250 1,000 c.c. 1 gramme. 100 c.c. 5 grammes. 100 c.c. SPECIAL INVESTIGATIONS OF THE URINE, ETC. 269 A scale is provided which gives the percentage of purine bodies in the urine corresponding to the number of cubic centimetres of precipitate. The estimation of creatine.— Creatinine is a normal constituent of the urine, and about 1 gramme of it is excreted daily. It is the anhydride of creatine and is obtained from it by boiling with an acid. Creatine is not found in the urine under normal circumstances. Creatinine is supposed to be derived from either ingested or muscle creatine ; but modern experimental physiology is partly opposed to this view, for the daily output of creatinine is very constant whatever the diet of the individual. Excess of creatinine in the food is excreted as creatinine, and excess of creatine as creatine. The seat of formation of creatinine is held by some to be the liver, and in certain diseases of the liver creatine is found in considerable amount in the urine. It is stated that a neoplasm of the liver, whether primary or secondary in origin and however small, leads to an excretion of creatine. A similar excretion takes place with a liver abscess, but not with cirrhosis of the liver. Creatine may also appear in the urine in grave wasting diseases, or in any condition associated with acidosis. Should these statements be confirmed, and there is evidence in their support, the detection and estimation of creatine in the urine should in certain cases be a valuable aid to clinical diagnosis. In the absence of severe wasting a positive creatine estimation would be strongly in support of the diagnosis of hepatic neoplasm in doubtful cases of jaundice. The negative test is more reliable, since absence of creatine from the urine in cases of gastric or other abdominal carcino- mata would be in favour of operative treatment in so far as indicating that dissemination of the growth in the liver had not yet taken place. The actual estimation of creatine is comparatively rapid and simple, but requires a special apparatus. The estimation is performed in two parts. In the first part the creatinine is estimated. In the second the creatine which may be present is converted by hydrolysis with an acid into creatinine, and the total creatinine again estimated. The difference between the two estimations represents the creatine. The apparatus required for the estimation of creatinine is a 270 CLINICAL PATHOLOGY. somewhat expensive one, and is known as the Duboscq colori- meter. The colour estimation depends upon the fact that a layer of -~ potassium bichromate 8 mm. deep has the same colour as a layer of a solution made from 10 rngrni. of creatinine, picric acid and caustic soda 8*1 mm. deep. By a comparison of a solution of jr potassium bichromate with a solution of picric acid and caustic soda containing an unknown quantity of creatinine the amount of creatinine present can be reckoned from the depth of the solution, which corresponds in colour with the bichromate solution 8 mm. deep. The estimation of creatine is performed as follows : — A sample from a "2± hours specimen of urine should be used. Part 1. — To estimate the preformed creatinine. To 10 c.c. of urine in a 500 c.c. measure add 15 c.c. of a saturated aqueous solution of picric acid and 5 c.c. of 10 per cent, caustic soda. Shake well. Stand 5 minutes at room temperature. Make up to 500 c.c. with distilled water and mix. In one cylinder of the Duboscq colorimeter place «- potassium bichromate solution and set the glass plunger at 8 mm. depth. Place the treated urine in the other cylinder and by means of the screw move the plunger up and down until the colour of the two solutions is the same. Use davlight and take the mean of at least 6 readings. The calculation is reliable at readings between depths of 5 and 12 mm. The calculation is performed as follows : — When 8'1 mm. treated urine = 8 mm. of the standard solution, then 10 rngrni. of creatinine were present in the 10 c.c. of urine taken. Suppose the average reading to be 6 mm., then 10 c.c. of „ • , . 10X8-1 , the untie contains ^ rngrni. ot creatinine. Part 2. — To convert creatine into creatinine. Take 10 c.c. of urine in a small flask and add 5 c.c. of 3*5 per cent. HC1. SPECIAL INVESTIGATIONS OF THE URINE, ETC. 271 Heat at 120° C. in the autoclave for 30 minutes. Neutralise the HC1. exactly with strong caustic soda. Cool to room temperature; add 15 c.c. of the picric acid solution and 5 c.c. of the 10 per cent, caustic soda. Stand 5 minutes and wash out with distilled water up to 500 c.c. in a large measure glass. Repeat the colour estimation as in Part 1. Calculation : / NH * Minus /NH\, Creatine = NH-C V w f = Creatinine = NH-C \N-CHoCOOH vvatei \ XT I , * x N - CH 2 CH * ok (Molecular weight = 131) (Molecular weight = 113) m = M6. 113 Suppose reading of preformed creatinine =11 and of total creatinine . . . =8 Then preformed creatinine and total creatinine 10 X 8-1 11 10 X 8-1 The difference multiplied by 116 = creatine present in 10 c.c. of urine. Estimation of phosphates. A method of estimating the phosphates in urine finds a place in the majority of text-books and is given here. The estimation cannot be said to have any valuable bearing at present upon ordinary clinical medicine, and in the majority of cases the elimination of phosphates by the urine varies directly with that of uric acid. The phosphates can be estimated by the following volu- metric method, which depends upon the precipitation of the phosphates by uranium nitrate in the presence of acid sodium acetate. The indicator used is tincture of cochineal, which becomes green in the presence of excess of uranium nitrate. A standard uranium nitrate solution is used, 1 c.c. of which =0 005 gramme P20 5 . The acid sodium acetate solution is made by dissolving 100 grammes sodium acetate in a little water, adding 100 c.c. of strong acetic acid, and making up to 1 litre with water. 272 CLINICAL PATHOLOGY. The estimation is performed as follows : — Measure 50 c.c. of urine into a beaker. Add 5 c.c. of the sodium acetate solution. Add a few drops of cochineal tincture. Heat the urine to boiling, and run in the standard uranium nitrate solution so long as a precipitate is formed. Continue to run the uranium solution into the boiling urine drop by drop until the red colour becomes green. To calculate the result : — Multiply the number of cubic centimetres of uranium solution used by 0*005, and the result is the number of grammes of P 2 5 in 50 c.c. of urine. The average daily excretion of P 2 5 is from 2 to 3 grammes. Estimation of sulphates. — The sulphates of the urine are of two kinds — the inorganic, combined with sodium and potassium, and the organic or ethereal, combined with cresol, phenol, indole, scatole, etc. The inorganic sulphates are greatly in excess of the ethereal. The total sulphates are increased in febrile conditions and the ethereal sulphates with stasis of the intestinal contents and in suppurative processes. The most reliable methods of estimating the sulphates are gravimetric and are too long to be described here. The estima- tion of the total sulphates only has little significance. Estimation of chlorides. — The chlorides in the urine consist almost entirely of sodium chloride, and necessarily vary with the amount of salt ingested. The chlorides are diminished in all febrile conditions, and are very markedly diminished in lobar pneumonia. The diminution of the chlorides of the urine in pneumonia is more extreme than in almost any other condition, and is of diagnostic significance ; the return of the chlorides to normal or above the normal in this disease commences after the crisis. The amount of chloride in the urine can be roughly tested by filtering some urine into a test tube, adding a few drops of pure nitric acid and then silver nitrate solution until no more precipitate occurs. A thick, white, curdy precipitate of chlorides takes place in normal urine- In cases of pneumonia, only a faint cloud forms. SPECIAL INVESTIGATIONS OF THE URINE, ETC. 273 The chlorides can be estimated by the Volhard process. The principle of the method consists in adding to the urine an excess of a standard solution of silver nitrate and then estimating this excess by titrating the mixture with a standard solution of potassium sulpho-cyanide, iron alum being used as the indicator. The estimation is performed as follows : — Measure 10 c.c. of urine into a 100 c.c. measure. Add about 4 c.c. of pure nitric acid. N Add 20 c.c. of — silver nitrate solution. Make up to 100 c.c. with distilled water. Mix thoroughly and filter 50 c.c. of the mixture into a beaker. Add about 5 c.c. of a freshly-prepared solution of iron alum. Run in — potassium sulpho-cyanide solution until a reddish- brown colour which remains for 3 minutes is obtained. To calculate the result : — The number of cubic centimetres of ammonium sulpho- cyanide used corresponds to the number of cubic centimetres of silver nitrate in excess, and has to be deducted from the 10 c.c. added to the 5 c.c of urine (only half the quantity of the mixture of urine and silver solution was titrated). The difference in cubic centimetres represents the chlorides in 5 c.c. of urine, and this difference multiplied by '00585 = the grammes of chlorides, reckoned as NaCl, in 5 c.c. of urine. To obtain the percentage multiply by 20. The average daily excretion of chlorides is about 12 grammes. Cammidge's pancreatic reaction.— The position which this reaction holds among chemical aids to clinical diagnosis is a matter of considerable dispute. The reaction has been so widely used that it is desirable to describe it here. Cammidge's original reaction was a double one, consisting of two separate analyses described as test A and test B. From the nature of the crystals found in these two tests it was claimed that in the majority of instances not only could the existence of a pancreatic lesion be recognised, but also the nature of the lesion present. The original theory of the 18 274 CLINICAL PATHOLOGY. reaction appears to have been broadly as follows. An escape of the pancreatic juice leads to fat necrosis, with the splitting of neutral fats into fatty acids and glycerin. The fatty acids remain in the necrotic areas, and the glycerin is absorbed into the circulation and excreted by the urine. The original test was consequently devised to demonstrate glycerin in the urine by the production of glycerosazone crystals. The theory was subsequently considerably altered — the tests A and B were abandoned and replaced by a single process known as test C. The nature of the crystals found in the test was reconsidered, and the clinical significance given to the results of the test was modified. The present theory of the reaction appears to be as follows. The crystals produced result from the presence of a sugar complex, which on hydrolysis with hydrochloric acid yields a substance having the reaction of a pentose. The crystals, therefore, of a positive reaction are supposed to be pentosazone crystals (see also page 303). The pancreas is stated to contain five times as much pentose as any other organ in the body, and when partial disintegration of the pancreas occurs as the result of disease the Cammidge crystals will be obtained in the test. On this theory a mere blocking of the pancreatic secretion will not yield a positive urinary reaction, nor will fibrosis of the pancreas apart from active disease. Cammidge is consequently of opinion that a positive reaction is evidence of active degeneration, such as occurs in acute liEemorrhagic or in chronic pancreatitis, and a negative re- action contra-indicates active degeneration, but does not exclude old pancreatitis nor malignant disease of the pancreas. In 75 per cent, of malignant cases the reaction is negative. Cammidge does not claim that the test is always positive in cases of pancreatitis, nor that a negative test definitely excludes pancreatitis, but insists that the result must be con- sidered in conjunction with the clinical findings, with other urinary tests, and in particular with an examination of the fasces (see page 321). A considerable experience of the test has not convinced me of its practical value in diagnosis, and there can be no doubt that positive reactions of marked intensity may be obtained in a considerable variety of affections. At the same time it must be allowed that an abundant deposit of the SPECIAL INVESTIGATIONS OF THE UBINE, ETC. 275 crystals is an interesting abnormality and lias some meaning, however cryptic this may be at the present. Test C is performed as follows : — Preliminary. — Filter a portion of a 24 hours specimen of urine. Test for albumin. — If albumin is present in amount more than a mere trace, measure out 50 c.c. of the nitrate, and add a few drops of acetic acid. Boil. Cool. Filter, and make up nitrate to 50 c.c. Test for sugar. — Either Fehling's or Nylander's test, care- fully performed, must be completely negative. If there is any reduction on standing about 50 c.c. of the albumin-free urine must be mixed with yeast, fermented for from 12 to 24 hours, and filtered. Stage 1. — Measure 20 c.c. of the clear albumin- and sugar- free nitrate into a small flask with an inverted filter funnel placed in its mouth as a condenser. Add 1 c.c. of strong HC1. Boil on the sand bath for 10 minutes from the commence- ment of ebullition. The boiling should not be too vigorous, and the flame should be turned low for the greater part of the time. Stage 2. — Cool under the tap. Make up contents to 20 c.c. with distilled water. Slowly add 4 grammes of lead carbonate ; shake gently at first and more thoroughly later. Stand, and shake occasion- ally until no more gas comes off. Filter through a paper moistened with distilled water. Stage 3. — Add 4 grammes of powdered tribasic lead acetate. Shake thoroughly for some minutes and allow to stand. Filter through a moistened filter paper. Stage 4. — To the clear and almost colourless filtrate add 2 grammes of powdered sodium sulphate. Shake thoroughly for several minutes. Bring slowly up to the boiling point on a sand bath, shaking from time to time. The excess of lead is removed at this stage, and it is important that the shaking and heating should be done care- fully. Stage 5. — Cool under the tap and filter. Measure 10 c.c. of clear filtrate. Make them up to 18 c.c. with distilled water. 18—2 276 CLINICAL PATHOLOGY. Add 0*8 gramme of phenyl hydrazine hydrochlorate, 2 grammes of powdered sodium acetate, and 1 c.c. of 50 per cent, acetic acid. Boil in a flask with a funnel condenser on the sand bath for 10 minutes from the commencement of ebullition. Do not boil too vigorously. Filter hot through a filter paper moistened with boiling distilled water into a 15 c.c. measure. Should the filtrate fail to reach the 15 c.c. mark, make up to 15 c.c. with hot distilled water. Stand for from 4 to 5 hours or longer at room temperature or in the ice chest. Examine the filtrate for the appearance, solubility, and amount of crystal formation. The typical crystals, examined under the microscope, are of the osazone type and more circular and tuft-like than glucosa- zone crystals (see Plate XI.). Eun under the cover-slip 33 per cent, sulphuric acid ; the crystals should be dissolved in from 10 to 15 seconds. The crystals have to be distinguished from the coarse yellow needles which may be deposited if the excess of lead was not removed in stage 4. In a strongly positive reaction the deposit of crystals may occupy half the bulk of the filtrate. In a completely negative reaction the filtrate remains clear. In all cases the deposit should be examined microscopically and chemically. The Bacteriology of the Urino -genital Tract. The variety of pathogenic organisms commonly met with in the urino -genital tract is not very great, and in general the bacteriological examinations should be conducted on the lines indicated in the section dealing with bacteriology, where a further account of the organisms will be found. The special precautions to be attached to the investigation of this tract are considered under the heading of each organism. The gonococcus. — In the male the recognition of the gonococcus in a purulent urethral discharge is commonly a simple matter, and in the very great majority of cases the causative organism of an acute urethritis is the gonococcus. Occasionally some other organism is the cause of the inflam- mation, and in some cases a discharge of pus from the urethra SPECIAL INVESTIGATIONS OF THE URINE, ETC. 277 has some external origin, as for example a prostatic abscess, and may be caused by the colon bacillus or a staphylococcus. In cases of chronic gleet, so long as there is a considerable urethral discharge, gonococci commonly remain fairly numerous. Gonococci may in exceptional cases produce a urethritis associated with the causative organism which lasts for years. Not infrequently a history will be given of a urethritis which cleared up some years previously and which has again recurred. If a thick purulent discharge loaded with gonococci be found, it is extremely probable that the patient has exposed himself to a second infection. The latency of the gonococcus in the urethra is a most important question, and the examination of the urethra for evidence of such infection is a procedure requiring the co-operation of the surgeon and the pathologist. If a slight morning discharge is present, or if pus cells and prostatic threads are found in the morning specimen of urine, or even if the patient can detect no discharge at all, the urethra may still be infective. In all cases in which the patient seeks advice as to infectivity, and particularly with a view to matrimony, a thorough search has to be made for the gonococcus as well as for clinical evidence of urethral inflammation. The patient should come for examination in the early morning under instructions to hold his water until he has been investigated. The orifice of the urethra should be first examined, and if any discharge is found film preparations should be made from it and cultures taken on serum agar. The urine should then be passed and examined by the naked eye for prostatic threads. The urine should then be centrifuged and the deposit searched for pus and epithelial cells, and if these are present gonococci should be carefully looked for. The anterior urethra should next be thoroughly irrigated with sterile water. The patient should then be placed on his hands and knees and vigorous massage of the prostate performed. Several minims of clear or turbid prostatic fluid can often be made to flow out from the urethra, and this is collected and examined for the presence and nature of the cells and organisms. The urinary examination alone for gonococci should never be depended upon, since it is only exceptionally that gonococci can be recognised in a urinary deposit. If a purulent morning discharge is absent, if there is no pus in the urine, and if the prostatic fluid is clear and 278 CLINICAL PATHOLOGY. contains only mononuclear cells, the patient, in the absence of clinical signs or symptoms, is probably free from gonorrhoea. If any of these abnormalities are present the gonococcus may or may not be found. All cases of residual urethritis or prostatitis after gonorrhoea are by no means due to the gonococcus. Failure to find the gonococcus at one examina- tion does not, however, exclude the possibility of infectivity. A further search should be made and conducted on the same lines, but after the passage of a large-sized sound and the irrigation of the urethra with strong silver solution. The inflammatory cells resulting from these procedures should be searched for gonococci. If no gonococci are found the patient is as certainly free from infection as a reasonably careful investigation can prove. The examination of the female genito-urinary tract for gonococci is less commonly successful, even in the acute stage, and the special precautions to be adopted have been previously described (page 122). The spirochaeta pallida. — The demonstration of the spiroclaeta pallida has been described under the account of . that organism, and it is only reasonable that the clinical diagnosis of every genital chancre should be controlled by a bacteriological examination. In dealing with a chancre of the penis help in obtaining the clear serous exudate from the base of the cleaned ulcer may often be derived from the use of a small Bier's cup. The cup should have an orifice of about the size of a sixpence, the edge should be lightly smeared with vaseline, the rubber ball flattened and the cup firmly placed over the ulcer. On releasing the ball a quantity of serum is often sucked out, and the spirochgetes present in this may be very numerous. Spirochgetes, other than the syphilitic organisms, are not commonly present in genital chancres, but those of the refringens type may be very numerous in extensively ulcerated sores of the " soft " variety. Ducrey's bacillus. — To this organism is accredited the causation of the soft chancre. The bacillus may be found in the ulcer and in the buboes. It has been inoculated experi- mentally from the chancre in human beings, but the disease has not been reproduced in animals. The organism is a very small Gram-negative bacillus, which does not grow on the SPECIAL INVESTIGATIONS OF THE URINE, ETC. 279 ordinary media. On blood agar it grows in small, round, glistening colonies. The tubercle bacillus. — The method for the detection of tubercle bacilli in the urine has been described in Chapter XL, page 157. A 24 hours specimen of urine is preferable, but if the bacilli are abundant they may readily be found in a catheter specimen. It may be necessary to determine if one or both kidneys are tuberculous, and the question is sometimes solved by an examination of ureteric specimens from the two sides. Tubercle bacilli may be found on one side only or on both sides, but a negative examination is not very weighty evidence against tuberculous nephritis. Such specimens should also be examined for the presence of pus, care being taken not to confuse the small round mononuclear epithelial cells with pus cells. If no pus is present the kidney is probably healthy. Whatever the nature of the urinary specimen examined, a single failure to find the bacilli does not greatly outweigh clinical evidence. Two or more examinations may be required before the bacilli can be found in cases where they are scanty. It is not an infrequent experience to spend half an hour over a slide and to find only one group of bacilli. It is practically useless to examine any specimen for tubercle bacilli if no pus is present ; even if acid-fast bacilli are found, in the absence of pus there will be grave doubt as to their nature. The colon bacillus. — A catheter specimen, carefully taken after cleaning the urethral orifice, is essential for the detection of colon bacilli in the female. In the male a catheter specimen is preferable but not essential. If there is an objection to the passage of a catheter the glans penis should be thoroughly cleansed, and the patient should be instructed to pass the first portion of urine into an ordinary receiver, and the next portion into a sterile wide- mouthed flask fitted with a wool plug. When the specimen is obtained proceed as follows : — Pour approximately 3 to 4 c.c. of urine into a broth tube. Set aside the remainder of the specimen for further investigation. Incubate the culture at 37° C. for from 12 to 24 hours. Examine the broth culture for general turbidity, and microscopically for the presence of the bacilli, 280 CLINICAL PATHOLOGY. If a growth of bacilli has taken place, prepare two Petri dish plate cultures, one of which contains agar and the other MacConkey's medium. Take a platinum loop of the broth culture and make a series of streaks, first on the agar plate and next (without recharging the loop) on the MacConkey plate. Incubate the plate cultures and the original broth culture till the next morning. Examine both plates, and in particular the MacConkey plate. If large red colonies are present on the MacConkey plate and all are apparently of the same nature, subculture from them into the following media : — Litmus milk. Neutral red broth. Broth. Gelatin slope. Litmus dextrose. Litmus mannite. Litmus lactose. Incubate the sub-cultures until the next morning, but remember to place the gelatin slope in a separate crate at a temperature of from 18° to 22° C. Examine the sub-cultures for the following changes : — Acid and clot in milk ; green fluorescence in neutral red broth ; indole in broth, as shown by the production of a rose- pink colour on the addition of nitric acid ; growth on gelatin without liquefaction ; acidity and gas formation in the three litmus carbohydrate media. If all these changes are present the organism is the colon bacillus. If the changes have not yet occurred, reincubate the tubes for another 24 or 48 hours, or longer if necessary. The agar plate should also be examined for the presence of cocci in addition to the bacilli. Colonies of staphylococcal or streptococcal nature should be examined microsopically, and if cocci are present sub-cultures should be made in broth or on agar and the cultural character of the organisms further investigated. No cocci will be found on the MacConkey plate. If bacilli of the colon type are found on the agar plate and no growth has taken place on the MacConkey plate, this should be reinoculated from the original broth culture. If no growth occurs after reinoculation, colon bacilli are absent. If colonies of more than one type or colour are present on the MacConkey plate, a characteristic discrete colony of each type must be transferred to broth and sub-cultures from each SPECIAL INVESTIGATIONS OF THE URINE, ETC. 281 broth tube made on the following day into the series of media given for the colon bacillus. The catheter specimen of urine must on every occasion be examined generally as well as bacteriologically . Very little infor- mation is derived from the cultivation of a colon bacillus from the urine, and such information as is given may be totally misleading. Colon bacilli in the urine are not by any means the necessary explanation of an obscure fever or even of definite urinary symptoms. All bacterial investigations must be considered in conjunction with the general examination of the urine and the clinical condition of the patient. The further examinations of the urine to be invariably made are simple, and concerned with the appearance, the reaction, the presence and amount of albumin, and the nature of the deposit. The following types of urinary infection may be recog- nised : — In the latent type colon bacilli may exist in the urine of men or women, but more frequently the latter, without pro- ducing any signs or symptoms of disease or any changes in the urine. In such cases the urine is normal, no bacilli are seen in the centrifuged deposit, and a growth of the colon bacillus is obtained on culture. This class of case constitutes the "colon carrier." The condition is liable to pass into the acute inflammatory stages, particularly if pregnancy or some other complication arises. In the acute type the patient has marked vesical and often renal symptoms, has high fever, sometimes with rigors, and may present all the aspects of extremely serious illness. The urine is turbid on passing, the turbidity being due to the enormous numbers of bacilli. Pus cells are present, but they may be scanty, and red cells may be found. A trace of albumin is present. Such patients practically always recover from this severe stage under treatment and often, apparently, in spite of treatment. They, however, frequently pass into the chronic type. In the chronic type, which may persist for months and may be apparently chronic from the onset, some other pre- disposing cause, such as pregnancy, obstinate constipation, hemorrhoids, or an ischio-rectal abscess, is often present. The urinary symptoms are subacute or absent, a mild and inter- mittent pyrexia is present, and the urine is acid, containing a 282 CLINICAL PATHOLOGY. trace of albumin and a considerable naked-eye deposit, due to pus and bacilli. The presence of colon bacilli, therefore, in a culture taken from the urine is insufficient evidence that the bacilli are actively producing disease, and if no pus is present it is almost certain that they are not. Even if pus and bacilli are present it does not follow that the ultimate diagnosis has been reached. Infection by the colon bacillus is readily superimposed upon some other lesion, such as that of tuberculosis or calculus. The bacillus proteus is in such instances often present in addition. In all cases of coli infection of the urine associated with a considerable degree of pyuria it is a wise pre- caution to search the urine for tubercle bacilli also. The treatment of coli infections by autogenous vaccines is worthy of trial in selected cases. The bacillus proteus. — The examination for this organism is conducted in exactly the same manner as that for the colon bacillus. The bacilli grow well on the MacConkey plate, and are recognised by the yellow colour of their colonies, the more slimy nature of their growth, and their somewhat character- istic and offensive odour. The cultural character which mainly distinguishes them from the colon bacillus is the power of liquefying gelatin. Bacilli which in other respects are cul- turally identical with the colon bacillus may grow in yellow colonies on the MacConkey plate. Pure infections of the urinary tract by B. proteus occur, but the organism is more frequently found in association with other bacteria and other lesions of the tract, such as neoplasm of the bladder or renal calculus. In cases of proteus infection the urine is commonly alkaline. The typhoid bacillus. — This organism is examined for by the same routine. The cultural characters are given under the description of the bacillus. The bacillus should be further identified by serum agglutination tests, using both the serum of the patient and that from a known case of typhoid fever. The typhoid bacillus may exist in the urine without pro- ducing symptoms, and in such cases is a dangerous source of infection to others. Bacilluria following typhoid fever is common, but is in the majority of cases due to the B. coli. SPECIAL INVESTIGATIONS OF THE URINE, ETC. 283 Staphylococci : Streptococci : Diplococci.— Any of the ordinary pyogenic cocci may be found in the urine, but in the great majority of cases they come from the lower part of the tract and are of little importance. The specimen for examination mast be a catheter specimen, and preferably should be withdrawn after thorough irrigation of the urethra with sterile water. The urine should be first added to broth, and, if the growth in the broth tube appears to be purely coccal, plate cultures should be made on two agar Petri dishes. The colonies on agar are examined in the usual way. Any of the three varieties of staphylococci may be present, and may produce a cystitis associated with a purulent and often alkaline urine. Staphylococcal cystitis is rarely primary, and is more often superimposed upon some other lesion, such as a urethral stricture or prostatic enlargement. Streptococci are less frequently met with, if the urethral lavage has been thorough. A long chained streptococcus, of low pathogenicity to animals, is the variety most commonly found. Pneumococci in the urine are often recorded and extremely rarely found. The organism commonly confounded with the pneumococcus is a paired coccus met with frequently in the urinary tract. This diplococcus is Gram-positive and morphologically resembles the pneumococcus, but is readily distinguished by its cultural characters. The coccus grows abundantly in all media, producing an acid reaction. Litmus milk is characteristically clotted and completely decolorised in about 12 hours. Abundant growth in pin-point colonies occurs on gelatin without liquefaction. The coccus is non- pathogenic to mice. The organism is more commonly met with in specimens from women and children than from men, and in the majority of cases gives rise to no symptoms. It may, however, be found in pure culture in cases of cystitis or pyelitis, and it has been recovered from the general circulation. Other organisms. — M. melitensis can, in the great majority of cases of Malta fever, be isolated from the urine during the early period of the disease. The cocci grow slowly and in small colonies upon agar. Bacilli of the influenza type are occasionally met with in association with a purulent urethral discharge in which no 284 CLINICAL PATHOLOGY. gonococci can be found. Such unusual infections may follow sexual intercourse. Diphtheroid bacilli are frequently found in the urine, and are practically always derived from the lower urethra. The appearance of these organisms in the cultures is evidence of urethral contamination. The bacilli are apparently harmless. In conclusion, any of the organisms of the colon-typhoid group not previously mentioned may be found in the urine, and bacilli of this group which are difficult to exactly classify are fairly frequently met with. SECTION Y. THE ALIMENTARY SYSTEM. CHAPTER XX. The Mouth— The Stomach. CHAPTER XXI. The Pancreas — The Liver — The Spleen — The Peritoneum. CHAPTER XXII. The Faeces. CHAPTER XXIII. The Parasitology of the Faeces. CHAPTER XX. the mouth the stomach. The Mouth. The laboratory investigations of the buccal cavity are mainly bacteriological in nature. Examinations of the chemical composition of the saliva or of its cellular content have very little practical bearing. Oral sepsis and intestinal toxaemia have borne the odium of all the ills which man may suffer. There can be no question that a thoroughly septic condition of the mouth, leading to an absorption of offensive pus, may set up con- siderable disturbance, of which the most direct effect is gastric or intestinal in nature. More remote lesions due to the absorption of toxins into the circulation are also possible, but difficult to prove. Of these the most probable is a variety of osteo-arthritis, which more particularly, attacks the phalanges, and is very commonly associated with some local septic lesion such as pyorrhoea alveolaris. The bacteriological investi- gation of the mouth is reasonable in such cases, and is occasionally profitable. The practitioner should, however, be warned against the indiscriminate use of vaccines prepared from organisms of the buccal cavity. Nothing is more simple than the preparation of such a vaccine for any and every condition, and nothing would be more futile were it not for the mental effect of a hypodermic injection upon a confiding patient. Pernicious anaemia, the leukaemias, arthritis of every variety including gout, and almost every form of gastric and intestinal disorder, have been attributed to oral sepsis. I have even known prolapse of the kidney explained by the same cause. We can be certain at any rate of this, that all such diseases readily develop in the absence of any oral sepsis, and if present may progress in spite of its cure. These views are not of course opposed to the rational treatment of a septic mouth, nor can it be imagined that the absorption of pus from infected gums is anything but harmful. THE MOUTH— THE STOMACH. 287 Pyorrhoea alveolaris is readily recognised by the exudation of pus from the sockets of the teeth. In severe cases the majority of the teeth are loose and almost floating in a purulent bed. The teeth themselves may show little or no signs of caries. In order to make a cultural examination the mouth should be well washed out with clean and preferably sterile water, and a loop of pus as it exudes after pressure on the crown of a tooth be taken in a platinum wire. If little discharge is present, a sterile wool swab similar to that used for diphtheria cases may be rubbed against the root of the tooth. If it is advisable to extract a tooth, cultures may be taken from the socket. The platinum wire or swab should be rubbed over the surface of an agar or nasgar slope, or preferably by a series of streaks over two agar plates. Films of the pus should also be made. The cultures are incubated till the next morning, and the nature of the colonies investi- gated by the hand glass and microscopically. Sub-cultures should be made from each variety of colony on the plate and their full cultural characters investigated. The predominant organism in the pus films and in the plate cultures should be noted. In the films of pus numerous spirilla and fusiform or beaded bacilli can often be seen, but they do not grow in the cultures. If a vaccine is required it should be made from the predominant organism of greatest virulence, and in a case of doubt a mixed vaccine can be prepared. The organism most commonly found and most frequently predominant in the plate cultures is a short-chained streptococcus of the brevis type. This organism grows in short chains of 6 to 10 members, and very constantly acidifies and clots litmus milk. It appears to be more closely allied to the pneumococcus than to the streptococcus pyogenes, but differs from both in being practically non-virulent to mice and rabbits. Other organisms which may be obtained are other varieties of streptococci, micrococcus catarrhalis, and less commonly the pneumococcus. Staphylococci are usually present in addition, and almost any of the pathogenic organisms, including the bacillus coli, may be occasionally isolated. Thrush is an infection of the buccal cavity by the oidium albicans. It is common in children, but occurs also in adults, and particularly in febrile patients the care of whose mouths has been neglected. A white, flaky membrane is present and 288 CLINICAL PATHOLOGY. may spread over the entire buccal cavity, including the palate and the tongue. The membrane is usually detachable with- out leaving a raw surface. In children the membrane has to be distinguished from particles of milk left in the mouth after feeding. In films made from the membrane an abundant interlacing mycelium is usually found, together with a few of the yeast-like oval cells. In cultures the cellular element of the organism may predominate. The condition is readily cured by ordinary antiseptic treat- ment. Diphtheria produces a membrane which is most commonly confined to one tonsil, and is removed with difficulty, leaving a bleeding surface. There may be little constitutional dis- turbance. The methods of taking and examining swabs and cultures from suspected cases of faucial diphtheria have been already described (p. 129). The organisms are often difficult to identify in swab preparations, but are readily recognised after from 8 to 12 hours' incubation on blood serum. Vincent's angina consists in an ulcerative stomatitis, often with membrane formation. The condition is most frequently met with in young, ill-nourished children, and is accompanied by severe general symptoms. In films made from the ulcerated surface the organisms associated with Vincent's name are very numerous. Pus cells may be scant}' and lying on a. background of innumerable spirilla of varying shape and size. The fusiform bacilli are less numerous and of similarly variable size, some being stout and cigar-shaped, others long, thin, and beaded. It is by no means certain that these organisms are the essential cause of the stomatitis. They are very numerous in almost any septic mouth, and in Vincent's angina other pyogenic organisms, such as strepto- cocci and staphylococci, are present in addition. Follicular tonsilitis is characterised by swelling of one or both tonsils, and by numerous small, round yellow spots of suppuration in them. The organisms found in the pus are commonly staphylococci or streptococci. Streptococcal inflammation may produce swelling and hyperemia of the tonsil only, and these organisms are usually associated with the angina of scarlet fever as well as with the tonsilitis which almost invariably precedes by 10 days or a fortnight an THE MOUTH— THE STOMACH. 289 attack of rheumatic fever. The streptococcus associated with scarlet fever is often a long-chained one, while that obtained from the rheumatic tonsil is usually of the brevis type. Streptococci may also set up a membranous tonsilitis closely resembling that of diphtheria, but usually associated with severer constitutional disturbance. The distinction between the two conditions is readily made on bacteriological grounds. The Stomach. Laboratory investigations into the functions of the stomach are mainly chemical in nature and are principally concerned with an analysis of the gastric juice. The diagnosis of a condition associated with gastric symptoms as " dyspepsia " has little real meaning and carries us no further than a diagnosis of " anaemia " in other conditions. A careful analysis of the gastric contents associated with the clinical examination of the patient enables us in the great majority of cases to recognise the condition present and the treatment which should be adopted. Of all the methods of investigation into the composition of the gastric juice, that of the " test meal" analysis is the most important. The Test Meal. — The sole difficulty in the procedure lies in the withdrawal of the test meal, in reality a very simple manoeuvre. The passage of an oesophageal tube is uncomfort- able, but free from pain or danger in careful hands. The objection of the patient to its passage varies inversely with the tact and confidence of the practitioner. The meaning of the results obtained by the analysis of gastric contents will be discussed later. If the various precautions indicated below are properly observed, the results of the analysis may be depended upon. It must be very clearly understood, however, that these results considered alone are of practically no value in diagnosis, but must be read in conjunction with the signs and symptoms of the patient. It is a most elementary error to suppose that absence of free hydrochloric acid in a test meal means that carcinoma of the stomach is present. There are even circumstances in which absence of free acid is opposed to the diagnosis of carcinoma. A carefully taken history of the case read in conjunction with the analysis of the test meal will usually lead to a correct diagnosis, but all other methods of investigation should be freely made use of. p. 19 290 CLINICAL PATHOLOGY. The following is the technique of gastric analysis : — (1) Lavage of the stomach. — It is not necessary to wash out the stomach before giving the meal in the majority of cases, and indeed a more reliable result is obtained if lavage is omitted. In cases of obvious dilatation of the stomach with retention of food the stomach should be emptied and washed out with water a few hours before giving the meal. (2) The test meal should be of a simple character. It is most important that in any series of observations the same type of meal should be given, and that in any single observa- tion the nature of the normal result obtained from such a meal should be known. The figures given here are those obtained from gastric contents after a test meal which is practically that of Ewald. The meal consists of two large cups of tea, with milk and sugar if desired, and two rounds of toast lightly buttered. The actual bulk of the meal is unimportant, since the quantity of gastric juice accommodates itself to the bulk of material in the stomach. The quantity given, however, should be considerable in order to facilitate the subsequent with- drawal. (3) Removal of the gastric contents. — These should be removed exactly 1 hour after the test meal has been given. A clean rubber oesophageal tube, the outside of which has been moistened in hot water, is passed gently down the oesophagus into the stomach. The passage is aided by swallowing movements on the part of the patient, who may be lying in bed with his head propped up on pillows, or sitting up. Attached to the tube by a glass junction should be a second piece of tubing, terminating, if desired, in a glass filter funnel, and the whole must be sufficiently long to allow the funnel to be held well below the level of the stomach. As soon as the tube is in the stomach, that is to say has passed freely at least 18 inches beyond the teeth, and the resistance of the stomach wall is felt, depress the funnel over a clean specimen glass or other receptacle and tell the patient to strain. The contents usually flow out quite readily. If the flow does not commence, alter slightly the position of the oesophageal tube by withdrawing it a little or pushing it further in. The flow can sometimes be aided by flattening out a few inches of the tube with the fingers of the THE MOUTH— THE STOMACH. 291 two hands and releasing first the portion nearest the stomach, thus creating a partial vacuum to suck the contents out. It is never necessary to use a pump. The straining movements of the patient, aided if necessary by cautious pressure of the hand on the epigastrium, are sufficient to expel the contents. It is wise to have a clean wide receiver ready in case the stomach contents are vomited during the passage of the tube, but this can usually be avoided if the patient is told to refrain from straining until the tube is in its place. After the contents have been with- drawn the stomach can be washed out if such treatment is indicated. Precautions. — All medicines must be countermanded for several hours previous to the giving of the test meal. No water should be added to the test meal, and none passed down the tube to start the flow of gastric contents. (4) The examination of the gastric contents. — Note the amount obtained and the appearance. The microscopic examination of slides made from the contents is of little value. Some stress has been laid upon the presence of bacilli and sarcinse in film preparations, but bacteria seem to be commonly present in most gastric contents. Sarcinse are perhaps most common in cases of dilated stomach with gastric fermentation. Filter the contents through a filter paper moistened with distilled water. With the filtrate perform the routine examination described below. The materials required are a porcelain evaporating dish, a burette, a beaker, a 10 c.c. pipette, decinormal caustic soda solution, Topfer's solution, phenol-phthalein solution, absolute alcohol, phloroglucin, and vanillin. Topfer's solution consists of a 0*5 per cent, solution of dimethylamidoazobenzene in absolute alcohol. Phenol-phthalein solution is of 0'5 per cent, strength in 50 per cent, alcohol. The procedure is as follows : — (a) Perform Giinzburg's test for free HC1. In a porcelain evaporating dish place a pinch of phloro- glucin and a pinch of vanillin. Dissolve in about 3 c.c. of absolute alcohol. Add about 3 c.c. of the filtered test meal. Evaporate slowly on a sand bath without boiling. 19—2 292 CLINICAL PATHOLOGY. A rose-pink colour forms on the dish in the presence of free hydrochloric acid. A reddish-brown colour of the residue after complete evapora- tion is due to charring of the proteids, and must not be confounded with the rose-pink of a positive test. The exact proportions of Gunzburg's reagent are phloro- glucin, 0*5 gramme ; vanillin, 0*25 gramme ; absolute alcohol, 10 c.c., to which are added 10 c.c. of the filtered test meal. The reagent does not keep in solution and must be freshly prepared. It is not necessary to measure the ingredients, nor to use such large quantities. (b) Estimation of the acidity. — Wash out a clean N burette with a few cubic centimetres of — NaOH and then fill with ~ NaOH. Wash out a clean 10 c.c. pipette with a few cubic centimetres of the test meal filtrate, and then measure 10 c.c. of the filtrate into a large clean beaker. Dilute the filtrate with about 4 times its volume of distilled water. Add 2 drops of Topfer's solution. In the presence of free HC1 the mixture turns red. Read the level of fluid in the burette. N Carefully run in — - NaOH until the red colour becomes yellow. The end-point is often blurred, and the change of colour passes gradually from red, through an orange colour, to a lemon -yellow tint. As soon as the last trace of pink has gone and the fluid is definitely yellow, take the reading of the burette. Add 2 drops of phenol-phthalein. N . . Continue to run in — NaOH until a faint, permanent pink colour is produced. Again read the burette. The result is calculated as follows : — The difference between the original level of the soda solution and the first reading gives the number of cubic centimetres of N -.-pr-NaOH required to neutralise the filtrate to Topfer's solu- THE MOUTH -THE STOMACH. 293 tion. In the presence of a positive Giinzburg's reaction this amount may be taken, with certain reservations, as the equiva- lent of the free HC1 present in 10 c.c. of the test meal. The calculation may be made in terms of HC1 ; thus if 5 c.c. of decinormal soda were required for 10 c.c. of the nitrate, 50 c.c. would be required for 100 c.c. of nitrate : 1 c.c. of decinormal soda is the equivalent of 1 c.c. of decinormal HC1.* The molecular weight of HC1 is 36 - 5 grammes, therefore 1 c.c. of -^ HC1 contains -00365 gramme of HC1, and 50 c.c. contains 50 X '00365 = - 1825 gramme, which is the amount con- tained in 100 c.c. of the test meal. The difference between the original level of the soda solution and the second reading N gives the number of cubic centimetres of — NaOH required to neutralise the filtrate to phenol-phthalein. This amount is known as as the " Total Acidity," and includes the acidity due to free HC1, to protein HC1, to any organic acids which may be present, and to acid salts. The total acidity is not reckoned in terms of HC1, but is commonly given as the number of N cubic centimetres of ^ NaOH required to neutralise 100 c.c. of the filtered test meal. Thus, if 7 c.c. of soda were used for 10 c.c. of the nitrate, the total acidity is recorded as 70. The fallacies of the estimation. Presence of free HC1. — For reasons that need not be entered into here, there is little doubt that free HC1 may be present in exceptional test meals, and yet Giinzburg's test may be negative. Topfer's reagent may give a red colour with substances other than free hydrochloric acid. There is no certain chemical test in ordinary use for the demonstration of free HC1 in a test meal. If Giinzburg's test is positive free HC1 is certainly present ; if the test is negative it is advisable to presume that free HC1 is absent. In a normal test meal Giinzburg's test is positive. Amount of free HC1. — With the exception of the estimation of the inversion of cane sugar by the polariscope, there is no accurate method for the determination of the * A normal solution of a monobasic acid or alkali contains the molecular weight of the substance per litre. 294 CLINICAL PATHOLOGY. amount of HC1 in a test meal. The polariscope method is too difficult for use as a routine clinical method, and is not described here. Willcox lays stress on the amount of " active " HC1 as estimated by the Volhard process. By " active " or available HC1 he means the sum of free HC1 and HC1 combined with protein. To arrive at this measurement, the total chlorides present in 10 c.c. of the test meal are estimated as described under the estimation of chlorides in the urine (page 273). A second 10 c.c. are evaporated to dryness and then heated over gauze to drive off the free and protein HC1 ; the residue is dissolved in water, and the remaining or inorganic chlorides are similarly estimated. The difference between the total and the inorganic chlorides gives the "active" HC1. The result almost invariably corresponds closely with the total acidity, and appears to give a figure for the "active" HC1 considerably in excess of what is actually present. The Topfer acidity, as described above, is a roughly accurate measure of free HC1 under certain limitations. The reagent gives a red colour with lactic acid in excess, but not in small amount, as well as with other substances which may occa- sionally be present. It does not react with protein HC1 nor with acid salts. Briefly, when the free HC1 is in normal amount or in excess lactic acid is practically absent, and the Topfer acidity is a fairly accurate estimation of free HC1. If the HC1 is diminished or absent, the Topfer acidity may record considerably more free HC1 than is present. A definite Topfer acidity may be present with a negative Giinzburg reaction. For clinical purposes we require to know if the free HC1 is normal, increased, decreased, or absent. The Topfer acidity read in conjunction with Gunzburg's test gives us this information in almost every instance. The normal Topfer acidity is the equivalent of 0*08 to 0"1 gramme of HC1 per cent. The total acidity. — The phenol-phthalein method is accurate, but includes a variety of substances. Alterations in the amount of the total acidity are of considerable clinical significance, and in the reading of a test meal result chief reliance is to be placed upon Gunzburg's test and the measure of the total acidity. The normal total acidity is from 40 to 50. THE MOUTH— THE STOMACH. 295 Summary of procedure. — Perforin Gunzburg's test for free HC1. If positive, add a few drops of Topfer's reagent to 10 c.c. of N nitrate. Run in r-r NaOH till the red colour becomes yellow. Calculate result in terms of HC1 (normal 0*08 to O'l per cent.). Add a few drops of phenol-phthalein, and continue to run . N in r-r NaOH till the colour becomes red again. Calculate total N acidity in terms of cubic centimetres of r-pr NaOH (normal 40 J 10 to 50). If Gunzburg's test was negative, the Topfer estimation may be omitted. The whole procedure takes about 10 minutes. The variations in disease. — Free HC1 may be absent, as shown by a negative Giinzburg reaction, in the following conditions. Carcinoma of the stomach is almost invariably associated with anegativeGiinzburgreaction. The free HC1 may be absent within a very few weeks or even a few days of the onset of symptoms. It is unfortunately the fact that an inoperable carcinoma may be found in an equally short period. In the rare form of carcinoma which appears in a patient who gives a long and definite history of previous "attacks" of gastric ulcer, free HC1 may persist. A positive Giinzburg reaction in a patient with a comparatively short history of carcinoma is very strong evidence against a diagnosis of gastric carcinoma. Carcinoma elsewhere than in the stomach may in a minority of cases, the majority of which are cachectic, lead to an absence of HC1. The total acidity is practically always low in gastric carcinoma. The average total acidity is 26. In severe anaemias, including pernicious anaemia, the gastric juice has a very low total acidity and no free HC1. Such conditions are benefited by gastric treatment as an adjuvant to treatment directed against the anaemia. In the rare condition known as achylia gastrica, the gastric juice is practically absent. Gunzburg's test is negative, the total acidity is in the neighbourhood of 10, and pepsin is absent. In such cases the passage of food through the 296 CLINICAL PATHOLOGY. stomach is very rapid, the amount of the test meal recovered from the stomach is small, and the meal may have to be withdrawn in half an hour or less. In cholelithiasis free HC1 is almost always absent, an important diagnostic point between gall stones and duodenal ulcer. In chronic gastritis, particularly of the atonic type, and in the later stages of alcoholic gastritis, free HC1 may be absent. These varieties of dyspepsia are rapidly relieved by the administration of pepsin and hydrochloric acid by the mouth. Free HC1 is almost invariably absent after a successful gastrojejunostomy has been performed, apparently because of the neutralisation of the gastric juice by admixture with the biliary secretion. This neutralisation is a most impor- tant and not too widely recognised effect of the operation for the cure of duodenal ulcer. In two such cases in which free HC1 persisted after the operation peptic jejunal ulcers followed. Free HC1 may be increased in the following conditions. Duodenal ulcer is almost invariably accompanied by a great increase in the free acid and in the total acidity. The average free acidity in a series of cases was 0*17, and the average total acidity was 69. Exceptionally the total acidity may rise above 100. So long as the gastric contents are kept neutralised by drug treatment in these cases the symptoms disappear and complications are most unlikely to occur. Simple gastric ulcer is accompanied by a similar but less marked increase in the acidity. The average of the free HC1 is 0'13, and of the total acidity 58. Higher readings are, of course, frequent. The acidity commonly falls very low, and the free HC1 may disappear, after a considerable hsematernesis. The fall in acidity is accompanied by an improvement in symptoms. Hyperchlorhydria or increase in the acidity occurs in the absence of ulceration, and may be very pronounced. It gives rise to symptoms which are readily alleviated by the administration of alkalies. Increase in the acidity is not uncommon in hysterical subjects and may be present in some forms of acute gastritis, particularly of the alcoholic type. THE MOUTH— THE STOMACH. 297 (c) Further investigations of the test meal. Microscopic investigation has been already referred to. It is remarkable how rarely anything of practical importance is made out by this means. Lactic acid, if present in sufficient amount to give the ordinary tests, is abnormal. The presence of lactic acid is practically always associated with a negative Giinzburg reaction and a total acidity of moderate amount, that is from 30 to 40. The demonstration of lactic acid in a test meal is no real additional evidence of carcinoma. Lactic acid is commonly present in carcinoma cases, as well as in chronic gastritis with diminution or absence of HC1. To test for lactic acid proceed as follows : — To 10 c.c. of the filtered test meal add about 20 c.c. of ether. Mix thoroughly by repeated inversion in a separating funnel. Allow to stand for half an hour. Evaporate down the ether extract in a beaker standing in hot water (not over a flame). To the residue add about 3 c.c. of distilled water. With the watery solution perform Uffelmann's test as follows : — Fill a test tube one-third full with 1 in 20 carbolic acid. Add 1 drop of ferric chloride. Dilute the mixture with water until the amethyst blue colour is just transparent. Add the watery solution of the ether residue to the mixture. In the presence of lactic acid the amethyst blue of the reagent becomes decolorised and changed to a canary yellow colour. The test is distinctive, but not very delicate. Pepsin. — The pepsin content of a test meal is of con- siderable importance and is readily estimated. Pepsin appears to vary roughl ywith the amount of the free HC1 and the total acidity. It is, however, rarely absent except in cases of achylia gastrica. The edestin method for the estimation of pepsin is a simple and very reliable one. Edestin is a crystalline albumin obtained from eggs. It is converted by pepsin into edeston. 298 CLINICAL PATHOLOGY. Edeston is soluble in sodium chloride. Edestin is pre- cipitated by sodium chloride. Pure edestin can be obtained from Merck. To perform the estimation : — Get ready the following solutions. (A) ^ HC1 30 c.e. Distilled water ... 70 c.c. Edestin . . . . - l gramme. The edestin should pass readily into solution, but if it does not the mixture may be heated and if necessary filtered. (B) A saturated solution of NaCl. (C) The filtrate of the test meal. Into each of 10 clean test tubes measure 1 c.c. of the edestin solution. Number each test tube from 1 to 10. To No. 1 add 1 c.c. of the test meal. Mix. Take 1 c.c. of the mixture and add to No. 2. Mix. Take 1 c.c. of the mixture and add to No. 3, and so on to No. 10. Leave at room temperature for half an hour. Add a few drops of NaCl solution to each tube. In the tubes which remain clear peptic action is complete. With normal gastric juice the last clear tube will be 6, 7 or 8. Rennin is considered by some authorities to be identical with pepsin, and is allowed by all to vary in amount directly with the quantity of pepsin present. The rennin can be estimated by the coagulating action of the gastric contents on milk. To perform the estimation proceed as follows : — Label 10 clean test tubes from 1 to 10. Into each tube measure 1 c.c. of distilled water. Into tube 1 measure with a pipette 1 c.c. of the test meal filtrate. Mix. Take 1 c.c. of the mixture and add to tube 2. Mix again and take 1 c.c. of the mixture and add to tube 3. Continue the process to tube 10. The filtrate is thus pro- gressively diluted by half in each tube. To each tube add 5 c.c. of fresh milk. Stand the tubes in a crate in water at room temperature for 2 hours. Stand in a water bath at 37° C. for 5 minutes. Remove and examine the tubes for coagulation of the milk. THE MOUTH— THE STOMACH. 299 With normal gastric contents coagulation will have occurred up to tubes 6, 7 or 8. Blood should not be present in a test meal, but may be found in considerable amount in cases of carcinomatous, and less often of simple gastric ulcer. Blood in quantity visible to the naked eye may be confirmed by microscopic examination ; blood in less amount should be tested for as follows : — To 5 c.c. of filtered test meal in a test tube add 2 c.c. of acetic acid. Shake with an excess of ether. Allow to stand. Pour off clear ether. Add to the ether 10 drops of a freshly- prepared alcoholic solution of guaiacum resin and 20 drops of a 5 per cent, solution of hydrogen peroxide. In the presence of blood the mixture becomes coloured blue of greater or less intensity. Bile may be present in the test meal in cases of jaundice, and after the performance of a gastrojejunostomy. The bile pigments should be tested for in the usual way. Other gastric investigations. — (1) The motility of the stomach. Sahli's desmoid test. — The test does not involve the use of a stomach-tube and depends upon the fact that gastric juice alone can digest raw catgut, which is quite insoluble in the pancreatic juice. The test is not precisely one of the motility of the stomach, but a general test of the gastric digestive function. The test is performed as follows : — A rubber membrane of the finest Para rubber, 0*2 mm. thick, is used as a wrapper. In it is placed a methylene blue pill made according to the following prescription : — Methylene blue (medicinal) . . 10"0 grammes. Pulv. glycyrrhizae . . . 10"0 „ Bismuth subnitr. . . . 100*0 „ Glucose syrup (sat.) q.s. Divide into 200 pills. The edges of the membrane are closed firmly, but gently, by winding round them a piece of the best catgut 0*3 mm. thick. During the ordinary mid-day meal, and immediately after 300 CLINICAL PATHOLOGY. soup, the patient is instructed to swallow the sac with the aid of a little water. The patient is told to pass water at ,5 p.m., at 7 p.m., and on rising the following morning. The samples of urine are kept separate. If the methylene blue appears in the urine of the same evening or of the following morning digestion is considered to be normal. If the methylene blue fails to appear, either gastric digestion is incomplete, or the stomach is hypermotile. A delayed reaction taking place on the afternoon or evening of the following day points to incom- plete digestion in the stomach associated with hypoacidity and gastric stasis. The iodopin test. — This test is considered to bear directly upon the motility of the stomach. It is performed as follows : — A quarter of an hour after a test breakfast the patient is given a teaspoonful of iodopin. Subsequently the saliva is tested every 15 minutes for iodine. The patient is provided with a series of papers which have been soaked in a starch emulsion and instructed to salivate upon them at regular intervals. Normally the iodine appears in the saliva in from 60 to 75 minutes. It cannot be said that either of the above tests are particularly satisfactory. Nor can the information derived from them compare with the results of an accurate investiga- tion of a test meal. Both methods, however, as well as numerous others of a similar kind, are fairly extensively used. The fasting stomach. — The motility of the stomach can be estimated by examination in the fasting condition. The ordinary evening meal is taken and no other food until 10 o'clock on the following morning, when a tube is passed and the contents, if any, are removed. Under normal conditions the stomach should be quite empty within 7 or 8 hours of an abundant meal. If particles of food are found the next morning motor insufficiency is present. If a considerable quantity of fluid is withdrawn containing a definite percentage of hydrochloric acid and no particles of food, hyperacidity is present. A little fluid containing a faint trace of acid is not abnormal, since it may be induced by the passage of the tube. The test for motility of the stomach may be combined with the examination of the test breakfast. The ordinary dinner is given in the evening, and with the THE MOUTH— THE STOMACH. 301 dinner half a dozen or more raisins are swallowed whole. At 9 o'clock the following morning the test breakfast is given. At 10 o'clock the breakfast is withdrawn and examined in the ordinary way. If the raisins or fragments of them are present in the gastric contents, hypomotility can be diagnosed. (2) Examination of the vomit. — The points to be investigated in vomited material — without considering the detection of poisons — are the following : — Blood should be examined for and, unless in very obvious amount, tested for by the method given above. Among the more important conditions associated with hsemateniesis are cirrhosis of the liver, gastric ulcer, simple and malignant, and rarely duodenal ulcer. Small quantities of blood in the vomit are of little importance, and may be produced by the act of vomiting from a tiny ruptured vessel. Larger amounts may come from elsewhere, as from the nose, or lungs, or mouth, and appear after being swallowed. Profuse haemor- rhage from the gastric mucous membrane may follow minute erosions in the absence of macroscopic ulceration, and is usually accompanied by hyperacidity. Bile is of little importance, and is likely to occur with any form of vomiting if prolonged. Faecal vomiting is characteristic of intestinal obstruc- tion. The vomit is of a brownish-black colour resembling that of altered blood, and of a faecal odour. Faecal vomiting is very occasionally met with in purely functional cases, and it may happen with such patients that the odour and colour of a turpentine or soap enema may appear in the vomit. Mucus in large, tough masses may be seen in the vomit in cases of chronic catarrhal gastritis with hypoacidity. It is found in the morning vomit of chronic alcoholic patients. The acidity of the vomit is in the majority of cases scarcely worth estimating ; but the expulsion of a quantity of acid fluid some hours after a meal is evidence of hyper- secretion. (3) The bacteriology of the stomach.— Although it is held by some authorities that many gastric disorders are due to bacterial infection, the bacteriological investigation of the stomach contents is at the present of little value. The anti- septic action of the gastric juice is no doubt slighter than was 302 CLINICAL PATHOLOGY. formerly supposed, and micro-organisms of any and every kind can, on occasion, be found even in highly acid gastric contents. The presence of the Oppler-Boas bacillus in the test meal or the vomit is still held by some to be characteristic of gastric carcinoma. The bacillus is long, thin, and may occur in chains. It is often associated with sarcinse, and is not frequently found in cases of gastro-stasis with hypoacidity. Among the methods recommended for examination of the bacterial content of the stomach is the washing of the fasting stomach. A sterile tube is passed, and the stomach is washed out into a bowl with sterile water. The water is examined microscopically and culturally. As might be expected, it is quite unusual to obtain a sterile fluid by this method. The organisms found may come from the stomach, from the oesophagus or mouth by contamination of the end of the tube, or from the swallowed saliva. The organisms most commonly obtained are streptococci, staphylococci, bacilli of the coli or proteus group, and sarcinse. The significance to be attached to any of them is probably slight. CHAPTER XXI. the pancreas — the liver — the spleen — the peritoneum. The Pancreas. There are no satisfactory clinical methods for investigating the functions of the pancreas and the efficiency of the pancreatic juice. The pancreatic reaction of Cammidge has been described in the section on the urine. It is of very doubtful clinical value, and recently further experimental evidence has been adduced against it. It is stated that in test C the glycuronic acid is incompletely removed, and that the Cammidge crystals are derived from glycuronic acid, which is known to occur in the urine in variable amount under very varying circumstances such as are not necessarily connected with pancreatic disease. The investigation of the pancreatic activity in fat metabolism will be described in the chapter on the faeces. Pancreatic efficiency is also investigated by a method similar to the " desmoid " test of Sahli for gastric efficiency. The test is performed by enclosing a little salol in a gelatin capsule hardened with formalin. The capsule is given with the ordinary morning breakfast or with the test breakfast. The gastric juice is unable to dissolve the capsule, which passes unchanged through the stomach, but is readily digested by the pancreatic juice. The salol is then tested for in the urine in samples taken after 2, 3, 4, 5, and 6 hours. The test for salol in the urine is to add a few drops of perchloride of iron, which gives an amethyst blue colour in the presence of salol. In cases of pancreatic insufficiency the appearance of the salol in the urine is much delayed or may not occur. In cases of diarrhoea with a normal pancreatic digestion the salol reaction appears early. In health the salol appears in the urine in about 5 hours. Pancreatic cysts, and the methods of testing ferments in them, have been mentioned in the section on pancreatic fluids. 304 CLINICAL PATHOLOGY. The Liver. The test for new growths of the liver, or abscess of the liver, by demonstration of creatine in the urine has been already described. In acute yellow atrophy of the liver, and in phosphorus poisoning, crystals of leucine and tyrosine are to be looked for in the urine. In cases of biliary obstruction the patient is, as a rule, obviously jaundiced and the condition may be confirmed by the demonstration of bile pigment in the serum. The pigments are also present in the urine, but absent from the stools. The serum in such cases is often markedly bile-tinged at a period when clinical evidence of jaundice is lacking. Puncture of the liver is occasionally performed in cases of suspected liver abscess or of hydatid cyst. Except during the course of an operation, puncture of the liver should be performed cautiously and only in the area of an evidently superficial tumour. A fine hypodermic needle should be used, The fluid withdrawn from a tropical abscess is very typical in appearance, having a considerable resemblance to anchovy sauce. Unless a secondary infection has occurred, the fluid is odourless. Amoebae should be looked for in fresh preparations of the fluid, but are rarely detected, since they seem to be mainly confined to the lining wall of the abscess. After the abscess has been opened and drained, the amoebae are likely to appear on the second or third day. The methods of detecting amoebae in the stools should be applied in the examination of " pus " from the liver. Dried films made from the pus show little besides necrotic cells and amorphous debris. Cultures are almost invariably sterile. The nature of the fluid of a hydatid cyst has been described in the section on puncture fluids. The characteristic hydatid hooklets should always be sought for. Bile is occasionally obtained by puncture through the abdominal wall of a distended gall bladder ; it is more frequently and justifiably received in the laboratory from the operating theatre. In cases of distension of the gall bladder due to a calculus impacted in the cystic duct, the fluid is often colourless or merely tinged with bile pigment ; proteids are scanty, and mucin is usually present in consider- able amount. If no distension of the gall bladder is present, PANCREAS— LIVER— SPLEEN— PERITONEUM. 305 the normal bile is greenish in colour and of a glycerine-like consistency. When inflammation is present, or gall-stones are found in addition, the bile may be either paler or darker in colour, and is almost always more syrupy and ropy in consistence. More rarely the gall bladder may be filled with pus, and actual gangrene of the walls may be present. The association of gall-stone formation with microbic infection of the bile passages is undoubted. The path by which the organisms reach the gall bladder is in dispute, but the character of the bacteria commonly found, and the direct communication between duodenum and gall bladder would point to an intestinal infection as the most obvious route. The bile in such cases should be examined by cytological and cultural methods. Gall-stones, if present, should be culturally examined and their composition investigated. In about 20 per cent, of cases with gall-stones the bladder is found to be sterile, and formerly considerable importance was attached to the antiseptic properties of the bile in preventing the spread of organisms from the alimentary tract. It is probable that the antiseptic action of the bile salts is of importance, but it appears to have little effect upon organisms of the colon and typhoid groups. This action has been taken advantage of in the preparation of MacConkey's bile salt medium. The growth of cocci is completely inhibited by the presence of the sodium taurocholate in the medium, while the colon and typhoid bacilli grow abundantly. The antiseptic action of the bile is possibly diminished under various pathological conditions, and organisms other than those of the coli-typhoid group may be found. The following are among the organisms most commonly met with in the gall bladder : — The bacillus coli is the organism most frequently present in inflammatory conditions of the gall bladder, either alone or in association with gall-stones. There is no difficulty in recovering and identifying it in cultures made from the bile by the ordinary methods. The typhoid bacillus may be present in the gall bladder many years after an attack of typhoid fever. The association of typhoid fever with the subsequent production of gall-stones is well known. The detection of typhoid bacilli in the gall p. 20 306 CLINICAL PATHOLOGY. bladder is of great importance apart from the production of calculi, since the latency of the organisms in this situation is probably responsible for the majority of instances of " typhoid carriers." The organisms readily pass down the intestinal tract and are excreted in the faeces. Such a typhoid carrier occupying the position of a cook or a domestic servant is a serious menace to the community with which he or she is associated. The detection of typhoid bacilli in the bile is a simple matter, if the bile is available during an opera- tion for gall-stones. Typhoid carriers can otherwise be recognised by the detection of the bacilli in the stools and by examination of the serum for the presence of typhoid agglutinins. Streptococci, staphylococci, and much more rarely pneumococci, are among other organisms which may be obtained in cultures from the bile. Gall-stones may be either single or multiple ; if multiple they are usually facetted. They may be found in the gall bladder, cystic duct or common duct, or they may have escaped into the intestine. Barely they may have ulcerated through into the peritoneal cavity or through the anterior abdominal wall. Exceptionally small calculi may be found in the finer hepatic ducts in the substance of the liver. The great majority of gall-stones consist mainly or entirely of cholesterin. Pure cholesterin calculi are of a translucent yellow colour and very light. The stones not infrequently con- tain in addition to the cholesterin varying amounts of biliary pigment, either bilirubin or biliverdin. More rarely small calculi are found consisting entirely of biliverdin or bilirubin with a small admixture of calcium. Calcium carbonate and phosphate calculi are common in some animals, but very rare in man. The following tests for cholesterin should be applied to a gall-stone after powdering a portion of it. (1) Dissolve some of the powder in ether. Evaporate down the ether extract in hot water (not over a flame). Dissolve a small portion of the residue in a little absolute alcohol. Allow a few drops of the alcoholic extract to evaporate at room temperature on a microscope slide. PANCREAS— LIVER— SPLEEN— PERITONEUM. 807 Examine the slide for the typical flat crystals of choles- terin. The crystals consist of rhombic plates notched at one angle (Fig. 18). (2) Dissolve another portion of the ether residue in pure anhydrous ether. Allow a few drops to evaporate on a slide as above. Cholesterin crystallises from ether in the form of fine needles. (3) Dissolve another portion of the ether residue in a small quantity of chloroform in a test tube. Add an equal volume of concen- trated sulphuric acid and mix. The chloroform be- comes coloured a deep red and rises to the surface of the mixture. The sulphuric acid shows a brilliant green fluores- cence. This test is known as Salkowski's reaction. Bile pigments in gall-stones are recognised by their colour and by their reaction to Gmelin's test. The tests for carbonates, phosphates, and calcium are conducted in the same manner as described under the analysis of urinary calculi. The bacteriological investigation of gall-stones is conducted as follows : — If the stone has been removed by the surgeon from the bile passages and placed direct in a sterile receptacle no preliminary treatment is necessary. If, however, the surface of the stone has become contaminated it should be placed on a clean dish and washed repeatedly with sterile water. The stone is then placed in a sterile mortar and crushed. Culture tubes are inoculated with portions of the 20—2 Fig. 18. — Cholesterin Crystals. 308 CLINICAL PATHOLOGY. crushed calculus. The remainder can be used for chemical analysis. The bacteria found in gall-stones are precisely similar to those present in the bile. A considerable percentage of gall- stones, however, are sterile. The Spleen. Owing to the remarkable changes which may take place in the spleen in certain blood diseases, the part which it plays in them is apt to be over-estimated. There is little evidence that enlargement of the spleen in the primary anaemias is anything but a secondary phenomenon, or that in adult life the spleen takes any prominent or special share in blood formation. We are not concerned with the various theories advanced as to the functions of the spleen, but it may be mentioned that, apart from any possible controlling action on the iron metabolism of the body, almost the only certain function of the spleen is to act as a reservoir for the portal circulation during digestion. Tumours in the left hyphochondrium continue to furnish the clinician with interesting problems for diagnosis. If such a tumour be present several pathological investigations may be necessary. It is not intended to imply that all these investigations should be performed in any case of splenic enlargement. The clinical examination of the patient will almost always indicate which investigation is necessary. Examination of the cells of the blood.— In all cases of splenic enlargement some examination of the blood must be made, and this in a considerable percentage of cases will establish the diagnosis. In myeloid leukaemia, among the primary blood diseases, there is almost always a considerable enlargement of the spleen, and the enlargement is often so great that the spleen comes to occupy more than half the abdominal cavity. In lymphoid leukaemia the spleen is as a rule moderately enlarged, but occasionally the enlargement is insufficient for detection by clinical means. In pernicious anaemia the spleen is enlarged and commonly of such a size as to be just palpable. PANCREAS— LIVER— SPLEEN— PERITONEUM. 309 Enlargement of the spleen from any of the above three causes is readily recognised by the ordinary examination of the blood. In chlorosis there is no evident enlargement of the spleen, and if considerable splenic enlargement is present with an anaemia of the chlorotic type some other cause for the splenomegaly must be sought. In erythremia a considerable increase in the size of the spleen is usual. Patients with this condition are, as a rule, cyanosed, and the blood shows great excess in the number of red cells. Marked enlargement of the spleen in small children may be associated with active rickets or with congenital syphilis, and with little change in the blood ; or these diseases may be absent and the blood show the pronounced, if variable, changes of the splenic anaemia of children. Occasionally marked enlargement of the spleen in children may occur in the absence of other obvious disease or of changes in the blood. In primary splenomegaly or splenic anaemia of adults, and in Banti's disease, the size of the spleen is usually very con- siderable, and may even rival that found in myeloid leukaemia. The changes in the blood in this condition are not diagnostic, but if a considerable anaemia of the secondary type is absent and there is no leucopenia, the diagnosis of primary spleno- megaly is probably incorrect. In acholuric family jaundice the size of the spleen is con- siderable. The condition may be recognised by the abnormal fragility of the red cells in dilute saline. In acute inflammatory conditions a slight enlargement of the spleen is frequently met with. The blood shows an increase in the total leucocytes, with a relative increase in the polynu- clear neutrophils and large hyalines. Examination of the blood serum. — The moderate enlargement of the spleen which occurs in typhoid fever, and the more marked enlargement present in Malta fever, may be confirmed by the demonstration of the specific agglutinins in the serum. Enlargement of the spleen is often considerable in congenital syphilis, and in the amyloid degeneration which may follow congenital or acquired syphilis. The spleen may be palpable 310 CLINICAL PATHOLOGY. in the secondary stage of syphilis. A positive Wassermann reaction will be found in the serum. The parasitology of the blood.— Definite enlargement of the spleen may follow infarction in the course of infective endocarditis, and the nature of the disease may be established by cultivation of the blood. In relapsing fever the spirillum is present in blood films. In malaria a moderate enlargement of the spleen is usual, and the parasites can nearly always be found in film preparations. The enlargement of the spleen which accompanies malaria may exceptionally persist for a con- siderable time after the patient has left the malarial district : in such cases no parasites may be found in the blood. A very considerable enlargement of the spleen without the typical signs of malaria and in the absence of malarial parasites in the blood is probably due to some other cause. A past history of malaria is not in itself sufficient to account for a tumour in the left hypochondrium. Spleen puncture should be performed in cases of splenic enlargement which may possibly be the result of kala-azar. The puncture is without risk if performed with a fine hypodermic needle. Sufficient material can usually be with- drawn to demonstrate the Leishman-Donovan bodies. The urine must always be carefully examined in all cases of tumour in the left hypochondrium. A renal swelling on clinical examination may very closely resemble an enlarged spleen, and the detection of pus in the urine may serve to clinch the diagnosis. If the spleen is enlarged from amyloid disease the kidneys will probably also be affected, and the urine will contain albumin, often in considerable amount. Cirrhosis of the liver is often associated with a hard and palpable spleen. The diagnosis as a rule is readily made on clinical grounds. Hodgkin's disease is frequently associated with a splenic enlargement which may be considerable. None of the exami- nations just described are likely to throw any light upon the diagnosis. A general enlargement of the glands, however, is nearly always present, and it is justifiable to remove a single superficial gland for histological investigation. Tuberculosis, particularly of the generalised glandular type, may lead to considerable splenic enlargement. Labora- tory investigations, in the absence of a recognised complement PANCREAS— LIVER— SPLEEN— PERITONEUM. 311 deviation test for tuberculosis, are mainly negative. The diagnosis is often arrived at by a process of exclusion, and commonly rests between tuberculosis and Hodgkin's disease. Tumours of the spleen, whether primary or secondary, are extremely rare. Cysts of the spleen and gummata of the spleen are likewise in the nature of pathological curiosities. Renal tumours, tumours of the stomach or intestine, and tumours or cysts of the omentum, mesentery, or pancreas may all closely resemble splenic tumours on physical examination. It is evident that a very large number of diseases may be accompanied by splenic enlargement. In the majority of them, however, the pathological condition is fairly obvious on clinical grounds and the enlargement of the spleen is of secondary importance. Particularly is this the case in slight enlargement of the spleen to palpation. Great enlargement of the spleen is rare, and the most usual causes are myeloid leukaemia, splenic anaemia, kala-azar, the splenic anaemia of children, and rarely chronic lymphoid leukaemia. These diseases can nearly always be recognised by a blood examina- tion or by spleen puncture. Moderate enlargements of the spleen are more difficult of diagnosis, and the commonest causes are tuberculosis, Hodgkin's disease, and in children congenital syphilis and rickets. The Peeitoneum. The cellular nature of peritoneal exudates has been sufficiently indicated in the section dealing with puncture fluids. The bacteriological examination of exudates obtained at operation may be further considered in reference to the abdominal lesion responsible for the inflamed peritoneum. Gastric ulcer. — Perforation of a gastric ulcer may lead to a localised abscess or to a generalised peritonitis. A number of different organisms may be found in the exudate, but in the majority of cases the bacterium which predominates in film preparations, and often in cultures, is a streptococcus. The streptococcus most often found differs in some respects from streptococcus pyogenes. It has been referred to as a strepto- diplococcus because the chain arrangement depends upon a succession of paired cocci ; but this appearance is common to the majority of streptococci. It is feebly Gram-positive or 312 CLINICAL PATHOLOGY. even Gram -negative, and is of very low virulence to animals. The predominance of a streptococcus in cases of perforated ulcer helps to distinguish them from lesions lower in the gut, where the exudate is almost entirely bacillary. A very similar streptococcus is often present in the pus of a perinephric abscess. It must not be supposed that the presence of a streptococcus in the peritoneum in a case of perforated gastric ulcer indicates that the ulcer was due to streptococcal inflammation. Streptococci of a similar nature may be found frequently in the gastric contents in the absence of ulceration, and, whatever may be the cause of gastric ulcer, the effect of perforation is to give to the organisms which may have been in the stomach access to the peritoneum. The same warning must be applied to all bacteriological finding in peritonitis following visceral lesions. Among other organisms which may be found in this condition are staphylococci and B. coli. Duodenal ulcer. — The bacteriology of the peritoneum after perforation of a duodenal ulcer is similar to the fore- going, but streptococci are less commonly, and staphylococci and colon bacilli more commonly, met with. The ileum, — The most important variety of perforation of the ileum is that which follows a typhoid ulcer, the most common site of perforation being within the last foot of the ileum. Failure to find the typhoid bacillus in these cases is common, and is no evidence against the specific cause of the intestinal ulceration. Colon bacilli, staphylococci, and streptococci are recovered more frequently than the typhoid bacillus, and possibly in the majority of cases take a greater share in the peritoneal inflammation. Similar organisms (with the exception of B. typhosus) are found in the peritoneum after perforation of the ileum from injury or other causes. The appendix. — In cultures made from the lumen of an inflamed appendix, from a localised appendix abscess, from the general peritonitis accompanying a diseased or perforated appendix, and from the residual and remote abscesses which may follow appendicitis the predominant organism found is in the great majority of cases the colon bacillus. It is open to doubt, however, whether anything approaching this propor- tion of appendicular inflammation is produced by the same organisms which cause the peritonitis. There is no evidence PANCEEAS -LIVER— SPLEEN— PERITONEUM. 313 of any specific cause of appendicitis, and it is possible that a variety of organisms may be responsible, and that as soon as a communication has been established between the lumen of the gut and the peritoneal cavity the colon bacillus rapidly multiplies and takes the predominant share in the subsequent peritonitis. There is abundant evidence that the colon bacillus in pure culture is able to set up a virulent peritonitis. In addition to the colon bacillus other organisms are frequently present in film preparations. Long, thin and sometimes beaded bacilli are often found, and are probably the cause of the offensive smell so often met with in the pus of an appendix abscess. These organisms do not grow in aerobic cultures, and not always under anaerobic conditions. They are probably identical with the organisms found in stinking empyemata and in some cerebral abscesses, and their role appears to be mainly that of saprophytes. Their normal habitat is the alimentary or respiratory tract. Staphylococci and strepto- cocci are commonly met with in film preparations in association with the colon bacillus, particularly in localised appendix abscesses. They may also be isolated in culture, and are occasionally present in pure culture. The bacillus pyocyaneus is less frequently met with, and is practically never found in association with the colon bacillus. The latter organism appears to be unable to exist in the presence of pyocyaneus. Lesions of the large intestine may originate a peritonitis as after perforation of a simple or a malignant ulcer. The organisms found in the peritoneal exudate are of much the same type as those met with in appendix cases. The pelvic organs in the female may cause peritonitis following suppuration in one of them. A pyosalpinx rarely ruptures, but when it does peritonitis usually follows. The pus is almost invariably sterile, and very occasionally the gonococcus can be identified in film preparations after con- siderable search. Recovery in such cases is the rule. In the case of suppuration in ovarian cysts or tumours a peritonitis of the ordinary type is set up, and colon bacilli, streptococci, and staphylococci are found in the exudate. Not infrequently the pneumococcus is isolated in pure culture from the pus in the neighbourhood of an ovarian abscess, and the prognosis appears to be favourable. 314 CLINICAL PATHOLOGY. Post operative peritonitis is now fortunately of rare occurrence, but even in the most careful hands occasional instances of operative infection occur. In the pus of such cases the streptococcus pyogenes may be isolated in pure culture and a rapidly fatal course is to be expected. Less virulent staphylococci are found in some cases, and in others colon bacilli or B. pyocaneus. Intestinal obstruction. — The commonest variety of intestinal obstruction is that which follows strangulation of a hernia. In cultures made from the general peritoneal cavity staphylococci are frequently obtained, and less com- monly colon bacilli. The more damaged the gut, the more frequently are colon bacilli obtained, and the more serious is the prognosis. A considerable amount of free fluid is often found in the hernial sac. In the majority of such cases this fluid is quite clear, contains few cells other than endothelials, and cultures from it remain sterile. If the gut in the sac is gangrenous, organisms are more likely to be obtained in culture. In cases of obstruction due to other causes the probability of obtaining sterile cultures from the peritoneum depends upon the chronicity of the process. The more acute the case, the more commonly are organisms found. "Idiopathic peritonitis." — This term was formerly applied to cases of peritoneal inflammation without any recognisable focus in the intestine. An important organism met with in such cases, and often in pure culture, is the pneumococcus. Pneumococcal infection of the peritoneum is more common in children than in adults, and females are more frequently affected than males. A pneumococcal infection of the lung may be present, but more commonly no such local focus is found, and the organism appears to have entered the peritoneum from the general circulation in much the same way as it may enter a synovial cavity. In young girls the pneumococci appear in a certain percentage of cases to enter by way of the genital tract. The organisms may be present in pure culture in the interior of the uterus, and are among the causative bacteria to be met with in cases of ovarian suppuration. Streptococcal peritonitis due to causes other than intestinal may develop as a terminal infection in several chronic PANCREAS— LIVER— SPLEEN— PERITONEUM. 315 diseases, of which chronic nephritis is the most common. Peritonitis may also develop as part of a general streptococcal septicaemia with a primary focus elsewhere. More rarely a streptococcal peritonitis may be present as a local disease and without any traceable source of infection. The prognosis in those cases in which pneumococci are found in the peritoneum is much more favourable than those associated with streptococci. The streptococcal cases are almost invariably fatal. Tuberculous peritonitis. — In tuberculous peritonitis with effusion the fluid is commonly quite clear, and the pre- dominant cell is a small lymphocyte. Mixed cellular fluids are, however, common in the peritoneal cavity, and a percentage of lymphocytes is found in other than tuberculous conditions. Secondary infection of a tuberculous peritonitis is fairly frequent, and the fluid may be turbid or purulent and con- tain a considerable percentage of polynuclear cells. Staphy- lococci are often found in cultures taken from such cases. Tubercle bacilli are usually difficult to find, but may be very numerous, and should be sought for by the ordinary methods. If the bacilli cannot be found the fluid can be proved to be tuberculous by injection into a guinea-pig. CHAPTEK XXII. THE F.^CES. It is remarkable how much the clinical investigation of the faeces has fallen behind the physiological knowledge of the excreta. Notwithstanding the interest attached to numerous intestinal disorders and the importance attributed to the con- dition known as intestinal toxaemia, the usual investigation of the stools consists of little more than a perfunctory inspection. The main reason of this neglect is that very few simple clinical investigations are known to throw any direct light on the condition of the patient. Elaborate chemical analyses of the excreta require that the patient should be dieted in a manner impossible in the ordinary hospital ward, and the analyses themselves are too prolonged for ordinary routine work. A certain number of minor investigations, however, such as are within the scope of every-day work, are of considerable use in some diseases and essential for diagnosis in others. Such methods of examination as are given here are divided into naked-eye, chemical, microscopical, and parasitological investigations. In any investigation it is most advisable that the ordinary stool of the patient should be examined when available, and not the stool which follows the administration of a purgative or the giving of an enema. The naked-eye investigation. — The amount and the appearance of the f aeces naturally vary considerably in health. The quantity passed by a healthy man on an average diet in the 24 hours is said to be about 150 grammes. The colour of the stools is very variable, and the colour in health is due to the presence of the pigment stercobilin. Stercobilin is very similar to the urinary pigment urobilin, and is considered to be identical with it. The two bodies give the same chemical tests. Urobilin, or a very similar sub- stance, can be artificially produced from bile pigment, and there is little doubt that the stercobilin of the faeces is formed from bilirubin in the alimentary canal. Unaltered bile pigment THE FAECES. 317 is never present in the faeces in health. The colour of the faeces may be darkened by an excess of stercobilin, or after the taking of medicines containing iron, manganese, or bismuth. The blackening of the stools as the result of drugs is due to the formation of the sulphides of the metals in the alimentary tract. Blood coming from the lower part of the bowel may be of the usual red colour, but if from the upper part it is black, and the stools resemble closely those which follow the taking of iron or bismuth and can only be distinguished by a chemical examination. Green stools may under abnormal circumstances be due to the presence of unaltered bile pig- ment. Yellow stools follow the administration of senna, santonin, and rhubarb. The colour of the faeces is lost if the bile is prevented from entering the intestinal canal, and the characteristic " clayey " stools of jaundice are passed. Light- coloured stools, due to a diminution of stercobilin, are passed in various conditions associated with diarrhoea. They do not necessarily contain an excess of fat, but cannot be distinguished by the naked eye from the " fatty " stools suggestive of pancreatic disease. The odour of the faeces is due mainly to the presence of indole and scatole. The offensiveness varies with the nature of the food — being much greater with a meat than a carbo- hydrate diet — and with the amount of intestinal putrefaction. The consistence of the stool naturally passed is of great importance. The formed stool of health is replaced by the more or less watery evacuation of diarrhoea from a large number of causes. An unformed stool in an adult on an ordinary diet is always abnormal, irrespective of the number of the motions in the 24 hours. Abnormal ingredients. — Numerous abnormal sub- stances in the stools can be detected by the ordinary inspec- tion. In cases where abnormal substances are to be expected the examination is greatly facilitated by repeatedly washing the stool in tap water through a tine sieve and inspecting the residue. The following are among the abnormal substances to be looked for. The larger animal parasites are easily detected with the naked eye. They will be described subsequently. Mucous shreds and mucous casts are of considerable importance, and are readily seen in the residue of the stool 318 CLINICAL PATHOLOGY. floated in water. They are commonly met with in numerous varieties of colitis, and consist as a rule of thin, ragged mem- branes of varying size composed of mucin and fibrin. Large mucous casts of the alimentary tract are less commonly seen, and may appear as long, twisted strands which bear some resemblance to tape-worms. The strands are not regularly segmented, and should be readily distinguished from parasites. Portions of undigested food, and particularly orange and banana fibre, closely resemble mucoid shreds, but can be differentiated under the microscope. Small balls of banana fibre have some microscopic resemblance to worm segments filled with ova. Shreds of epithelium, and rarely particles of malignant growth, may be met with and should be reserved for micro- scopical examination. Pus and blood may be present in amount obvious to the naked eye, but in nearly all cases the macroscopic evidence should be confirmed by other means. Gall-stones are to be carefully looked for in all suspicious cases, and are readily extracted from the faeces by the sieve pro- cess described above. Gall-stones have to be distinguished from scybalous particles of faeces, and after washing in water they must be chemically tested for cholesterin. Cholesterin is normally present in the faeces, but a solid macroscopic object in the faeces consisting almost entirely of this substance is certainly derived from the gall bladder. Faecal concretions are as a rule more friable and pultaceous, contain very little or no cholesterin, and often a considerable amount of car- bonates or phosphates. Intestinal sand is occasionally passed in considerable amounts in some forms of colitis. True intestinal sand is of a brownish colour, and is composed mainly of calcium car- bonate and calcium phosphate. Cholesterin is absent. The colour is due to stercobilin with often traces of bile pigment. False intestinal sand consists as a rule of particles of undigested vegetable fibres, and most commonly follows the ingestion of pears. Chemical examinations. — The reaction. — The reaction of the stool to litmus paper has no particular significance. The normal stool may be either faintly acid or faintly alkaline. Marked acidity or alkalinity is abnormal. THE F.ECES. 319 Stercobilin is probably identical with urobilin, and can be detected in the faeces as follows : — To a watery extract of the faeces add sulphuric acid in the proportion of 2 grammes to the litre, and solid ammonium sulphate. Filter and wash the precipitate with warm, saturated ammonium sulphate solution. Dry on a water bath. Extract with a boiling alcoholic solution of ammonia. To a portion of the extract add a few drops of a 10 per cent, solution of zinc chloride. The solution becomes fluorescent in the presence of stercobilin. Acidify another portion with acetic acid, and examine spectroscopically for the urobilin spectrum (page 224). A simpler and more direct method is the following : — Extract a portion of the fasces with chloroform. Pour off the tinted extract, and add hydrochloric acid con- taining a little nitric acid. Examine with the spectroscope. The chloroform in this process extracts the chromogen, and the acid mixture converts the chromogen into stercobilin (or urobilin). Stercobilin may by these tests be demonstrated even hi almost colourless stools. Stercobilin is absent in cases of complete occlusion of the bile ducts. It is present if the occlusion is incomplete, how- ever deeply jaundiced the patient may be. The test is thus a satisfactory one for the demonstration of complete occlusion of the bile ducts. Stercobilin is also stated to be usually absent in carcinoma of the pancreas. Bile. — Bile pigment is normally absent from the faeces. It is present in some cases of diarrhoea, and traces are found in conditions associated with jaundice. Bile pigment may be tested for by Gmelin's test applied to a watery mixture of the faeces, or a particle of faeces may be mixed with a concentrated solution of mercuric chloride, covered and allowed to stand for 24 hours. The normal stool coloured by stercobilin turns red. If bile pigment is present green particles appear. Bile acids may be tested for as follows : — Extract the faeces with alcohol. Dissolve the residue in dilute caustic soda and perform Pettenkofer's test, thus : — 320 CLINICAL PATHOLOGY. Dissolve a fragment of cane sugar in the solution of the residue in a test tube. Kun in about 5 c.c. of concentrated sulphuric acid to the bottom of the tube and gently shake. A slowly-developing purple colour forms from the line of junction of the two fluids. Excess of cane sugar in the mixture must be avoided, since it is charred by the acid and so masks the reaction. Blood pigment. — Blood present in considerable amount and coming from the lower part of the tract may be detected by the microscope or by its spectrum. Blood in small amount or from the upper part of the tract cannot be recognised under the microscope, and is best examined for by one of the tests for so-called " occult "blood. By occult blood is meant blood present in minute traces, such as may come from an oozing gastric, duodenal, or malignant ulcer. Various delicate tests capable of revealing very minute traces of blood are employed, and are of some assistance in the diagnosis of alimentary conditions associated with haemor- rhage. Before performing such a test it is necessary to put the patient on a blood- free diet for 48 hours. The test may be performed as follows : — Rub up a fragment of faeces in 4 c.c. of water in a mortar. Or, if the faeces are liquid, mix thoroughly 2 c.c. of faeces and 2 c.c. of water. Add 2 c.c. of glacial acetic acid. Pour into a test tube and shake thoroughly. Add 5 c.c. of ether. Invert the tube slowly and gently twenty times ; if the inver- sion is roughly done the ether will not separate on standing. Allow to stand and pour off 2 c.c. of the ether extract into a 10 c.c. measure. Test for haematin acetate thus : — Add \ c.c. of glacial acetic acid. Add 2 c.c. of a saturated solution of benzidin in rectified spirit. The benzidin solution must be freshly prepared. Add 2 c.c. of a five- volume solution of hydrogen peroxide. Mix. In the presence of blood the mixture becomes blue of a greater or less intensity. With a very minute trace of blood the colour is greenish. THE F^CES. 321 Albumin. — In health no proteid reaction can be obtained in the faeces on an ordinary diet, but a considerable quantity of albumin can be detected in the stools in numerous conditions associated with diarrhoea. To test for albumin : — Add to a small portion of fasces a considerable quantity of water acidified with acetic acid. Mix thoroughly, and allow to stand. Filter several times. Test the filtrate for albumin by the ordinary tests. Fat. — The normal faeces contain both neutral fats and fatty acids. The determination of the amount of fat in the stools and the form in which it is present may be of assistance in numerous forms of intestinal disorder, and is most frequently required in the investigation of pancreatic disease. It will be remembered that the action of the pancreatic juice upon the neutral fats in the duodenum is to split them into fatty acids and glycerin, a process of hydrolysis known as saponification. The saponification is aided by the admixture of bile. The flow of the pancreatic juice is determined by secretin, which is produced in the duodenal mucous membrane on contact of the acid contents of the stomach. The secretin passes into the blood and is carried to the pancreas. The fat-splitting ferment of the pancreas is of course the lipase. The fatty acids split off during saponification combine in the intestine with the alkalies to form soaps. The soaps are the sodium and potassium salts of the higher fatty acids, and become reconverted into fatty acids on the addition of an inorganic acid. The fatty acids or soaps are absorbed from the intestine, and are built up again into fats in the tissues. The neutral fats and free fatty acids are soluble in ether; the soaps are insoluble in ether. These elementary chemical points are necessary for the understanding of the methods of estimating fatty bodies in the fasces and the interpretation of the results. The simple method of estimating the fats described by Cammidge is sufficiently accurate for clinical purposes, and is advocated by him as a means of diagnosis in biliary and pan- creatic diseases. Under normal conditions the total fat forms from 20 to 25 p. 21 322 CLINICAL PATHOLOGY. per cent, of the dried faeces, and consists of neutral fat, and soaps representing the free fatty acids, in about equal pro- portions. If the entry of the pancreatic secretion into the duodenum is prevented, as in pancreatic calculus or carcinoma of the pan- creas, or if the pancreatic secretion is poor, as in chronic pancreatitis, the fats are imperfectly dealt with ; consequently an excess of fat is found in the stools, and since the neutral fats have been insufficiently split up by the deficient pancreatic juice, they are present in excess of the fatty acids. If the biliary secretion is interfered with and the pancreatic secretion is normal, the neutral fats become converted into fatty acid and glycerin, as under normal conditions : but saponification and absorption of fat is interfered with ; conse- quently the total fat is again in excess, but the fatty acids are in excess of the neutral fats. A similar state prevails in some varieties of diarrhoea and in other conditions which interfere with intestinal absorption. The estimation of the fats is performed as follows : — A sample of the usual stool is taken, the patient being given an ordinary mixed diet. The faeces are placed in a porcelain evaporating dish and heated, first over the water bath, and finally on the sand bath in a fume cupboard. The faeces are stirred occasionally with a glass rod and the heating process is continued until they are thoroughly dry. The process takes some hours as a rule, and may be completed by leaving in a drying chamber over night. When cool the dried faeces are practically inoffensive. The dried faeces are powdered into as fine a dust as possible in a mortar. It may be impossible to powder very fatty stools. Two samples, each of 0*5 gramme, are carefully weighed out. Two clean and absolutely dry Schmidt- Werner tubes are prepared, and labelled A and B. Each should be provided with a 10 c.c. mark. Into the lower bulb of each place 0*5 gramme of faeces. Wash the residue into the A tube with 1 in 3 hydrochloric acid, and fill up with the acid to the 10 c.c. mark. Wash the residue into the B tube with distilled water, and fill with water to the 10 c.c. mark. THE F^CES. 323 In each tube all the faeces must be collected with the fluid in the lower bulb. The A tube is then heated in boiling water for 15 minutes and is rotated from time to time. By this process the fatty- acids are split off from their bases and rendered soluble in ether. Cool the A tube. Fill both tubes to the 50 c.c. mark with ether, and cork them securely. Slowly invert and rotate each tube forty times. Allow the tubes to stand for 30 minutes or until all the residue has collected into the lower bulbs. While the tubes are standing weigh carefully two small dry evaporating flasks labelled A and B. Note the weight of each. Into each appropriate flask measure 20 c.c. of the clear ethereal extract from the tubes : the measurement is most conveniently made with a pipette, but care must be taken to keep the nozzle of the pipette under the surface of the fluid. Note the amount of ether left in each tube. Evaporate the ether extracts by holding the flasks in a stream of hot water. Dry the residue by heating, without charring, on a water bath in the fume cupboard. Unless this step is thoroughly done the error in the subsequent estimation is considerable. After cooling weigh each flask. The difference in -the weight of each represents the weight of fat extracted by 20 c.c. of ether from 0'25 gramme of dried faeces, supposing no ether to have been lost from the tubes and consequently the level of the remaining fluid to be at the 30 c.c. mark. The percentage of fat in the dried faeces is thus given by multiplying the result by 400. The yield from the A tube may be reckoned as the total fat, since it includes the ether-soluble neutral fats and the fatty acids which have been split off from their bases by the hydro- chloric acid. The yield for the B tube may be reckoned as neutral fat. The difference between the two tubes represents the fatty acids. In 13 cases of carcinoma of the pancreas in which the faeces were thus analysed by Cammidge the total fat ranged from 21—2 324 CLINICAL PATHOLOGY. 50 to 90 per cent, of the dried fasces, and of this the neutral fat formed 40 to 60 per cent., and the fatty acids 9 to 33 per cent. An estimate of the amount of fat present in the fasces from a naked-eye inspection is extremely misleading, and often the so-called fatty-looking stools of alleged pancreatic diarrhoea are found to be merely pallid stools with a deficiency of stercobilin and a normal amount of fat. Some chemical estimation is required, and the method of Cammidge requires little apparatus, takes up much less actual time than might be supposed from the above description, and is . sufficiently accurate to detect the gross changes which may occur in disease. The deductions to be drawn from the results are to be carefully correlated with the clinical evidence and all other available modes of examination. Taken by them- selves estimations of this character are of very little value. Other chemical investigations. — Mucin appears to be a normal constituent of the stools. It may be tested for by adding to a watery suspension of the stool an equal volume of lime-water. The mixture is allowed to stand until the next morning and is then filtered. A little acetic acid is added to the filtrate and a white cloud of mucin, insoluble in excess of acid and increased on boiling, is produced. Peptone is absent from the fasces in health, but is present usually in cases of typhoid fever, and in all stools containing pus. Peptone is absent in catarrhal jaundice, but usually present in cirrhosis and malignant disease of the liver. Peptone in the faeces may be detected as follows : — Mix some of the fasues with water. Boil and filter while hot. Test for albumin — if absent, Mix the filtrate with neutral lead acetate and filter after standing. Acidulate filtrate with hydrochloric acid. Add phospho-tungstic acid until no more precipitate forms. Filter at once. Wash the precipitate on the filter with 5 per cent, sulphuric acid until a colourless filtrate is obtained. Wash the precipitate off the filter paper into a dish with the minimum amount of water. Mix the filtrate with barium carbonate till alkaline. Heat on a water bath for 15 minutes. THE MCES. 325 Test for peptone by the biuret test as follows : — Add caustic potash and drop by drop a 10 per cent, solution of copper sulphate. A bluish pink or violet colour indicates peptone. If albumin is present in the original nitrate it must be removed as follows : — A solution of acetate of soda is added, and next one of perchloride of iron. The mixture is then exactly neutralised with caustic soda, boiled, filtered, and allowed to cool. Hydrochloric acid is then added and the process described above is performed. Cholesterin is present in the faeces in health and can be extracted from them. It practically never occurs in a crystalline form. Microscopical investigation.— Numerous points of con- siderable importance may be found in the course of a micro- scopic examination of the faeces. In order to make the examination prepare in a test tube a turbid suspension of the faeces in normal saline. Shake thoroughly. Allow the suspension to stand. Pipette up the deposit, and examine it on a slide with a cover-slip over it in exactly the manner employed for a urinary deposit. If the ova of intestinal parasites are being looked for a similar mixture should be made in a large test tube, and a little carbolic acid may be added to remove the odour. After standing the supernatant fluid is decanted, and the sediment is again shaken with saline. The process is repeated two or three times and the ova sought for in samples of the sediment. The general microscopic view of a faecal suspension is at first confusing. The numerous particles of many varieties of food remnants, and the general debris lying among the fauna and flora of the intestinal canal, provide somewhat of a diagnostic debauch. The following are among the substances which should be looked for : — Vegetable cells and fibres. — These may occur in a great variety of shape and size. Spiral forms are extremely numerous, and may be found as free spirals or as long cells containing an evenly-wound refractile spiral. Other forms appear as long and sejDtate divisions marking off the usually 326 CLINICAL PATHOLOGY. empty cells. Some of the cells may contain chlorophyll, and others starch granules. Starch granules should not be present in great numbers ; they may be detected by their appearance and by their blue reaction on running a solution of iodine and potassium iodide under the cover-slip. Fat. — Fat globules should only be present in small numbers in a normal stool. They are present in large quantities after the injection of an oil enema. They are readily recognised by their shape and refractility, and if necessary by their chemical reactions. Fat occurs more commonly in the form of sheaves of colourless pointed crystals. These sheaves of fatty acid crystals dissolve on warming, and in ether. The soaps may appear in the form of coarser crystals and dissolve on warming, but not on the addition of ether. When an excess of fat is present in the faeces fatty acid crystals are usually found, but a chemical estimation is necessary in order to determine whether fat is actually in excess or not. Muscle fibres are nearly always found in the faeces of a person on a normal diet. Their amount and the degree of their digestion naturally vary with the amount of meat taken ; but under ordinary conditions the relative number of undigested muscle fibres is a considerable guide to the activity of the digestive juice and the suitability of the diet to the individual. Muscle fibres as seen under the microscope are of a yellow colour, and are certainly recognised by their fine transverse striation. The striation can be perfectly well seen with a Jth-inch objective, particularly if the diaphragm of the condenser is partly shut down. Under normal condi- tions few fibres will be seen on any one slide, and in the great majority of these the striae will be almost or quite invisible. In conditions associated with alimentary disorders the fibres are very numerous and their striation well marked. Elastic fibres can be often detected in the faeces, and are recognised by their shape, their curved form, and their double contour. Red blood corpuscles can as a rule only be recognised in the faeces under the microscope when the haemorrhage has come from the lower part of the intestinal canal. With quite pro- fuse haemorrhage from high up in the gut, as in duodenal ulcer or even in typhoid fever, recognisable red cells are rarely met THE F^CES. 327 with. Keddish-brown pigment masses are seen in such cases and can be recognised as of probable blood origin, but the chemical test for blood should always be applied. The sulphides of the metals, and particularly of bismuth and iron, give an appearanc3 to the stools similar to that produced by altered blood. Undsr the microscope the sulphides appear as black amorphous masses. Bismuth may occasionally appear in the stools in the form of black crystals of a shape similar to that of haemin crystals. Leucocytes are absent, or almost completely absent, from the stools in health. In the numerous conditions associated with a catarrhal state of the intestines there is practically no increase in the number of leucocytes found. Actual pus in the stools is very rarely seen, and any considerable increase in the leucocytes points to actual ulceration of the gut. Pus if present indicates that an abscess has discharged into the intestine, as for example an appendix abscess into the rectum. Pus cells have here, as elsewhere, to be differentiated from epithelial cells, and in cases of doubt stained preparations should be made. Epithelium. — Epithelial cells, more or less degenerated, are practically always found in the faeces. They may be squamous, or less often columnar, and most commonly are fairly small, more or less fusiform cells. They occur singly and less often in small plaques. In cases of intestinal catarrh epithelial cells are usually much increased in number. Crystals. — The commonest inorganic crystal to be found is that of calcium oxalate, and the envelope forms are those most often seen. The crystals are derived from the food, and are most abundant after a vegetable diet. Other crystals which may be present are calcium carbonate and calcium sulphate rarely, calcium phosphate and triple phosphates more commonly. These crystals are recognised in the same way as in urinary deposits. Bacteria are always present in considerable numbers in the stools. The majority of the organisms are bacilli, and in Gram-stained films the Gram-negative bacilli predominate in normal conditions ; cocci are also present as a rule. Spirilla and vibrios are less common, but may appear in small numbers in health. The varieties of bacteria and their significance will be considered in the next chapter. 328 CLINICAL PATHOLOGY. Ova of parasites are among the abnormal objects to be looked for in the stools. They will be described in the account of the intestinal parasites. Debris.— Amorphous particles derived from the food form a considerable part of the slide preparations made from the faeces. They are of variable size, and may appear as discrete particles or in clumps. CHAPTER XXIII. THE PARASITOLOGY OF THE FiECES. The animal parasites. — A considerable variety of animal parasites may on certain occasions and in certain countries infest the human alimentary tract. Only three species are, however, commonly distributed in this country : namely, Oxyuris vermicular is, Ascai'is lumbricoides and Tcenia saginata. Other species, such as ankylostoma, are common in certain circumscribed areas. Others again may be imported by their human host from other countries, or may occur only fortuitously in man. The following is a brief account of the more important parasites, arranged zoologically. Platy helminths. — These are worm-like, flattened dorso- ventrally, and frequently hermaphroditic. They are divided into three classes : — Class 1, Turbellaria, are not parasitic. Class 2, Trematoda (or flukes), are parasitic in man and unsegmented ; in all the life-cycle from ovum to adult is complex, requiring an intermediate host, and asexual multi- plication takes place outside the body from the sexually- produced egg. Class 3, Cestoda (tape-worms) are parasitic, are elongated, flattened, and segmented ; the mode of development is com- paratively simple. Trematodes. — A considerable number of flukes are parasitic to man, but none which produce symptoms are native to this country. The flukes may conveniently be divided on clinical grounds into those which inhabit the biliary passages, the bronchi, and the blood-vessels. Liver flukes are numerous, the most important being Fasciolopsis buski, common in China and India, and Clonorchis sinensis, an extremely frequent parasite of Eastern peoples. Usually no symptoms are produced, but there may be enlargement of the liver with jaundice and ascites. The ova 330 CLINICAL PATHOLOGY. are found in the faeces. They are roughly oval, but with one end narrower than the other, and are of a yellowish colour. They have a shell with a lid at one end, and contain round retractile globules. The bronchial fluke, or Paragonimus westermani, is the cause of endemic haemoptysis in China, Japan, and the Philippines. It measures about 10 mm. long and 5 mm. broad. Infection is diagnosed by the ready detection of the ova in the sputuni. More rarely the parasite is found in the intestine and the ova in the faeces. The intermediate host is probably a mollusc, but is undetermined. The young flukes or cercariae are probably taken into the mouth with the food and by some unknown route reach the lungs. The blood flukes, of which the most important is Bilharzia hcematobia, are bisexual trematodes. Bilharzia is a common parasite of Egypt and parts of South Africa. The adult worm lives in the veins without pro- ducing much change in them. The symptoms are consequent upon the passage of the ova through the mucous membrane into the rectum or bladder. The diagnosis is determined by the presence of the spined eggs in the urine or the faeces. The ova differ from those of other trematodes in having no lid. The single spine is terminal in the case of bladder infection and lateral in ova found in the faeces. The different ova may be derived from two different species of parasites. Ova may be found some years after the patient has left the infected district. They contain a ciliated embryo. The adult male is from 15 to 18 mm. long and 3 to 5 mm. broad. The female is longer and thinner, being '20 mm. long and only 0*25 mm. broad. The male has two suckers, the anterior of which is terminal. It is a flat worm rolled longitudinally upon itself to form a hollow tube, within which the female is clasped. Both life history and mode of infection are unknown, but there is no question that Fig. 19. — Bilharzia Hsematobia. Ovum. Male and Female. Natural Size. THE PARASITOLOGY OF THE FAECES. 331 water plays an important part in each case. The ciliated embryo in the urine on reaching water bursts its capsule and swims about busily. An intermediate host is probably neces- sary. The infection is thought to take place by entry of the parasite into the urethra or anus, or possibly through the skin while bathing. A smaller trematode also inhabiting the portal veins of man, and common in the East, is Schistosomum japonicum. The male similarly contains the female in a gynsecophoric canal. The ova are found in the faeces and contain a ciliated embryo, but have no spine. Cestodes. — The tape-worms are divided into two groups by the character of their heads, namely, Dibothriocephaloidea and TceniidcE. The heads of the former are provided with two elongated slits, the latter have four round suckers and a rostellum, which in some species is armed with hooklets. All the worms consist of a head or scolex, from which arises a series of segments or proglottides. Each proglottis is bisexual. The dibothriocephaloidea. — The most important human parasite of this class is Bothriocephalus latus. Bothriocephalus latus is not met with in Great Britain, but is common in Iceland, Switzerland, and parts of Germany. The adult worm lives in the intestinal tract of man, and feeds upon the intestinal contents. A severe anaemia is pro- duced in the host, probably by the action of a toxin excreted by the worm. The parasite is very large and long, and may grow to 25 or 30 feet in length. The head is long and narrow, and is attached to the gut wall by two long slit-like suckers. The genital pore opens upon the flat surface of the pro- glottis. The ova are enclosed in shells fitted with a lid, and at the time of passage in the faeces are immature. The shells are almost colourless. Within the ovum a six-hooked embryo forms, with a ciliated capsule around it. Embryo and capsule are called the oncosphere. The ciliated embryo escapes in water and enters the intestinal tract of a pike or other fish. Here the ciliated envelope disappears, and the embryo makes its way through the intestinal wall into the muscles and, losing its hooklets, develops slit-like suckers and an unsegmented worm-like body. This larval form is eaten by man or other host and develops into the adult segmented worm. 332 CLINICAL PATHOLOGY. A similar and even larger tape-worm is met with in Japan. It may attain a length of 12 yards, and is known as Diplogonoponcs grandis. The tseniidse are represented by several species which may be parasitic to man, and three are common. Man is the definitive host in the case of Tcenia solium and Tcenia saginata, but for the former may act as the intermediate host. Man is the intermediate host for the echinococcus. The ova of the tseniidas ripen in the proglottides, and at the time of passage in the faeces are in the oncosphere stage. At this stage the embryo has a head armed with six hooklets, and is contained in a thick, radially striated, but not ciliated capsule. The oncospheres are swallowed by man or sheep in the case of the T. echinococcus, by cattle with the T. saginata, and by pigs with T. solium. The capsule is dis- solved in the intestinal canal of the intermediate host, and the hooked embryo pierces the gut wall and reaches the viscera. Here the embryo becomes encysted, and its hooklets drop off. The cyst or Fie. 20.-Head of TMa c 3^ercus becomes lined with Solium. Hooklet. Head g erm cells > from whlch scollces Natural Size. develop. The echinococcus cysts develop secondary or daughter cysts, and the scolices grow in these. The cysticercus is ingested by a flesh-eating animal ; the scolices are set free, attach themselves to the gut wall, and grow into adult worms. Taenia solium, the pork tape-worm, reaches a length of about 10 feet. The ripe proglottis is about 10 mm. long and 5 mm. broad. The genital pores, as is the case in all the tseniidse, open laterally. The uterus has about 10 lateral branches. The rostellum has 4 suckers and a double circle of hooklets. Man becomes infected by eating imperfectly-cooked and " measled " pork. The infection is much commoner in Germany than in Great Britain. Man occasionally acts as the intermediate host, and becomes infected by swallowing the oncospheres ; such infection may occur when human faeces are used as manure, or by auto-infection. THE PARASITOLOGY OF THE F.ECES. 333 ! The adult form can be recognised by the passage of the onco- spheres and the segments in the faeces. The head of the worm, which is a comparatively minute object, must be particularly sought for, and the rostellum, with its suckers and hooklets, is readily recognised with a low magnification. The cyst con- tents of the cysticercus infection are searched for the small hooklets, which are pointed, slightly curved, and not barbed. This cysticercus is known as the Cysticercus celluloses. Taenia saginata is the commonest of all human tape worms. It is larger and longer than T. solium and may attain a length of 20 feet. The ripe proglottis is longer and broader than that of T. solium. The uterus has from 20 to 25 main branches. The head has 4 suckers but no hook- lets, and there is usually a more or less circular deposit of black pigment in the anterior part of the head. The cysticercus occurs in the muscles of the ox and is known as Cysticercus bovis. Man is probably never affected by the cysticercus, but is always the definitive host. Persons most liable to be affected are those who eat raw or lightly-cooked beef, the small cysticerci in which can easily escape notice. The adult worm in the intestine produces few symptoms other than of a subjective nature. The diagnosis rests with the detection of the head, the proglottides, or the oncospheres in the fasces. Taenia echinococcus. — The echinococcus, or hydatid, is a small tape-worm only 4 or 5 mm. long. It is composed of a variable number of segments, usually 4, sometimes 3 or 5. The mature ova are contained in the last segment. The head is provided with 4 suckers and a double row of hooklets. The adult tape-worm is found in dogs, wolves, and jackals ; and the period between ingestion and the passage of mature ova is about 40 days. The oncospheres may be deposited on vegetables or grass, and from this source man or sheep may become infected. In man the cysticercoid form only is found, and the tumours may take three or more years to develop. Pig. 21.- nata. Head of Taenia Sagi- Head ^Natural Size. 334 CLINICAL PATHOLOGY. Human infection is common in Australia and other sheep- raising countries. In England it is perhaps most common in the eastern counties, and a considerable number of cases are met with in the Cambridgeshire district. Persons who have never been out of London may occasionally be infected, possibly through the dangerous medium of a dog-infested watercress bed. The cysticercus may be found in almost any region of the body in man, and occasionally numbers of cysts may be passed in the faeces following the rupture of a parent cyst into the gut. The cysts may be quite small, translucent, round bodies not unlike grapes. Their nature is certainly determined by the finding of barbed hooklets or well-formed Fig. 22. — Taenia Echinococcus. Scolices and Hooklets. scolices within them, but it is not uncommon to find numbers of well -developed but sterile cysts. Nemathelminths. — These are round, unsegmented worms, usually tapering at both ends. They are divided into three classes : — Class 1, Nematoda, commonly parasitic in man. Class 2, Nematomorpha, not parasitic in man. Class 3, Acanthocephala, very rarely parasitic in man. Only the nematodes are considered here. Nematodes. — The nematodes include a considerable number of different families, not all of which are parasitic, while others are very commonly parasitic in man. Strongyloides intestinalis is a very common intestinal parasite in tropical countries. It passes through a parasitic and a free-living form. The adult parasitic form lives in the mucous membrane of the small intestine. It is a cylin- drical worm 2*2 mm. long with a pointed tail. Only one sex, the female, is found. The eggs are deposited in the intestinal-mucous membrane and develop into larvae, which THE PARASITOLOGY OF THE F^CES. 335 leave the host in the faeces. Here they develop further, cast their skins, and become sexually mature. The females are 1 mm. long, and the males shorter. The sexes copulate, and the female lays eggs, from which larvse are hatched which develop into the parasitic form, and cannot reproduce them- selves unless again reintroduced into the human intestine. The larvae of the parasitic form are very frequently found in the stools of perfectly healthy persons, and do not appear to produce any symptoms. FUariid.Ee. — The varieties of these nematode worms occur- ring in the blood have been described in the chapter on Fig. 23. — Trichocephalus Dispar. Ovum. Female. Natural Size. the parasitology of the blood. The Dracuncidus medinensis, or guinea-worm, is considered in the chapter on the skin. Trichotrachelidae. — In man only two parasitic species of the nematode worms are known. They are Trichocephalus dispar and Trichinella spiralia. Trichocephalus dispar, or the whip-worm, is a bisexual worm with a simple developmental history. The female is about 50 mm. long, and the male is somewhat smaller. The anterior two-thirds of the worm consists of a thin filiform process. The posterior extremity of the male is curved upon itself, and ends in a rounded projection in which the vas deferens opens. The eggs are oval, and have a thick brown capsule with an opening at each end. The parasite is much more commonly found in the inhabitants of tropical climates than in temperate countries. No ill effect is produced by the, 336 CLINICAL PATHOLOGY. worms. Infection takes place by ingestion of the ova, and no intermediate host is required. Trichinella spiralis is viviparous, and requires two flesh- eating hosts for development. The females measure about 4 mm. in length, and the males half that size. Both have a pointed anterior extremity, but taper to it gradually. The posterior extremity of the male has two short caudal appen- dages, between which the cloaca lies. Trichinosis in man is rare in Great Britain, but common in Germany and America. The usual host is the pig, which may become infected by eating dead rats. The rats carry on the infection by eating each other. Man becomes infected by eating imperfectly-cooked pork. The larvae are encysted in the pork, and when swallowed are set free in the stomach. They become sexually mature in Fia. 24. — TTicHnella Spiralis. Larva Encysted in Muscle. the upper part of the small intestine, and copulate. The males die, and are passed in the faeces. The females bore through the gut wall into the lymphatic spaces, where they pass their larvae. The larvae pass into the circulation, and come to rest in striated muscle. Here they become encysted between the muscle fibres. The female dies after a vigorous productive period of about 7 weeks. . The infection is a serious one, and the migration of the larvae into the muscles is accompanied by considerable disturbance and local pain. A well-marked eosinophilia is present in the blood. Strongylidse — By far the most important of these worms found in man is the ankylostoma (uncinaria). Ankylostoma duodenale is a nematode worm which has a very wide distribution, and causes serious symptoms. There are two distinct species of ankylostoma, A. duodenale and A. americanum. The latter has not yet been met with in THE PABASITOLOGY OF THE F.ECES. 337 this country. In A. duodenale the female is 10 to 13 mm. long and the male is somewhat shorter. The anterior extremity of each is rounded, and the buccal cavity is guarded by 4 incurved spines. The posterior extremity of the female is pointed, while that of the male ends in a mem- branous expansion from which 2 long spicules protrude. The eggs are oval, with a very thin transparent and colour- less shell. Segmentation of the contents has commenced when they reach the faeces, 2 or 4 cells being visible. In A. ameri- canum the worms are smaller and the buccal cavity has no spines. The worms are extremely widely distributed in Fig. 25. — Ankylostoma Duodenale. Anterior and Posterior Extremity of Male. Posterior Extremity of FemaLe. Ovum. Female. Natural Size. tropical and subtropical countries, but in Great Britain are con- fined to places where suitable climatic conditions exist for the development of the ova. The infection is found amongst workers in the metalliferous mines of Cornwall, having been intro- duced from foreign countries. An extremely severe anaemia, accompanied by a well-marked eosinophilia, follows the infection. The adult worms live in the small intestine of man, being firmly attached by their heads to the mucous membrane. The ova pass out in the stools. The larvae hatch out under favourable circumstances in about 2 days. The full-grown larva is produced from the ovum in about 10 days, and after moulting three or four times becomes a sexless individual moving p. 22 338 CLINICAL PATHOLOGY. freely in a chitinous sheath. The ova are not infective ; the full-grown larvae are. Man becomes infected in one of two ways ; either the larvae are swallowed, a method which is probably the less common, or they enter through the skin, causing a local erythremia or urticaria, then pass to the lungs, causing bronchial catarrh, and finally find their way to the stomach. The diagnosis is made by the finding of the ova in the faeces. If the live ova are incubated they will be found to contain larvae in about 24 hours. The adult worms are rarely seen in the faeces. Ascaridae. — The ascaridae include two members which are commonly parasitic to man in this country, namely, Ascaris lumbricoides and Oxyuris vermicular is. Ascaris lumbricoides is the common round worm of man, and has an extremely wide geographical distribution. The female is from 20 to 40 cm. long, and the male about two-thirds that length. The heads of both sexes have 3 prominent lips, 2 ventral and 1 dorsal. The tail of the male worm is strongly curved in a ventral direction, and 2 fine spicules extrude from it. The worms are commonly found in pairs in the small intestine, and in the great majority of cases cause no symptoms. They occasionally, however, wander from the intestine, and may make their way up the common bile duct into the gall bladder or into the liver, and more rarely have been found in the pancreatic duct. The worms are only from time to time passed in the faeces, but the diagnosis can readily be made as a rule by finding the ova, which have a characteristic appearance. The ova are fairly large, measuring "05 by *07 mm., and are more round than oval. The un segmented ovum has a thick, transparent, colourless shell, which it does not completely fill. The shell is nearly always coated with a rough, granular albuminous material of a brown colour. The unfertilised ova contain retractile globules. The embryos develop in a moist atmosphere at room temperature Fig. 26. — Ascaris Lurnbricoid.es. Posterior Extremity of Male. Head Enlarged and Natural Size. Ovum. THE PARASITOLOGY OF THE FAECES. 339 in from 30 to 40 days, and can remain alive for long periods. The life-history is simple ; the ova containing the embryos are ingested, the capsules are dissolved, and the free larvae reach maturity in the intestine in about 5 weeks. Somewhat similar, but smaller, ascaridae infest dogs and cats, and are occasionally found in man. Oxyuris vermicularis is vulgarly known as the thread- worm, and is a common parasite of man and particularly of children of the poorer classes. The female is about 10 mm. long, and the male about half that length. The tail of the female is straight and pointed ; that of the male is curved and rounded. The two sexes live and copulate in the small intestine. The fertilised females leave the males and travel downwards to form large congregations in the caecum, appendix, Fig. 27. — Oxyuris Vermicularis. Male. Female and Natural Size. Ovum with Embryo. and ascending colon, where they reside until the ova are nearly mature. The females then set out on their travels again, pass down the rectum and appear at the anus. The ova are deposited on the mucous membrane and skin of the anus and perineum. The wanderings of the worms, the irritation the ova and of the larvae, which may escape from them, cause considerable perineal itching. The child scratches its perineum, and conveys the ova to its mouth or nose or to the face of its neighbour. The ova loose their capsules in the stomach, and the freed embryos reach maturity in about a fort- night. Fertilised females containing mature eggs are often swept out with the faeces, and the diagnosis is made by detecting them in the stools. The free ova are rarely found. The ova are of about the same size and shape as those of ankylostoma, and are enclosed in a thin capsule. They are, however, distinctly 22—2 340 CLINICAL PATHOLOGY. flattened on one side, and at the time of passage contain a well-developed embryo. Amoebae. — Amcebic dysentery is practically confined to tropical or almost tropical countries, and consequently has a much narrower geographical range than bacillary dysentery. Amoebic dysentery is common in India and Africa, and in many parts of these countries is the form of dysentery most fre- quently met with. Very rare examples of tropical abscess of the liver, possibly due to the Amoeba dysenterica, have been met with in patients who have never been out of Great Britain. The diagnosis of amoebic dysentery rests with the detection of the causative organism in the stools. In the large majority of cases the organisms are extremely numerous in the acute stage and readily found. All that is necessary is to place a drop of the blood-stained " mucus "on a slide with a cover-slip over it and examine with a ^-inch objective. A warm-stage and hanging-drop preparation is not essential, but the amoebic movements are more certainly seen by this method. The stool should always be examined within an hour or two of being passed, and large doses of ipecacuanha should be with- held until the examination is made. On a cold day the ordinary slide preparation should be warmed, and in any case it should be examined immediately after it has been made. If the amoebae are few in number a second piece of the mucus should be picked out and mounted in a drop of 1 per cent, watery methylene blue with a cover-slip over it. The advan- tages of this method are that the leucocytes and epithelial cells are immediately stained blue, but the amoebae resist taking the stain, while retaining their mobility for a considerable time. The clear, retractile bodies of the amoebae stand out very prominently on the blue background, and can be detected with a f-inch objective, the higher power being reserved for confirmation of the diagnosis. Amoebae of more than one variety are to be met with in the fasces. One is a perfectly harmless and normal inhabitant of the intestinal tract, and has to be distinguished from the dysenteric organism. The harmless amoeba is known as the Amoeba or Entamoeba coli : the dysenteric organism is called the Entamoeba histolytica. The Entamoeba coli, which is found in the upper part of the large gut, is of comparatively small size, and has a well-defined nucleus and little clear ectoplasm. THE PARASITOLOGY OF THE F.ECES. 341 The Amoeba histolytica is a large amoeba measuring from 25 to 35 fju in diameter, and has an abundant, clear, refractile ecto- plasm, and a poorly-defined nucleus which stains badly with the ordinary dyes. The distinction between the two amoebae is not of very great practical importance in a dysenteric district, since the harmless amoeba is very rarely met with, and if amoebae are found in considerable numbers in any stools the case may safely be diagnosed as amoebic dysentery. Bacteriology. — The faeces are normally so infested with bacteria that the difficulty is, not to obtain a growth in culture media, but to isolate the pathogenic organisms from the non- pathogenic and to obtain them in pure culture. Some departure from the ordinary routine bacteriological methods is necessary, and some clinical advice is particularly required as to the class of organism to be sought for. The detection of organisms known to be pathogenic and to be normally absent from the intestinal tract, such as the cholera vibrio or the typhoid bacillus, is of diagnostic significance. The detection of organisms of atypical cultural characters and of doubtful pathogenicity should be amplified so far as possible by other methods, such as the investigation of the agglutinat- ing action of the patient's serum upon them, and the findings, unless strongly supported by such means, must be accepted with reserve. The detection of bacteria known to be found in the gut in health, such as the bacillus coli, has no diagnostic significance. - Film preparations of the faeces commonly give little bacterio- logical information, but they should be made in the case of very liquid stools in order to gain an idea of the type of the prevalent organism. In cases of cholera the vibrio may be present in enormous numbers in the films. The cultural investigation necessarily varies with the type of organism looked for, but in the majority of cases the following method will be found useful as a routine : — The faeces should be passed into an ordinary clean (not carbolised) bed-pan, and should be examined as soon as possible after they have been passed. In an ordinary broth culture tube take 10 to 12 loops of the faeces with a sterile platinum wire. Shake the broth tube thoroughly, without letting the fluid 342 CLINICAL PATHOLOGY. splash against the wool plug, and stand the tube aside for a few moments to allow the solid particles to settle. Take a loop of the supernatant fluid and plate on an agar plate. Incubate the broth tube for from -I to 6 hours at 37° C. Take a loop of the supernatant broth and plate it out on 3 MacConkey plates, without recharging the loop. Incubate the 4 plates till the next morning. Examine the agar plate for colonies of the staphylococcal and streptococcal type. Examine the MacConkey plates for yellow colonies in particular. Take sub-cultures of the suspected colonies in the usual way. The points to be aimed at in this process are to obtain plate cultures which are neither hopelessly overgrown nor contain merely one or two colonies, but which show a fail* number of discrete colonies on the majority of the plates. The more important pathogenic organisms to be met with are the following : — The typhoid bacillus. — The bacilli can be isolated from the fasces by the above method. The yellowish colonies are picked out from the MacConkey plates and two or three of them subcultured into broth. The broth cultures are then subcultured into the appropriate media. If bacilli are obtained which culturally resemble the typhoid bacillus it is wise to test them with the serum from a known case of typhoid fever as well as with the serum from the patient. Paratyphoid bacilli. — These organisms are looked for in exactly the same manner, and in all cases the agglutinating action of the patient's serum upon them should be investi- gated. The sera of animals inoculated against the various organisms can be obtained from a few reliable sources, and should also be tested upon them. The dysentery bacillus. — This organism is investigated in the same manner. Either the Shiga-Kruse or the Flexner type may be found, and serum reactions should always be investigated. The bacilli may be met with in cases of dysen- tery and colitis in this country, and have been isolated in some epidemics of infantile summer diarrhoea in America. A bacillus of the same class, which has been associated with THE PARASITOLOGY OF THE FAECES. 343 British epidemics of infantile diarrhoea, has the cultural peculiarity of failing to ferment mannite, and is known as Morgan's No. 1 bacillus. The cholera vibrio. — The recognition of the cholera vibrio is a comparatively simple matter in an epidemic, or in districts where cholera is known to be rife. Sporadic cases of cholera are to be diagnosed with extreme caution, since the differentiation between the genuine cholera vibrio and other vibrios which may occasionally be present in the gut is a matter of considerable difficulty. The vibrios other than the cholera vibrio will be mentioned later. The examination of the faeces should be conducted as follows : — (1) Prepare a thin film of the faeces, and stain it with carbol- thionin. In an acute case the organisms are usually present in considerable numbers, and tend to appear in groups, all the members of which lie with their long axes in the same direc- tion, as trout lie in a stream. (2) Put several loops of the faeces into broth, and incubate for about 4 hours. Plate from the broth on to 3 gelatin plates, and incubate at from 18° to 20° C. for 24 hours. Examine the plates for colonies of the vibrio. These colonies are white with jagged outlines, and have the appear- ance of powdered glass on the surface of the plate. Film preparations from them show the vibrio. (3) Subculture from the gelatin plate into broth, on to agar slopes, and in gelatin stab cultures. Continue the incubation of the gelatin plate. After incubation for from 24 to 48 hours of the broth culture add to it a few drops of pure sulphuric acid. A rose-pink colour of nitroso-indole develops in the medium. The agar slope culture shows a yellowish irregular slimy growth. The gelatin stab culture shows a white streak of growth along the track of the inoculating wire, and at the surface of the medium a funnel-shaped depression of commencing liquefaction. The colonies on the gelatin plate slowly darken, and lique- faction takes place in the medium. (4) With the pure culture on the agar slope make a 344 CLINICAL PATHOLOGY. suspension of the vibrios in order to test the lytic action of an immune serum upon them (Chapter VIII., page 110). The crucial test of the true vibrio rests with the specfic action of the lytic serum upon it. It is not a practical necessity to perform this test in cases met with in an epidemic, but the notification of a case of cholera in a previously cholera- free district is a serious matter, demanding that every reason- able bacteriological precaution should be taken to ensure an accurate diagnosis. Among the organisms liable to be confounded with the vibrio of Asiatic cholera are the following : — The vibrio of cholera nostras described by Finkler and Prior may be found in considerable numbers in the stools of patients suffering from acute diarrhoea and vomiting with collapse, such as may occur in temperate climates in the summer months. In the majority of such sporadic cases, however, vibrios in the stools are either very scanty or absent. The Finkler-Prior vibrio is longer and broader than Koch's vibrio, and on gelatin plate culture forms round colonies with sharply-cut edges. Gelatin is liquefied rapidly. Similar vibrios, which may be found in the stools of healthy persons or patients with intestinal affections, have been isolated from cheese and from the water supplies of towns. Some of these vibrios closely resemble the cholera vibrio both on morphological and cultural grounds, and can only certainly be distinguished by serum tests. Inoculation of the cholera vibrio into the peritoneal cavity of guinea-pigs induces a typical toxic effect. Other vibrios are non-pathogenic to animals. Tubercle bacillus. — This organism may be present in the faeces in large numbers in cases of tuberculous enteritis, and in such cases pus is present in addition, and often blood. The bacilli are present in small numbers in the case of persons with pulmonary tuberculosis, and particularly of children, who are more likely to swallow the sputum. The faeces should be treated with antiformin and the bacilli looked for in the ordinary way. Occasionally the addition of antiformin to faeces produces a brilliant red colour ; in such cases the patients have been found to be taking " purgen " or phenol-phthalein, which turns red on the addition of alkaline antiformin. THE PARASITOLOGY OF THE F.ECES. 345 Other organisms. — Among other of the commoner organisms to be met with in the fasces are staphylococci and streptococci ; their significance is uncertain. Cocci are com- monly present in the normal fasces, and the streptococci met with are as a rule of very low pathogenicity for animals. In cases of ulcerative colitis the organisms commonly suspected and looked for as the causative agents are members of the coli- typhoid group, and it is quite possible that coccal infection may in some cases be overlooked. The cultural examination of colitis is aided by the passage of the sigmoidoscope. After thorough irrigation of the colon with sterile tap water a minute portion of the infected mucous membrane can be safely removed by a skilled operator and transferred to a culture tube. By such means streptococci may be found in pure culture in a small percentage of cases, and a vaccine prepared from the organism may be beneficial. The isolation of cocci from fasces by the ordinary methods may be difficult and can have no particular significance. SECTION VI. THE EYE AND SKIN. CHAPTEE XXIV. The Eye and Conjunctival Sac — The Skin. CHAPTER XXIV. the eye and conjunctival sac — the skin. The Eye and Conjunctival Sac. The cytology of the conjunctival sac does not materially differ from that of other parts of the body. In the more chronic infections of the conjunctiva large epithelial cells are commonly met with, and in the acute inflammations the ordinary polynuclear cells of suppuration. In the rare con- dition known as " spring catarrh " the exudate consists almost entirely of eosinophil cells. The bacteriology of the eye is of considerable importance, and in a variety of affections much assistance is obtained from a careful bacteriological investigation. The normal conjunctiva, being exposed to air contami- nation, is rarely sterile, and some acquaintance with the organisms present in health is necessary. The bacillus known to ophthalmologists as the xerosis bacillus is the organism most frequently met with, and is present in the majority of cases examined. The xerosis bacillus is no longer believed to play any essential part in the production of the condition the name of which it bears. It is a diphtheroid organism, and will be subsequently referred bo under that name. The diphtheroid bacilli met with in the eye are of more than one variety, but the great majority of them belong to a type which grows readily on the ordinary solid media, such as agar or blood serum, but merely maintains its existence in broth and other liquid media. It does not acidify litmus dextrose broth. Next to the diphtheroid bacilli a white staphylococcus of low virulerice is the organism most frequently met with. Exceptionally, more virulent organisms, such as staphylococcus aureus, streptococcus pyogenes and the pneumococcus may be cultivated from the apparently normal conjunctiva. The occasional finding of virulent organisms in normal 348 CLINICAL PATHOLOGY. cases is of considerable importance, and it is a reasonable precaution to make a bacteriological examination in all cases before conducting such an operation as that of cataract extraction. It is an essential precaution if any inflammatory condition of the lids or conjunctiva is present, however mild. Conjunctivitis- — The commonest and most widely-spread variety of acute conjunctivitis is that due to the Koch-Weeks bacillus. The disease has a short incubation period of about 24 hours, is extremely contagious, begins as a rule in one eye and almost invariably spreads to the other, is associated with redness and swelling of the lids and conjunctivae, and runs a variable course, often lasting from 2 to 4 weeks. The diagnosis can be sufficiently confirmed by film preparations. The minute, slender Gram- negative bacilli closely resemble in appearance the influenza bacillus, from which there is no practical necessity to identify them. They are of very variable length, and tend to appear in small clusters in the films. They are both intra- and extra- cellular. It is fortunate that the bacillus can be thus identified in film preparations, since their growth on culture media is extremely difficult to obtain. Special media containing some form of blood serum are required, and the organism is frequently outgrown by diphtheroid bacilli or other bacteria. Diplo-bacillary conjunctivitis. — The causative organism of this disease is known as the Morax-Axenfeld bacillus. The condition is widely distributed, and is more chronic than the Koch-Weeks' infection. It is infectious, almost always affects both eyes, and is associated with a characteristic redness of the angles of the palpebral fissure. The infection may in untreated cases last for years. A nasal catarrh is sometimes present in addition, and the causative organism may be found in the nasal discharge. In films made from the pus the bacilli are found as stout rods of moderate length with rounded ends, the great majority of them being in pairs and outside the cells. Occasional chains are present. The occurrence of Gram-negative bacilli of this nature in film preparations is sufficient to establish a diagnosis. A growth of the bacilli in the form of small, translucent colonies on serum agar can often be obtained. Gonorrhoea! conjunctivitis has been already referred to EYE AND CONJUNCTIVAL SAC— SKIN. 349 in the description of the gonococcus. The causative organism is as a rule numerous in the conjunctival secretion, and the condition is readily recognised in film preparations. Trachoma. — The cause of this disease is unknown, and numerous parasites, subsequently discredited, have been from time to time described. Other varieties of conjunctivitis. — A purulent con- junctivitis may be set up by any of the ordinary pyogenic bacteria, and the investigation of the exudate should be conducted on the ordinary lines. In no case should a diagnosis of the organisms be made from film preparations alone. Mixed infections are not infrequent. In a series of investigations dealing with this class of infection the staphy- lococcus albus was found twenty-eight times, staphylo- coccus aureus twenty-seven times, a streptococcus ten times, an intermediate staphylococcus three times, the diphtheria bacillus twice, and an unclassified coccus once. Diphtheroid bacilli were also present in about 60 per cent, of the cases. In addition to these organisms the pneumo- COCCUS may exceptionally be found as the cause of a primary conjunctivitis, and it has been described as a common cause owing to the improper identification of the organism by film preparations only. Epidemics of conjunctivitis due to the pneumococcus have been recorded. The diphtheria bacillus is a rare cause of conjunctivitis, being usually met with in children and in association with a nasal discharge containing the same organism. The diphtheria bacillus should be identified in this situation, not only by its morphological and cultured characters, but also by animal inoculation. Diphtheritic infection of the conjunctiva is associated, as elsewhere, with mem- brane formation, and is readily amenable to serum treatment. Membranous conjunctivitis is less commonly due to the diphtheria bacillus than to other organisms, of which the most important is the Streptococcus -pyogenes. Streptococcal infection of the conjunctiva is the most virulent type met with, and a small percentage of cases proceed rapidly to panophthalmitis in spite of all treatment. Less commonly staphylococci may produce a membranous conjunctivitis. The lids. — The bacteriology of the lids is practically identical with that of the skin, and all the commoner varieties of inflammation associated with the pyogenic cocci are met 350 CLINICAL PATHOLOGY. with. Molluscum contagiosum is occasionally encountered here, as elsewhere, on the exposed surfaces. The lachrymal sac. — Inflammations of the sac are set up by the ordinary pyogenic organisms, of which a streptococcus is perhaps the most common. Here again pneumococci may be present, but have been described more frequently than is probably correct owing to an undue reliance upon film prepara- tions. The micrococcus catarrhalis is another organism occasionally associated with this condition. The cornea. — The main organism concerned in the pro- duction of the serpiginous corneal ulcer is the pneumo- COCCUS. The infection is, however, not a truly specific one, and other organisms, such as the staphylococci and strepto- cocci, may be present in pure culture. The identification of the pneumococcus has been in some cases a matter of con- siderable doubt, and when cultures have been taken the organism has been depicted as growing in long chains of 30 members or more, a condition practically not met with among pneumococci from other sources. Bacilli of the Colon group, B. proteus and B. pyocyaneus, are occasionally found in corneal ulceration, as well as in exceptional cases of conjunctivitis without corneal infiltration. Aspergillosis of the cornea has also been recorded on numerous occasions. The chronic infective granulomata. — Tuberculosis, leprosy and syphilis may each effect the eye. The methods of recognising the causative organism are the same as those adopted for these diseases in other parts of the body. Endogenous infections. — Metastatic abscess in the eye may arise in the course of a general infection produced by any of the pyogenic organisms. An interesting variety of metastatic ophthalmitis occurs in epidemics of cerebro-spinal meningitis, and a similar condition, producing a disease known as " pseudo-glioma," has recently been demonstrated to be caused by the meningococcus, and to occur in cases with no history of meningeal symptoms. The coccus has been recovered from the interior of the eye after excision. The orbit.— The bacteriology of acute inflammatory con- ditions of the orbit calls for no particular description. Infection of the orbit arises by direct extension from adjacent parts in the great majority of cases. The most important sources of infection are the accessory sinuses of the nose. EYE AND CONJUNCTIVAL SAC— SKIN. 351 The Skin. There is scarcely any pathological investigation which may not from time to time be required for patients whose main complaint is of some skin lesion. The following are among some of the changes more particularly associated with diseases of the skin : — The blood. — An eosinophilia, often of marked degree, is frequently associated with many widespread dermal lesions. In the specific fevers associated with skin eruptions, and particularly in small-pox, chicken-pox, and scarlet fever, a considerable eosinophilia is the rule. In the two former diseases the eosinophils diminish in number and finally dis- appear when the bullae suppurate. Among other diseases associated with a vesicular eruption dermatitis herpetiformis is almost constantly accompanied by a considerable eosino- philia. In films made from the bullae in these cases, as well as in small-pox and chicken-pox, numerous leucocytes are present, and the great majority of them are eosinophils. In pemphigus chronicus, on the other hand, eosinophils are absent from the blebs and there is no eosinophilia in the blood. The cytological character of dermal exudates is thus of some assistance in differential diagnosis. Secondary infection of skin vesicles, however, very readily occurs, and it is necessary to examine the fluid as soon as possible after the vesicle has appeared. Cases are not infrequently met with in which vesicles have been deliberately produced by the patient either by the aid of blistering fluid or some cruder device. In these mechanical effusions the great majority of cells present are of the epithelial type, and the presence of such cells is strongly suggestive of an artificial lesion. Leukaemia is very rarely associated with considerable leuksemic infiltrations of the skin. These infiltrations have, however, on occasion been so considerable as to merit the name of " tumours," and the patients have first come under the observation of a skin clinic. Rare as the condition is, it is advisable in all cases of multiple skin tumours of doubtful origin to make an examination of the blood. The blood changes present may be either those of myeloid leukseinia or of the lymphatic variety. Pernicious anaemia is nearly always accompanied by a 352 CLINICAL PATHOLOGY. lemon-yellow colour of the skin, but in so far as the patient is concerned this is a matter of secondary importance. A rare condition may be met with known as hemochromatosis, in which the patient seeks advice upon the discoloration of his skin. This may be of a deep brown or almost black colour, and the change may have taken place in a few weeks or months. The discoloration is associated with an abnormal destruc- tion of red cells and the liberation of pigment from them. The blood as a rule shows all the changes typical of advanced pernicious anaemia. Syphilis. — In some continental clinics all cases of syphilis are treated by the dermatologist, and in this country numerous patients present themselves for examination principally for a skin syphilide. Consequently an examination of the Wasser- mann reaction in the serum is frequently required, and should be performed in all cases of syphilitic disease with skin lesions as well as for patients with rashes of a doubtful nature. The spirochete should also be looked for in the primary sore, and may be found in the condylomata and other secondary lesions. The parasitology of skin diseases. — The skin is peculiarly liable to infection both by animal and vegetable parasites, and a complete description of all such organisms as may be found in the skin can only be given in a book devoted to dermatology. A brief account only of the more important skin parasites is given here. The majority of them have been mentioned in the section on bacteriology. Animal parasites— Acarus scabiei is the parasite of scabies, or vulgarly "itch." The diagnosis of this common condition should be confirmed by the very simple examination needed to demonstrate the causative parasite. On the skin, and usually between the fingers, is seen a thin, greyish black raised line about \ inch long, forming the burrow produced by the female acarus as it travels from the surface along the skin. The burrow is dissected out with a surgical needle, and at the extremity furthest from the point of entrance is found a small black speck just visible to the naked eye, and evident under a hand magnifying glass as the female acarus. At intervals between the female and the surface are found the ova. The male acarus does not leave the surface of the skin, and is in consequence rarely observed. The female acarus, examined with a low power of the microscope, is seen as a EYE AND CONJUNCTIVAL SAC- SKIN. 353 somewhat rounded oval body with 8 limbs. The anterior 4 limbs are armed with suckers, the posterior 4 with bristles. The male acarus is similar but smaller. The ova may be detected by dissecting out a portion of the burrow and mount- ing it in saline or weak potash on a slide with a cover-slip over it. The oval eggs with a more or less developed contained embryo must be distinguished from the epithelial cells of the skin with their angular shape and central nucleus. Pediculi. — These loathsome-looking parasites are remark- ably particular in their habitat. Different varieties affect different parts of the human body, and practically never trans- gress upon each other's domains. Moreover the pediculi of some animals will not pass to other species of animals : for example, the common body lice of dogs do not attack man. The special body lice of man are named according to their distribution, Pediculus corporis, Pediculus capitis, and Pedicu- lus pubis. Pediculus corporis infests the trunk and the body clothing. It is the largest of the human pediculi, being about 3 mm. long and readily visible to the naked eye. The ova are laid upon the hairs or the clothing. Pedicidus corporis, as is the case with the other pediculi, has 6 legs armed with short claws. It attacks adults more commonly than children. Pediculus capitis is confined to the hairy scalp. It has a similar shape to Pediculus corporis, but is not so long. The numerous white ova or " nits " are attached to the hairs and form very conspicuous objects, while the adult parasites can readily be seen on close inspection moving among the hair roots. The infection is extremely common, especially among children with long hair. Adults, particularly women of the lower classes, are by no means exempt. Pediculus pubis is shorter and considerably stouter than either of the two former species. It is about 1^ mm. long. Its shape has earned it the euphonious name of " crab louse." The ova are brown, and are attached to the hairs in the same manner as those of Pedicidus capitis. Its range is almost entirely confined to the pubic hairs, but the hairs of the axillae and the eye-lashes may be exceptionally infected. The spread of this parasite is usually by sexual intercourse. Leptus autumnalis is an extremely common larva which p. 23 354 CLINICAL PATHOLOGY. attacks the human skin. Its vulgar name is "harvest bug." In many country districts the parasite is very numerous on grass lands in July and August, and attacks through thin clothing any part of the human body which may come in contact with the ground. The minute parasite buries its head in the skin. The common flea and the bed bug are objects sufficiently familiar to need no particular description. The reaction of the individual attacked is very variable, and the extreme swelling in a susceptible person may be very puzzling if the central puncture left by the bite be not recognised. There seems to be both a natural and an acquired immunity to the poison, and persons who make a practice of harbouring these creatures may show little swelling and no evidence of irritation. Dracunculus. — The guinea-worm is a common source of disease in the tropics, and, since the sojourn of the parasite in the human body is about one year, occasional instances of infection are met with in this country. The cycle of development is by way of a small water Crustacea to the human being, in whom the adult worm reaches maturity. The male worm fertilises the female and disappears. The fertile female migrates into the tissues, and leaves the body by piercing the skin in the most convenient position for deposit- ing her embryos in water. The skin is thus most commonly punctured in the neighbourhood of the foot or, if the host be a water-carrier, in the back. The worm is about the thick- ness of a piece of string and some 30 inches long. When the worm comes to the surface it is best removed by the time- honoured expedient of winding it on a stick a few inches at a time. The practice of injecting the worm with perchloride of mercury is less certain, since the dead worm is rendered more brittle and may have to be dissected out along its tortuous bed, or allowed to suppurate out. Fungi. — Microsporon Audouini is a common cause of ringworm of the scalp and body. It is peculiar to the human race and almost confined to children. The spores are closely attached around the shaft of a hair, and the short mycelial threads are scanty. Cultures on maltose agar produce round, white, downy colonies with a central tuft. Trichophyton megalosporon endothrix produces ring- EYE AND CONJUNCTIVAL SAC- SKIN. 355 worm of the scalp and body, and rarely of the nails. It is peculiar to the human race. The spores are arranged in chains within the hair. The colonies on maltose agar may be white, greyish yellow, primrose, or violet. Trichophyton megalosporon ectothrix is properly a parasite of ungulates, dogs, cats, and birds, but is transmissible to man. Ringworm of the body, beard, and nails is produced, the scalp being only occasionally attacked. The spores are arranged in chains, and the mycelium, which may be abun- dant, is jointed. There are numerous varieties of this fungus, which can be distinguished by the colour and appearance of their colonies. Achorion Schoenleinii is the commonest of the favus fungi, and affects the scalp, skin, and nails. It has a branched mycelium with numerous large spores. Cultures are brownish yellow, with a ridged surface. Microsporon furfur infects the hairy layer of the epidermis of uncleanly persons. The disease produced is known as pityriasis, or tinea versicolor. The parasite has an abundant branching mycelium, interspersed with clumps of spores. Microsporon minutissimum affects moist regions of the body, producing a lesion known as erythrasma. The fungus consists of an abundant mycelium composed of very fine long, unbranched threads. Spores are scanty. The diagnosis of these fungoid diseases should always be confirmed by the microscope. The simplest method of demonstrating the mycelium and spores is to remove an affected hair, or to scrape off the epithelial scales of the skin or the parings of infected nails, and mount them in liquor potassae on a slide beneath a cover-slip. Other fungi are rarely present in skin lesions met with in this country. Actinomycosis of the skin is very unusual, and the skin is practically only affected by extension from the deeper structures. The organism is to be sought in the yellow granules of the pus. Madura foot, or mycetoma, another cutaneous affection, is met with in India and East Africa, and is caused by a variety of fungoid organisms. Blastomyces, or yeast fungi, have been shown to produce a nodular infectious disease of the skin, and the organisms may become dissemi- nated through the system. The sporotrichia are organisms 23—2 356 CLINICAL PATHOLOGY. possessed with a regular septate mycelium and spore-bearing branches. They produce a granulomatous condition of the skin, simulating the lesions of tuberculosis and syphilis. The fungus grows slowly on appropriate media, and the serum of affected persons acquires agglutinins for a suspension of the spores obtained from a culture. The condition has, compara- tively recently, attracted considerable attention. Bacteria. — The Normal Skin. — The normal skin, like the normal conjunctiva, being an exposed surface, is in conse- quence rarely sterile. The organisms to be met with on almost any skin and in almost any part of the body are white staphylococci, sarcinae, and diphtheroid bacilli. These organisms are under ordinary circumstances non-pathogenic. It is possible that they play some part in the normal meta- bolism of the skin, and that in diseased conditions they may even become pathogenic. The number of these organisms present on the skin of different individuals varies considerably, and they are a common source of contamination in culture tubes, whether derived from the defective technique of the bacteriologist or from the skin of the patient. The diphtheroid bacilli and sarcinse are readily recognised, and their presence in films or in culture tubes may always be regarded as evidence of contamination. The staphylococci of the normal skin are almost always represented by a white variety which is very inactive in culture media, growing readily but producing little change. Less commonly more virulent organisms may be grown from the normal skin, and the majority of pathogenic species can on occasion be obtained. Staphylococcus aureus, or citreus, and the streptococci are the most important organisms met with, and were of particular importance to the surgeon before the introduction of rubber operating gloves. They are still of importance if present on the skin of the patient. Virulent organisms can maintain their existence on the skin without producing any harmful results upon their host. They may exist for considerable periods in spite of attack by all the ordinary methods of cleanliness, including antiseptics. The action of antiseptics applied to the skin rarely succeeds in destroying all bacterial growth without injury to the epidermis. A minute trace of a powerful anti- septic, however, will inhibit or delay the subsequent growth of EYE AND CONJUNCTIVAL SAC— SKIN. 357 organisms, and in cultures taken from a focus which has been exposed to antiseptic action it is common to find the visible growth of a freely-multiplying species delayed by two or three clays. Streptococcal infections of the skin are common, and the most notorious infection produced is erysipelas. Erysipelas proper is an acute inflammation of the dermis caused in the great majority of cases by the Streptococcus pyogenes. The cultural investigation is not always successful, since material may not be available for cultural purposes. The organisms spread along the superficial layers of the skin, but cultures made from recent bullse are in a considerable percentage of cases sterile. If cellulitis is present in addition, and incisions are made, the streptococci can be obtained in pure culture. The local lesion in erysipelas may provide the causative organism, but in cases of facial erysipelas particularly the local lesion may not be obvious, or may be situated in the nasal cavity and consequently form the habitat of numerous other bacteria. Impetigo contagiosa is a primary infectious disease most often met with in children, and produced by the Streptococcus pyogenes. The secondary impetigo produced by scratching in patients affected with scabies, pediculosis, or other irritative lesions may be streptococcal, or more commonly staphy- lococcal, in origin ; mixed infections are, however, frequent. The follicular impetigo of the hair follicles is essentially a staphylococcal disease. Pemphigus neonatorum is usually associated with a septic condition of the umbilical cord stump, and is a disease attended with a high mortality. The causa- tive organism may be a streptococcus or a staphylococcus. Staphylococcal infections of the skin are, as might be expected, extremely common, and very varied lesions are pro- duced. Follicular impetigo of the skin and of the scalp is a frequent infection, particularly among ill-cared-for children. The condition may be transmitted by one child to another, is liable to relapse, and often runs a prolonged course. The causative organism is nearly always the Staphylococcus aureus, but mixed infections are not infrequent. The lesions are readily inoculated by the patient from one part of the body to another. Staphylococcal inflammations of the eyelids or con- junctivae are often produced by auto-inoculation from an 358 CLINICAL PATHOLOGY. impetiginous focus on the finger or the face. In refractory cases benefit is often derived from vaccine treatment. It is preferable that an autogenous vaccine should be given in this and in all other staphylococcal infections of the skin. Boils are sequels of follicular impetigo, and due to the same organisms. They are prone to occur in the debilitated as well as among those in particularly vigorous health. Carbuncles are a more serious lesion produced by staphylococci, of which Staphylococcus aureus is the most frequent agent. Vaccine treatment is often of value as an aid to surgery, but should never be given to the exclusion of thorough surgical inter- ference. The majority of the cutaneous infections produced by staphylococci tend to run a chronic course and to recur, and attempted immunisation with vaccines is reasonable in most cases. Multiple subcutaneous abscesses due to staphylococcal infection are sometimes met with, and are not uncommon as a sequel to prolonged fevers. Typhoid fever is fairly often associated in the post-febrile stage with the appearance of numerous and widely-distributed subcutaneous abscesses. The Staphylococcus aureus or albus is nearly always obtained in pure culture, and I have never found the typhoid bacillus in these lesions. Subcutaneous abscesses may result from a general blood infection, and be associated with pus formation in other parts of the body ; but in the majority of cases the patient's general condition is good, and the abscesses appear to result rather from the local atrophic condition of the skin following fever and the prolonged stay in bed. The production of bed sores is a similar process in which pressure plays a prominent part. Whitlow is often of staphylococcal origin, and when accom- panied by a spreading lymphangitis is practically always due to the Staphylococcus aureus. Barber's rash, or sycosis, is one of numerous local skin in- fections, many of which are provided with special names and the majority of which are due to staphylococci. Seborrhcea may be produced by a variety of organisms, including staphylococci, and in view of the extraordinarily septic habits of many barbers, even in the most highly-gilded saloons, it is remarkable that more violent infections are not frequently transferred from one victim to another. Fortunately the EYE AND CONJUNCTIVAL SAC— SKIN. 359 highly-priced " tonics " applied after the operation usually contain some cheap and useful antiseptic. Acne vulgaris is an inflammation of the sebaceous glands, commencing as a rule at puberty and rarely lasting beyond the twenty-fifth year. The causative organism is a small Gram-positive one allied to the diphtheroid group, and a variety of them probably exist. They may be obtained from the comedones before suppuration occurs, and less commonly after it has taken place. Suppuration is usually the result of a mixed infection by the acne bacillus and the Staphylococcus albus. The result of vaccine treatment in these cases is very difficult to judge, owing to the natural variability of the disease and its tendency to sudden cessation apart from treatment. A very guarded prognosis should be given before commencing vaccine treatment, and it is wise to warn the patient that the scars of old lesions will not be affected by the vaccine. Old- standing cases with deep scarring are perhaps least often affected by vaccine treatment, while cases with comedones and no pus formation are the most favourable. In nonsuppura- tive cases doses of 5 to 10 million of the bacilli should be given, and in the suppurative cases the best results may be obtained with a coccal vaccine, or with a mixed vaccine of cocci and bacilli. Anthrax is a rare infection occurring among hide porters, wool sorters, and butchers. An account of the anthrax pustule and of the bacillus has been given in a previous chapter. Other pyogenic organisms which may affect the skin are numerous ; but the majority of such infections are not primarily dermal and spread to the skin by extension from the deeper tissues. Tuberculosis of the skin assumes many clinical forms, and on a pathological basis may be divided into at least two varieties. In one form the tubercle bacilli are present in the lesion, although as a rule in very small numbers. The bacilli are in a considerable percentage of cases of the bovine type, and the lesions which they produce have to be recognised mainly from their clinical features. Material is not ordinarily available for bacteriological examination, and the diagnosis has to be confirmed when necessary by removal of a portion of skin. The histological evidence of tuberculosis can sometimes be confirmed by detecting the bacilli in the 360 CLINICAL PATHOLOGY. sections, but the organisms are commonly very scanty, and often proof can only be completed by inoculation of portions of tissue into guinea-pigs. Diagnostic injections of old tuberculin, or Von Pirquet's reaction, are nearly always posi- tive, and a negative result to either test is strong evidence against tuberculosis. In the other class of tuberculous infections the bacillus appears to be entirely absent from the lesion, and to such conditions the term " tuberculides " is applied. It is supposed that the affections may be caused by the toxins of the bacillus which have been absorbed from a distant focus. Leprosy is characteristically associated with the formation of granulomata in the skin, and the diagnosis can nearly always be confirmed by the examination of swabs taken from the nasal secretion. The lepra bacilli are often numerous in this situation. Syphilis and the means of detecting the Spirochceta pallida, together with the importance of the Wasserman reaction, have already been mentioned. Another disease affecting the skin and produced by spirochetes is Yaws. This disease has some clinical features in common with syphilis, and is produced by a spirochete extremely like the Spirocliceta pallida. SECTION VII. THE KESPIRATOBY TEACT. CHAPTEE XXV. The Nose — The Sputum. CHAPTER XXV. the nose — the sputum. The Nose. The examination of the nasal secretion is mainly bacterio- logical, and since cultures taken from the nasal cavity are practically never sterile, the results of bacterial examination in disease must be critically considered. The methods of bacterial investigation do not materially differ from those previously described. It is advisable as a routine to use a cotton-wool swab similar to that employed for diphtheria cultures, and to plate direct from it on to two agar plates. A further culture may also be made into broth, and film preparations should be made in addition in all cases. The following are among the affections which may especially require investigation : — Nasal catarrh. — The usual attack of nasal catarrh is commonly left to run its course with or without the aid of domestic remedies. Some persons are so unfortunate as to suffer from repeated attacks at short intervals, and particu- larly during the autumn and winter months. Vaccine treatment has been very largely employed to meet such cases. Provided there is no local nasal condition such as can be rectified by the surgeon, and the patient is seriously embar- rassed by the attacks, it is reasonable to attempt to isolate the causative organism and prepare a vaccine from it. Excep- tional patients appear to derive actual benefit : others obtain mental comfort from the administration of a hypodermic injection : many are unaffected. On the whole the benefit to be expected is so uncertain that it is not justifiable to urge a course of vaccine therapy, or even to advise it, except as a last resort. The organisms which may be met with include the micro- coccus catarrhalis, streptococci, staphylococci, and THE NOSE -THE SPUTUM. 363 less commonly pneumococci and Friedlander's pneumo- baciUus. The vaccine is preferably made from the pre- dominant and most virulent organism. The selection of the causative bacterium is partly a matter of chance, and a mixed vaccine may be employed. Hay fever. — A standardised vaccine of pollen toxin has been given for this condition. Affected patients give an ophthalmic reaction to the pollen similar to the Calmette reaction to tuberculin. The method is still on its trial and is not described here. Diphtheria. — Nasal diphtheria is not infrequently met with in children, and the bacteriological examination is con- ducted in the same manner as in tonsilar diphtheria. Harmless diphtheroid bacilli, however, are more commonly met with in the nose than in the throat, and in cases of clinical doubt it is advisable to inoculate a guinea-pig with a culture of the suspected organism. Leprosy. — The examination of the nasal secretion in cases of leprosy has already been considered. The bacilli are almost invariably present in the nose in considerable numbers and in the early stages of the disease. There may be little actual discharge noticeable to the patient, and the secretion is often thick, crusted, and difficult to manipulate. The Sputum. General examination of the sputum. — Naked-eye observations. — The amount of the sputum is of importance in certain diseases, and in particular in cases of bronchiectasis and pulmonary tuberculosis. The amount may vary from a few cubic centimetres in the early morning to as much as a litre in the 24 hours. The odour is practically inoffensive in the majority of affections, but may be quite overpowering. Bronchiectastic sputum is nearly always offensive, as is also that from an abscess of the lung. If gangrene of the lung is present the odour is indescribable. The colour is whitish in early and mild catarrhal cases. It becomes yellow with advancing suppuration. It is red if blood is present. Blackish particles, visible to the naked eye, are nearly always present, and are due to inspired atmospheric carbon. 364 CLINICAL PATHOLOGY. The consistence and general appearance is some guide to the condition. The sputum in pneumonia is particularly viscid, and in tuberculosis of the lung the sputum may have a " nummular " character. Fibrinous casts, spirals, and shreds of solid tissue may in certain conditions be visible to the naked eye. Microscopical examination. — The sputum can be examined microscojjically both in the fresh state, by squeezing a portion between a slide and a cover-slip, and in stained film preparations. The stained films show in the majority of cases large epithe- lial cells and leucocytes of the polynuclear variety in varying proportions, fibrinous strands, and large numbers of organisms. The structures to be examined for are the following : — The cells. — Epithelial cells are nearly always present, but if they form the great majority of the cells in a film the " sputum " probably comes mainly from the mouth and upper air passages. Tubercle bacilli are rarely found in such speci- mens, which are liable to be produced by a patient to order and to be examined, with the misleading result that tubercle bacilli are stated to be absent. Pus cells are present in all sputa whether the underlying condition is tuberculous or not. Eosinophils are not recognised in carbol-thionin or methy- lene blue preparations ; they must be specially stained for, and are well seen in thin films treated with Leishman's stain in the ordinary way. Eosinophils may form the predominant cell in cases of genuine spasmodic asthma. Red blood corpuscles may be recognised in the fresh speci- mens and in the stained films. They stain a greenish colour with carbol-thionin, but are best fixed and stained by one of the blood stains. Elastic fibres.- — Before the discovery of the tubercle bacillus the presence of elastic fibres in the sputum was regarded as of great diagnostic importance. The fibres are now rarely looked for, but their presence in sputum is of significance, since they indicate that there has been actual destruction of lung tissue. Elastic fibres in small numbers may find their way into the sputum from the food, and it is advisable to instruct the patient to cleanse his mouth thoroughly before obtaining a sample of sputum. THE NOSE— THE SPUTUM. 365 Elastic fibres are found in tuberculosis, bronchiectasis, and pulmonary abscess. They are occasionally met with in lobar pneumonia apart from abscess formation. In actual gangrene of the lung the fibres are rarely found, probably because they have been dissolved locally by ferment action. Single elastic fibres are difficult to detect and are of little diagnostic importance, since they may have been introduced in the food, unless very particular care has been taken. Elastic fibres occurring in bundles which display an alveolar arrangement are more readily detected, and certainly come from the lung. The fibres vary in size, and have a wavy outline and double contour. If they are present in considerable numbers it is only necessary to mix a little sputum on a slide with 10 per cent, caustic potash, and to spread it out under a cover-slip. The fibres are more resistant to the potash than the other con- stituents of the sputum, and stand out as curved refractile threads. If the elastic fibres are few in number add to some sputum in a test tube an equal part of 10 per cent, caustic potash. Boil until the sputum is dissolved. Mix the solution with four times its own volume of water. Allow the mixture to stand for 24 hours, and examine the deposit for elastic fibres. Curschmann's spirals. — These spiral bodies are found in great numbers in the sputum of cases of spasmodic asthma. They are not found in asthmatic cases of old standing in whom advanced emphysema and bronchitis have occurred. The sputum in such cases is of the ordinary bronchial type. The spirals are present in the sputum which immediately follows a true spasmodic attack. They are not confined to asthma, but may be occasionally observed in cases of acute pulmonary tuberculosis. The spirals are visible to the naked eye, and appear as white twisted tenacious bodies in the sputum. Examined under the microscope they are seen to consist of a coarse central thread round which is wound a twisted meshwork of delicate fibrils. When straightened out the spirals may be 2 or 3 inches long. The fibrils appear to consist of a central thread of fibrin around which tendrils of mucin are wound. The spirals are often embedded in epithelial cells, and may contain in addition 366 CLINICAL PATHOLOGY. Charcot-Leyden crystals. Their source of origin is probably the smaller bronchioles. Charcot-Leyden crystals. — These are colourless, elon- gated, and sharply -pointed octahedral crystals. They are insoluble in water, alcohol or ether, and soluble in acids and alkalies. They are frequently found in the sputum, particularly after standing, of asthmatic patients, but are not diagnostic of this disease, and probably are of no particular significance. Fibrinous casts. — Small fibrinous casts of the finer bronchioles are occasionally detected in the sputum in lobar pneumonia, broncho-pneumonia, and rarely in bronchitis. They are whitish in colour, and of moderately firm consistence. Large fibrinous casts are practically confined to the very rare condition known as fibrinous, or chronic plastic, bronchitis. The fibrinous coagulum consists of a branched stem with numerous sub-divisions, resembling the leafless branch of a tree. Almost the complete cast of a bronchial system may occasionally be expectorated. The sputum in various diseases. — Bronchitis in the earlier stages is accompanied by a whitish, viscid, and scanty sputum, which later becomes yellow, copious, and obviously purulent. The sputum is rarely " nummular," but cannot be distinguished by its appearance from that of tuberculous cases. Bronchiectasis. — The sputum is copious, and is usually brought up in large quantities at a time, with considerable intervals between the attacks of expectoration. It is as a rule comparatively fluid, and almost invariably has a highly- offensive odour. Pneumonia. — At the onset of lobar pneumonia the sputum is very scanty or absent, and in exceptional cases there may be very little sputum throughout the course of the disease. Later the sputum becomes abundant and is characteristically tenacious. Owing to the intimate admixture of the blood and exudation in the pulmonary tissues the sputum is more or less evenly blood-stained, and is commonly described as " rusty " in appearance. Pulmonary tuberculosis. — In the majority of cases there is little in the appearance of the sputum by which tuberculous cases can be distinguished from other pulmonary conditions. The presence of blood in the sputum, but not intimately mixed THE NOSE— THE SPUTUM. 367 with it as in lobar pneumonia, is always suggestive of tuber- culosis. The blood is bright red as a rule, and may be in large amount, or the sputum may be streaked with blood. Nummular sputum is also indicative of tubercle of the lung with cavity formation. The nummular character is best seen by floating the sputum in water, when the round, more or less flattened, discs of muco-pus separate out and finally sink to the bottom. The only valuable evidence of tuberculous sputum is the finding of the tubercle bacillus. The bacilli are to be found in the very great majority of all cases of pulmonary tuberculosis associated with a considerable degree of expectoration. Influenza is associated in the early stages with sputum of the ordinary bronchial character, and in the later stages often becomes extremely profuse and very tenacious. The bacilli can be detected as a rule in ordinary film preparations. Asthma. — In cases of spasmodic asthma the sputum com- mences as the dyspnoea passes off, and small characteristic pellets are coughed up. The pellets contain eosinophil cells in large numbers, Curschmann's spirals, and often Charcot- Leyden crystals. Abscess. — In abscess of the lung the sputum may consist of pure pus with practically no mucoid admixture. In cases of any standing the smell is always offensive. The abscess may come from the lung itself or from the pleural cavity after rupture of an empyema into the lung. The material expectorated may be indistinguishable from the contents of a bronchiectatic cavity. In all such cases numerous fine, long bacilli are present and are probably responsible for the odour. A tropical abscess of the liver may rupture into the lung and give to the sputum an appearance of having been mixed with anchovy sauce. Amoebae may be detected in the sputum. Gangrene is associated with sputum of a green colour and extremely offensive odour. Gangrene of the lung may follow an injury, and is the rarest sequel of lobar pneumonia. Malignant disease of the lung is usually associated with haemorrhagic sputum. The expectoration has been pleasantly compared to red currant jelly. Malignant disease of the oesophagus is very commonly 368 CLINICAL PATHOLOGY. associated with a very profuse, pale, watery, and tenacious secretion. The occurrence of this material in the sputum pot is strong evidence of a malignant, as opposed to a spasmodic, stricture of the oesophagus. (Edema of the lung is accompanied by a copious white, frothy sputum. "When blood is present in addition the expectoration is commonly likened to prune juice. Infarction of the lung is accompanied by a bright red sputum intimately mixed with froth. Pneumoconiosis results in a brownish black sputum. The colour is due to particles of carbon in " anthracosis," or of sulphide of iron in " siderosis," or to lime dust in " stone- mason's lung." The parasitology of the sputum. — Infection of the lungs by the higher animal parasites is not common. In hydatid disease of the lungs or pleura the characteristic hooklets can sometimes be found in the sputum, and occasional small cysts may be coughed up entire. In cases of endemic haemoptysis met with in China and other parts of eastern Asia the ova of the Paragonimus westermani are to be looked for in the sputum. The parasite has been described in the section on the faeces. The amoebae of dysentery, after rupture of a liver abscess into the lung, are looked for in the sputum in exactly the same manner as in the faeces (p. 340). Failure to find the amoebae is not uncommon. The organisms are more likely to be met with 2 or 3 days after the rupture of the abscess has taken place. Bacteriology of the sputum. — In almost every pul- monary affection the sputum swarms with a variety of organisms, and, apart from the detection of the tubercle bacillus, the recognition of the causative bacterium is usually a matter of considerable uncertainty. The following procedure may be followed for the routine bacteriological examination of the sputum : — (1) The mouth should be cleansed as far as possible before the material is expectorated, but strong antiseptics must be avoided in the cleansing. (2) A sterile test tube fitted with a cotton-wool cork and a sterile glass filter funnel is provided, and the patient is directed to remove the cork from the tube, to insert the funnel, and to THE NOSE— THE SPUTUM. 369 expectorate down the funnel into the tube. The funnel is then removed and the tube re-corked. (3) Elaborate methods of washing, filtering, and teasing the sputum are in use ; but perfectly satisfactory results are obtained by siruply taking a loop of the sputum in a sterile platinum wire, and plating it out on two agar plates without re-charging the loop. Discrete colonies are almost invariably obtained in this way. A broth culture is preferably put up as a control at the same time. Film preparations are also made. (4) Incubate the plates till the following morning. (5) Examine the films and the plates for the predominant organisms. Pick off with the platinum wire sample colonies from the plate, and subculture them. (6) Subculture from the secondary cultures into the appropriate media organisms likely to be of pathological importance. Ignore colonies of sarcinae, spore-bearing bacilli, and in most cases staphylococci. If delicately-growing organisms of the influenza bacillus type are being sought for, substitute nasgar for agar plates in stage 3. Among the organisms to be looked for in the sputum are the following : — The tubercle bacillus. — The methods of examining the sputum for tubercle bacilli have been already described (page 155). If there is any urgency films may be made from the sputum direct, and stained by the Ziehl-Neelsen process. In the majority of cases it is preferable to carbolise the sputum and let it stand till the next day. In those cases in which tubercu- losis is strongly suspected and there is an appreciable quantity of sputum, yet no tubercle bacilli have been found by the carbolic acid process, a further sample of sputum may be treated by the " antiformin " method. It is, however, excep- tional to detect bacilli in a sample of sputum after treating with antiformin if they have not previously been found after carbolising. It occasionally happens, on the contrary, that scanty bacilli are found in the carbolised, and not in the antiformin, films. In all doubtful cases at least 15 minutes should be given to p. 21 370 CLINICAL PATHOLOGY. each slide before abandoning the search, and it may be necessary to examine the morning sputum on two or three occasions. If the patient with suspected pulmonary tuberculosis is a child who swallows the sputum it is worth while to examine the faeces for the bacilli by the antiformin method. The pneumococcus.— The diagnosis of lobar pneumonia by the demonstration of the causative organism in the sputum is rarely called for, and fortunately, since the bacterio- logical diagnosis is unsatisfactory. In films made from the sputum a variety of organisms are often present, and the pneumococcus can rarely be identified with any certainty. In cultures streptococci are often found in addition to the pneu- mococci, and the colonies of these organisms are so similar that it is difficult to separate them. Further, pneumococci may be obtained from the sputum in other conditions than lobar pneumonia. There is little question that the great majority of cases of lobar pneumonia are produced by the pneumococcus, as can be shown by lung puncture and by the examination of empyema fluids. A minority of cases, however, are due to streptococci and other organisms. Streptococcal pneumonia is commoner in children than in adults, and more often has a broncho- pneumonic than a lobar distribution. The recognition of the pneumococcus requires cultural investigation, and should not rest with the appearance of the organism in film preparations. Friedlander's pneumo-bacillus. — This organism is pre- sent in the sputum in some cases of pneumonia, as well as in other conditions. It may be found also in the mouth and in the nasal cavity. The bacilli are Gram-negative capsulated organisms, which usually appear as short rods with rounded ends. They occur in the films in small groups of 2, 3, and 4. The pneumo-bacillus is best isolated from the sputum by plating direct on two gelatine plates. The colonies appear as white, heaped-up points on the plates, and there is no liquefac- tion of the gelatin In gelatin stab cultures growth occurs along the track of the inoculating wire, and profusely in a round, heaped-up growth at the surface of the stab. The growth in gelatin stab cultures is described as " nail shaped." THE NOSE— THE SPUTUM. 371 Litmus milk is acidified and clotted. A copious and viscid growth is obtained on an agar slope. Both acid and gas are produced in the litmus carbo-hydrate media. The influenza bacillus. — The bacilli in the earlier stages of the disease may be found in very large numbers in the sputum. They may occur in considerable sized clumps out- side the cells, and a minority of them are intracellular. The minute " dew drop " colonies may be obtained in plate cultures on glycerine agar, or preferably on serum agar or nasgar. The colonies grow somewhat slowly, and the organisms, unless particularly abundant, are apt to be overgrown by other bacteria. Gram-stained film preparations of the sputum counterstained with carbol fuchsin are useful as a means of diagnosis. The minute red bacilli are sufficiently characteristic. Streptococci. — These organisms are very constantly met with in the sputum, but are usually not of the pyogenes variety. Perhaps the commonest bacterium met with in all samples of sputum is a short-chained streptococcus of the brevis type, which acidifies and clots milk and which resembles the pneu- mococcus in its cultural characters more closely than the Streptococcus pyogenes of acute inflammation. The organism differs markedly from the pneumococcus in its low pathogenicity to mice and rabbits. The Streptoccus brevis probably plays a part in a variety of pulmonary infections, but mainly as a secondary invader of the lung. It is an organism often pre- dominant in the contents of a bronchiectatic cavity. Staphylococci. — These organisms are frequently found in the sputum, and may be responsible for a percentage of bronchitis cases. S. aureus and S. citreus are much less commonly met with than the white staphylococcus, which is found in practically all sputa and has no diagnostic signifi- cance. Diphtheria bacillus. — This bacillus is rarely found in the sputum, but in cases of laryngeal diphtheria with a membrane spreading downwards portions of the membrane may be occa- sionally coughed up. Membrane is commonly expelled from a tracheotomy wound, and there is little difficulty in cultivating the bacillus from it. Actinomyces. — Actinomycosis of the lung is an uncommon condition, but the organisms may obtain primary lodgment 24—2 372 CLINICAL PATHOLOGY. in the lung or may be carried there from a distant focus. The characteristic granules can be found in the sputum or in the pus from an empyema, and should be examined in the ordinary way. The beaded Gram-positive streptothrix is sufficiently characteristic, but " clubs " are practically never met with. Other streptothrices are occasionally found, and some may be both Gram-positive and acid-fast, thus resembling the long and beaded forms of the tubercle bacillus. The diagnosis of a streptothrix infection from the sputum must be made with care, since streptothrix-like organisms may be present in the mouth as a contamination from the food, and it is advisable that the mouth should be thoroughly cleansed before the sample of sputum is obtained. The actinomyces organism occurs in tufts (see Plate IX.) of beaded filaments, and should not be confounded with the long, thin beaded bacilli often present in sputum, nor with the fine fibrinous filaments seen in all film preparations from sputum or muco-pus. The plague bacillus. — In the pneumonic forms of plague the causative organism is to be met with in large numbers in the sputum. The bacilli are present also in the septicemic form of plague, and in the bubonic form, but only in the latter condition when there is a pyaemia with metastatic abscesses in the lung. The diagnosis in a plague district may reasonably be made from the appearance of the bacilli in film preparations. The bacilli occur in the sputum in pairs, clumps, and short chains. The bipolar staining is best seen if the films are fixed in absolute alcohol before staining. Cultures of the bacillus should be obtained if possible, but the organism is often oul grown by the other bacteria of the sputum. Lung* puncture. — Puncture of the lung through the chest wall as a means of diagnosis has already been referred to, but since it is a proceeding not devoid of risk is rarely justifiable. The operation is not infrequently performed accidentally in the expectation of finding fluid in the pleural cavity, and the small quantity of blood-stained fluid removed from the lung is often sufficient for film preparations and for cultural processes. The bacteriological examination of fluid removed in this way is more satisfactory than similar investigations of the sputum. Pleural fluids. — The nature and mode of examination of these fluids has been described in the section on " Puncture Fluids." SECTION VIII. HISTOLOGY. CHAPTEE XXVI. The Examination of Sections — The Inflammations — The Degenerations. CHAPTEE XXVII. Neoplasms — Simple Tumours. CHAPTEE XXVIII. Carcinomata — Sarcomata — Other Tumours — Cysts. CHAPTEE XXIX. Histological Methods. CHAPTER XXVI. the examination of sections the inflammations the degeneeations. The Examination of Sections. The histological examination of tissues removed during life is among the most important of the methods of clinical pathology, and from the point of view of diag- nosis probably the most difficult. There is no other method of diagnosis which requires more experience for the interpretation of what one sees, and the necessary experience cannot be gained by reading, but comes from repeated examination of many types of the same pathological change. An acquaintance with the normal histology of the tissues is essential, and the student is advised not only to examine, whenever possible, type specimens of tumours and granulo- mata, but also to renew his acquaintance with sections of the normal human tissues. Methods of examining sections. — It is a common elementary crime of clinical medicine for the beginner, on being asked to examine a patient, to forget that he is provided with eyes and hands and to immediately clap a stethoscope on the chest. A very similar error is made in histological diagnosis. The impulse of the novice drives him to place his section on the largest available microscope and examine it at once under the highest possible power. The impulse is possibly fostered by a deplorable " examination " process vulgarly known as " spotting " sections. The most convenient micro- scope for the majority of histological work is a small one with- out a mechanical stage. The most suitable powers are a No. 2 eye-piece with a §-inch and J-inch objective. An ordinary hand magnifying glass is often of considerable assistance. Higher magnifying powers are occasionally, though rarely, required. The sections should first be examined with the naked eye, and points of considerable importance are frequently to be THE EXAMINATION OF SECTIONS, ETC. 375 made out. The naked-eye inspection can be amplified by- holding the slide against the light and examining it further with the hand glass. The distinction between the normal tissue and the abnormal is often obvious, and the general aspect of the section is of great value when considering the microscopic appearance of a series of " fields." The entire area of the section is then carefully gone over with the §-inch objective. Under this power the relation of the normal to the abnormal tissues is definitely made out, the grosser structures are recognised, and to a less extent the relation of the cellular elements to the connective tissue is observed. The ^-inch objective is used last and most sparingly. The portions of the section to be examined more particularly will have been indicated by the previous inspection. The points to be considered are the nature of the cells, their size, shape, and staining reaction, and the character of their nuclei. The rela- tion of the cells to each other and to the connective tissue network is further investigated. The observer who pays too much attention to the higher magnification and too little to the general examination under the lower powers is in danger of " not seeing the wood for the trees." Further, in considering the section, the observer must not lose sight of the patient. All the available clinical information concerning the case must be made use of, and the naked-eye appearance of the tissues removed must be considered. The following brief description of the various processes of morbid histology is not intended in any sense to give a detailed account of the various changes which may occur. It is only possible to indicate the variety of processes which may be met with, and the more important points to be considered in the microscopical diagnosis. The Inflammations. The inflammations may be divided into the acute and the chronic granulomata. The acute granuloraata are considered under the heading of " acute inflammation," and the chronic granulomata include the lesions of tuberculosis, syphilis, leprosy, actinomycosis, and glanders. Lymphadenoma, or Hodgkin's disease, is also conveniently considered here, since our ignorance of the essential nature of the condition prevents a more definite classification. A short account is 376 CLINICAL PATHOLOGY. also given of molluscum contagiosum, another disease of unknown aetiology. Acute inflammation. — By acute inflammation is meant the usual series of changes found in a tissue as the result of irritation by the toxins of pyogenic organisms, or less commonly by mechanical irritants. The word " acute " is used in the pathological rather than in the clinical sense to indicate a particular type of change. The processes involved may continue for a sufficient length of time to justify the clinical description of " chronic." The term " granuloma " is commonly used to describe inflammation accompanied by the formation of granulation tissue, and due to one of the specific diseases referred to here under the heading of " chronic granulomata." Granulation tissue is, however, a frequent product of the acute variety of inflammation. The typical changes of acute inflammation are most commonly produced by the ordinary pyogenic organisms, such as staphylococci and streptococci. The tissue most commonly removed for diagnostic purposes is granulation tissue from the more long standing varieties of acute inflammation. The histological diagnosis usually lies between the acute and chronic granulomata and some form of new growth, particularly sarcoma. The changes to be met with involve the special tissue elements affected, the blood-vessels, and the cellular elements. The tissue changes are mainly degenerative, and range from cloudy swelling to actual necrosis. Glandular cells, for example, stain poorly, both as regards their cytoplasm and their nuclei, and often show fatty change. Muscle cells stain homogeneously, and lose their striation. The changes of repair include the regeneration and multiplication of the cells of the less specialised tissues, and the linking of breaches by fibrous tissue. The stage of fibrosis, in which the cellular infiltration has largely disappeared, is less commonly seen in the clinical laboratory than the cellular granulomatous stage of still active inflammation. The vascular changes are often far from obvious. The earlier changes of dilatation, alteration in the rate of flow, and diapedesis of cells are necessarily for the most part lost at the moment of removing the tissue from the body ; thus a section taken through the red and obviously inflamed skin in THE EXAMINATION OP SECTIONS, ETC. 377 erysipelas may show practically no change in the actual vessels themselves. In the more chronic processes the tissues often show marked vascularity owing to the abundant for- mation of new, thin-walled vessels, which at first appear as mere clefts in the tissue meshwork lined with a single layer, or with a few layers only, of endothelial cells and filled with red corpuscles. Well-formed vessels are usually present in addition. The cellular changes are the most obvious and important in the histological examination. The cells are of several varieties, and are derived partly from the interior of the blood- vessels in the inflamed area, by active diapedesis of the leucocytes and passive exudation of the red cells, and partly from the local tissue cells. The cells may be scattered at random through the loose connective tissue of a granulation, or collected into circular areas, forming the microscopical abscesses of a local pyaemic condition, as may be well seen in some forms of the so-called surgical kidney. The cells other than red corpuscles consist mainly of the ordinary polynuclear neutrophil cells of the blood. The more acute the inflammatory process, the more abundant and predominant are the polynuclears. These cells, in common with the lymphoid cells shortly to be described, are referred to in many descriptions as the " small round cells " of in- flammation. It is preferable to give them the name by which they are known in their natural surroundings, and it is necessary to differentiate them from other varieties of " round cells." In a paraffin section stained with hematoxylin and eosin the polynuclear neutrophils are readily recognised under the J-inch objective. They appear as small round cells, with an eosinophilic cytoplasm and a bilobed, or less commonly trilobed, nucleus. Cells derived partly from the blood and partly from the tissues are " lymphoid " and " endothelioid " cells. The lymphoid cells are possibly of two varieties, derived in part from the lymphocytes of the blood and in part from similar cells of the tissues. They appear as small round cells, with very little cytoplasm and a relatively large deeply-stain- ing round nucleus. They are found only in very small numbers in the early stages of acute inflammation. Endothelioid cells, or as they are sometimes called 378 CLINICAL PATHOLOGY. epithelioid cells, are probably of numerous varieties. The majority of them are wandering phagocytic cells. They are derived in part from the large hyalines of the blood, in part from vascular and other endothelial membranes, and in part from the cells of the local tissue. They have probably a similar origin to the elongated tissue fibroblast. They aj)pear as large cells, often of a more or less angular shape. The cytoplasm is clear, relatively abundant, and takes the eosin dye faintly. The nuclei are comparatively small, faintly staining, and often show nucleoli ; they may be round or slightly indented. An important cell of inflammation, and one which is probably derived from the tissues, is the plasma cell. The origin and functions of the plasma cell are still in dispute and do not concern us here. It is probably mainly derived from the primitive perivascular lymphocytic cell of the neighbourhood. Special stains are in use for the demon- stration of plasma cells, but they are readily distinguished in the ordinary hematoxylin and eosin preparations. The cells are intermediate in size, between the lymphoid and endo- thelioid cells. Their shape is more or less that of an egg, and their cytoplasm is distinctly eosinophilic, staining a bright pink. Their nuclei are characteristically eccentric, and appear as if they were in the process of being squeezed out of the cell. Plasma cells are nearly always present in acute inflammatory processes, and are fairly abundant in those of some standing. In addition to the tissue changes of inflammation the exciting cause must often be looked for. The examination for organisms can be conducted by Gram's method of staining applied to tissues, or by a simple .process with carbol-thionin. Both these methods will be described subsequently. Organisms may be present in large clumps visible under the ^-inch, or even the §-inch objective, but it is necessary to use the oil immersion lens in order to determine their morphology. In sections through an anthrax pustule, for example, large masses of big Gram-positive bacilli are usually conspicuous. The exact nature of organisms can only exceptionally, as in the case of the tubercle bacillus, be identified in sections ; nor does the failure to demonstrate bacteria in sections invalidate other evidence of a bacterial infection. In examining a section of granulation tissue from an acute THE EXAMINATION OF SECTIONS, ETC. 379 inflammatory focus the points to be looked for are con- sequently the following : — A loose connective tissue basis enclosing an abundance of newly-formed blood-vessels. A scattered and irregular cellular infiltration, composed mainly of polynuclear neutrophils, with occasional plasma cells, few lymphoid cells, and an increasing number of endothelioid cells and fibroblasts, according to the duration of infection. Free red cells are usually present in the connective tissue spaces. The adjacent tissues of the part show degenerative changes in greater or less degree. Micro- 0. *> *■■ . %g jf$ n ** * 9jf, tic Fig. 28. — Acute Infective Granuloma. Showing Polynuclear and Plasma Cells. Drawn under £-inch Objective. organisms may be present in considerable numbers. The most characteristic changes of all are the presence of bacteria, and the preponderance of polynuclear neutrophils. Tuberculosis. — Tuberculosis produces the commonest and most widely spread lesions of the chronic infective granu- lomata. Almost any part of the body may be affected, but the changes are practically identical in all tissues. The characteristic histological process resulting from a tuberculous infection is the giant cell system or miliary tubercle. Other changes are either antecedent to this, or result from the spread or arrest of the infection. Thus in the 380 CLINICAL PATHOLOGY. very earliest stages there may be a proliferation of endothelioid cells, but no giant cell system, and in the later stages the giant cell systems may increase in size by enlargements of their caseous centres and coalesce with adjoining systems to form tuberculous foci of irregular shape and size. Caseation and fibrosis commonly proceed together, and both are features of a section through an advanced tuberculous lesion. The miliary tubercle is in a section just visible to the naked eye, and its zones can be made out under the magnification of a hand glass. The gross appearance of a section containing two or three such tubercles is very characteristic. The giant cell system has the following structure : — In the centre is a giant cell. Surrounding the giant cell is a zone of endothelioid cells. Surrounding the endothelioid cells is a zone of lymphoid cells. Blood-vessels are absent. As the tubercle grows larger its centre caseates. Two, three or more giant cells lie in the periphery of the caseous centre, and outside these again are the endothelioid and lymphoid cells. The caseous centre of the tubercle stains a bright pink with the eosin dye. The giant cell of a tuberculous lesion is very characteristic. It has an irregular and often angular shape, but the larger cells in particular are often rounded. Its centre is caseous and stains a pinkish colour with eosin. It is multinucleated and may contain 10 or more nuclei. The nuclei are usually confined to and arranged round the periphery of the cell. The larger the giant cell, as a rule, the more caseous its centre, and the more regular the arrangement of nuclei round its periphery. Smaller and less characteristic giant cells are often present in addition, and these may contain fewer nuclei, some of which have a nearly central position. The large giant cell is, with the exception of the demonstration of the tubercle bacillus, the most diagnostic histological feature of a tuberculous lesion. It is easily visible under the low power of the microscope, and may attain such a size as to occupy the greater part of the field of a ^-inch objective. Giant cells are present in a variety of other conditions. They occur in all the chronic infective granulomata, including syphilis, but are as a rule very scarce, comparatively small, and without the typical nuclear arrangement found in tuberculosis. In Hodgkin's disease giant cells, of a type to be subsequently THE EXAMINATION OF SECTIONS, ETC. 381 described, are numerous, but the nuclei are central and the cells do not at all resemble those of tuberculosis. Foreign bodies, such as ligatures, commonly have formed round them regular giant cell systems which very closely resemble miliary ..•■;■ ■■■' %y,,,; ■','$//:<. ' : « , ' : .• .. ■" .' ' Sj>»- W : --M/8i '/■&;:• ■>'' '•' ■■•.'•'''."•'•f : ; !'"."'. ■'■','.■■ v- .'" !', Fig. 29. — Tuberculosis of Kidney. Drawn under §-inch Objective. Fig. 30. — High Power Drawing of a giant Cell in Fig. 29. tubercles. The giant cells as a rule have nuclei placed cen- trally as well as peripherally, and a careful examination often discovers the foreign body, which may be engulfed within one of the giant cells. Portions of ligature in a section show as 382 CLINICAL PATHOLOGY. glistening threads, often faintly stained with the haeniatoxylin dye. Similar giant cells may also be found in lesions resulting from some chronic irritant, as at a point of pressure, the site of a chronic septic discharge, or the edge of a new growth. The giant cells of myeloid sarcoma are large cells with nuclei evenly scattered throughout their substance. In sections of tuberculous tissues removed during life regular giant cell systems are often absent. The tissues show areas of fibrosis, and irregular caseous areas merging into zones of cellular infiltration. Giant cells are to be looked for in the margins of the caseous areas. The cellular infiltration is com- posed mainly of lymphoid and endothelioid cells. Polynuclears and plasma cells are usually scanty or absent, but a few are often found at the periphery of a tuberculous area, and if a secondary infection has occurred they may be numerous. The histological appearance of a tuberculous lesion is in the majority of cases sufficient for diagnostic purposes. In some cases, however, it is desirable to examine for the bacillus. The method of staining the bacilli in section is described later. The number of bacilli present in the majority of tuberculous sections is extremely small, and to find a single bacillus on one slide is about as much as can be hoped for. Failure to find the bacilli is consequently little evidence against a diagnosis of tuber- culosis. The points in the histological diagnosis of tuberculosis therefore include the presence of giant cell systems in an earl} 7 lesion, with later areas of caseation and fibrosis, and a cellular infiltration consisting mainly of lymphoid and epithelioid cells. Syphilis. — The changes produced in the tissues by syphilis are varied, and include nothing so characteristic to the histologist as the giant cell system of tuberculosis. The certain diagnosis of a syphilitic lesion on histological grounds is often impossible, and one may only be able to state that the lesion is due to one of the chronic infective granulomata. Syphilitic tissues are not commonly removed during life, and a very brief account of the changes to be met with is given here. The tissue changes of syphilis consist, as in tuberculosis, of caseation and fibrosis. A diffuse and irregular fibrosis is a characteristic feature of many syphilitic tissues. The vascular changes in sj'philitic lesions are nearly always THE EXAMINATION OF SECTIONS, ETC. 383 very noticeable. The smaller arterioles have their lumina markedly narrowed by proliferation of the cells of the intima, and there is often fibrosis and thickening of the vessel walls, with an infiltration of lymphoid cells into the media, as well as into the adjacent tissues. The cellular exudation into the tissues is, except in the advanced fibrotic cases, very considerable. The cells consist of lymphoid, endothelioid, and plasma cells. The plasma cells are often very numerous, in contradistinction to what is com- monly found in tuberculous lesions. Giant cells are usually scanty or absent, and it is most unusual to find in syphilitic lesions large giant cells with peripheral nuclei such as are seen in tuberculomata. The main changes in a syphilitic lesion are the same in all stages of the disease, but differ some- what in detail. A section through the primary chancre shows a formation of new capillaries running at right angles to the ulcerated surface, a proliferation of the intima of the arterioles, and between the capillaries young fibrous tissue and fibroblasts, together with a large number of lymphoid cells and plasma cells, which spread also into the adjacent tissues. The mucous tubercles and condylomata of secondary syphilis are produced by the accumulation of cells and exuded fluid, together with a proliferation of the overlying epithelial cells. Tertiary syphilis is characterised by the formation of gummata consisting of circular caseous areas surrounded by fibrous tissue and an area of cellular infiltration. The points to be looked for in the sections of a syphilitic lesion are caseation, fibrosis, arterial changes, and particularly a proliferation of the intima of the arterioles, and a cellular infiltration by lymphoid and plasma cells. The Spirochceta pallida can be demonstrated in the primary and secondary, but not in the tertiary, lesions. Leprosy. — The cases of leprosy met with in this country are few, and all are imported. The lesions examined during life usually consist of nodules from the skin. The cellular infiltration is considerable, and the predominant cell is of the endothelioid variety. Caseation and fibrosis occur, and giant cells may be found in considerable numbers. The diagnosis of the condition on histological grounds is as a rule readily 384 CLINICAL PATHOLOGY. made by the demonstration of acid-fast bacilli in the sections. The bacilli are stained by the same method as that given for tubercle bacilli hi section (page 432), but 12 per cent, acid must be used for decolorising in iilace of 25 per cent. The bacilli are usually numerous in sections, and are often packed within large epithelioid cells. Actinomycosis. — Actinomycotic nodules are rarely met with in human pathology, but are exceptionally found in the skin, and not very infrequently the granuloraata may be found in the appendix or in scrapings from an abscess of the liver. The diagnosis of the condition rests upon the discovery of the organism, and the threads are most readily found in the pus, but may appear in large numbers and in clumps of consider- able size in the sections. The sections should be stained by Gram's method (page 432). The histology of the lesion is not particularly characteristic in the absence of proof of the causative organism. There is a considerable infiltration of the tissues forming the nodule by lymphoid cells, which are subsequently replaced by polynuclear neutrophils. The central portion breaks down to form pus. Giant cells and fibroblasts are only exceptionally present, but the inflammatory exudate may become encapsuled by fibrous tissue and the central area calcined. Glanders- — Glanders is properly a disease of equines, and is rarely conveyed from them to man. The glanders nodule has a central area of necrosed polynuclear cells, a middle zone of endothelioid cells with occasional giant cells, and an outer ring of young fibrous tissue. The nodules readily break down with the formation of ordinary pus. The diagnosis of the condition rests with the isolation of the specific bacillus. Hodgkin's disease (Lymphadenoma).— The aetiology of Hodgkin's disease is unknown. The condition has been classified among the blood diseases from its supposed relation- ship to lymphatic leukaemia, but the two conditions have practically nothing in common. It has been considered a form of new growth, allied to the sarcomata, owing to its usually rapid dissemination and fatal termination. The modern consensus of opinion probably is that Hodgkin's disease will prove to be a parasitic disease, and, in accordance with that view, it is conveniently considered here. The disease affects the lymphatic tissues, at first locally, as in THE EXAMINATION OF SECTIONS, ETC. 385 the glands of the neck, and later, as a rule, more or less generally throughout the body. The diagnosis of the condition can only be made with cer- tainty on histological grounds, and for this reason one of the discrete superficial glands may be removed under a local anaesthetic. The enlarged glands, if localised, are removed by the surgeon as a mode of treatment. The histological picture is a characteristic one. Under the low power of the microscope the general structure of the gland is seen to be completely altered. The distinction 8 » a H Oq % 4a ^m ?* ©%®®^ ® Jfcv Fig. 31. — Lymphatic Gland in Hodgkin's Disease. Drawn under \ -inch Objective. between cortex and medulla is lost and the germ centres have entirely or almost entirely disappeared. The gland tissue is composed of a delicate, but obvious, fibrous reticulum enclosing, comparatively to the normal gland, scanty cells. Scattered throughout the gland are numerous giant cells, fairly obvious under a low power. Under the J-inch objective the cellular content of the gland is seen to be very different from that of normal lymphatic tissue. Lymphoid cells are present, but in greatly p. 25 386 CLINICAL PATHOLOGY. diminished numbers. Endothelioid cells are numerous. The characteristic giant cells of Virchow are seen, and are quite unlike the giant cells of a tuberculous lesion. The Vii-chow cells are more or less oval, the cytoplasm resembling that of the endothelioid cells, but covering a considerably wider area. The cells are multinucleated, and commonly contain 2, 4, or 6 nuclei. The nuclei are placed in the centre of the cells, and are arranged in a ring, so that commonly 2 nuclei only are in focus at one time. The cells are some two or three times the size of an endothelioid cell, and are con- siderably smaller than the large tuberculous giant cells. In addition to the giant cells of Virchow a large number of eosinophil cells are nearly always present, and are fairly obvious in the ordinary hematoxylin and eosin sections. To display the eosinophil cells to greater advantage sections may be stained by Leishman's stain (page 433). The relative prepon- derance of cells and fibrous tissue differs in various glands. The fibrous reticulum may be greatly increased, producing a tough fibrous gland ; or only a fine fibrous network may be present with a soft and cellular gland. The main points to be looked for in the histological diagnosis of Hodgkin's disease are : — The replacement of the normal gland structure by a fibrous reticulum containing few lymphoid cells, many endothelioid cells, eosinophils, and the typical multinucleated cells of Virchow. Molluscum contagiosum. — Molluscum contagiosum is a not uncommon condition. It occurs in the form of small umbilicated nodules in the skin, is definitely contagious, and doubtless of parasitic origin. Molluscum contagiosum may be found in any part of the skin, and may be inoculated from one part to another. The tumours may develop, though rarely, on the penis, and be mistaken for chancres. Sections through a nodule have a very characteristic appearance. Under the low power the nodule is seen to con- sist of a number of more or less wedge-shaped lobules sejwated from each other by thin fibrous septa. Each lobule has an epithelial lining enclosing round or oval epithelial cells. The enclosed cells are more or less degenerated, and in their most advanced state become swollen homogeneous masses known as molluscum bodies. The molluscum cells have been considered THE EXAMINATION OF SECTIONS, ETC. 387 to be psorosperms, but the present view is that they are derived from the prickle cells of the skin and have become keratinised. Fig. 32. — Molluscum Contagiosum. Drawn under §-inch Objective. The Degenerations. Some of the commoner degenerations are not infrequently met with in tissues removed during life ; others are more often studied in the post-mortem room. The clinical pathologist may, however, meet with almost any pathological tissue, and the more important degenerations may be briefly recorded here. Cloudy swelling. — This is an extremely common condi- tion met with in inflammatory processes and in fevers. The epithelial and connective tissue cells are affected. In the earlier stages the cells become swollen, lose their outline, and stain poorly ; later they become granular. The nuclei stain feebly, and may ultimately disappear. (Edema. — The appearance of the cells in oedematous tissues is frequently misleading if the condition is not recognised. The cells stain badly, and become greatly distended with fluid* and misshapen. Cells oedematous but otherwise normal are apt to be mistaken for the large irregular cells of a carcinoma. Mucoid degeneration. — This form of degeneration is similar to the normal process met with in mucous tissues. It occurs also in catarrhal conditions and in some forms of new growth 25—2 388 CLINICAL PATHOLOGY. Mucoid degeneration is found in some varieties of sarcoma, and particularly in the myxo-sarcoma met with in the antrum of Highmore and other parts of the body. Carcinomata may also show the change, which is fairly frequently met with in carcinomata connected with the intestine. Sections of tissues with mucoid degeneration are often difficult to prepare, owing to the fact that the mucoid material swells up when the sections are floated in water. The mucin is found both in the cells and in the connective tissue fibres. The substance gives the reactions of mucin, being soluble in dilute alkalies and precipitated by acetic acid. Sections showing mucoid degeneration should be stained with carbol-thionin or van Gieson's stain (page 481). The mucin stains a reddish purple colour with carbol-thionin. Colloid degeneration. — Colloid is normally met with in the thyroid gland, and in the anterior glandular part of the pituitary body. Colloid, or a very similar substance, may occur as a result of degeneration in some new growths. It is found in the secondary deposits of thyroid carcinoma and in some ovarian tumours. Colloid, like mucin, swells up in water, but is not precipitated by acetic acid. It is often brittle and difficult to cut in paraffin, but sections are readily made by the gum method. In sections stained by van Gieson's stain colloid is a bright orange-red. Hyaline degeneration. — Hyaline is mainly found in the adventitia of the smaller arteries and in the connective tissues. The connective tissue fibres become swollen and semi-trans- lucent. The true hyaline change is probably not a common one. Van Gieson's stain, which gives a reddish orange with colloid and a bright rose-red with mucoid, stains hyaline a flesh-pink colour. It must be confessed, however, that absolute differentiation of these substances by van Gieson's stain is far from certain. Lardaceous degeneration. — It is not definitely settled whether the lardaceous change is a degeneration of the tissue cells or an infiltration of them by lardacein, or is due to some other substance which ultimately becomes lardacein. The change is practically confined to the middle coats of the smaller arteries. Lardaceous change may follow chronic suppuration, or syphilis, or both. THE EXAMINATION OF SECTIONS, ETC. 389 The change is not commonly met with in tissues removed during life, and the material may be recognised in paraffin sections by its staining reaction with aniline gentian-violet. The section can be stained as in the first part of Gram's method (page 432), differentiated in saturated oxalic acid solution, washed in water, and mounted in glycerine. The lardacein stains a bright rose-red and the tissues bluish. Fatty change. — Excess of fat may be found either as an increase of fat in normal situations, or as a deposition of fat in cells which do not normally contain it. Both fatty infiltration and fatty degeneration may occur together, the latter condition being the more important. In acute fatty degeneration the globules of fat are as a rule very minute. In the more chronic forms of fatty degeneration the globules are commonly large, and similar to those found in fatty infiltration as well as in normal fatty tissues. In the ordinary paraffin preparations the fat is dissolved out in the process of passing the tissue through alcohol, and the areas of the section previously occupied by globules of fat show as more or less circular empty spaces. The finer fatty changes cannot be recognised at all in such sections. To demonstrate fat in section the tissues must be treated by the gum process and the sections stained by one of the fat stains, such as Scharlach K. (page 425). Scharlach R. stains fat a bright red colour and leaves all other tissues unstained. The section may be subsequently counterstained with haema- toxylin, washed in water, and mounted in Earrant's solution. Calcareous degeneration. — A deposition in pathological tissues of calcium carbonate and phosphate is not uncommon in various conditions, and particularly in necrotic tissues, tuberculous foci, and in some malignant tumours. The main interest of the change to the clinical pathologist lies in the damage done to the section razor by the small gritty particles, and it may be necessary to decalcify such tissues. The calcareous nodules in hematoxylin and eosin sections stain a bluish colour. Pigmentation. — Excess of pigment in tissues may arise from a variety of causes. Extraneous pigment is frequently found in cutaneous tumours, and the pigment granules may be carried deeply into the tissues and into the neighbouring lymph glands by the wandering cells. Certain new growths, and 390 CLINICAL PATHOLOGY. in particular melanotic sarcomata, are deeply pigmented, and the brownish black particles are seen in large numbers mainly within the cells of the growth. Hematogenous pigmenta- tion occurs as the result of local bleeding and in the numerous general conditions associated with abnormal blood destruction. Pigment derived from the blood is often of a golden brown colour in section, and gives the free iron reaction (pag6 433). CHAPTER XXVII. NEOPLASMS — SIMPLE TUMOUKS. The neoplasms are conveniently divided into " simple " and "malignant" tumours. Each division can be sub- divided into tumours of epithelial or glandular structure, and tumours of the connective tissues. Only those tumours which are frequently or comparatively frequently met with can be mentioned here. Simple Tumours. Papilloma. — Papillomata are epithelial tumours, and consist of localised out-growths of epithelial surfaces. The cells forming the tumours are squamous or columnar, according to the nature of the epithelium from which they are growing. Papillomata are often multiple, commonly arise as the result of a local irritant, and can in some cases be inoculated by contact upon neighbouring tissues. Examples of inoculation may be seen in papillomata of the gut, the bladder, and the larynx. A papilloma of the skin reproduces the structure of the normal papillae, and if exposed to friction the epidermal cells multiply above the papilla forming a callosity, as in a corn. Increase and keratinisation of the horny cells produces a keratoma. A wart is a typical instance of a cutaneous papilloma. A papilloma of a mucous membrane, as of the bladder or intestine, is covered with a few layers of cells only, and being well supplied with blood-vessels is very apt to bleed. Papillomata may develop also from the epithelial lining of cysts, and are particularly common in ovarian tumours. The papillary processes involved frequently become branched, and the resulting tumour is consequently dendritic. Papillomata may undoubtedly become malignant and are then converted into (or replaced by) carcinomata, a transformation which appears to be particularly common in papillomata of the bladder. It 392 CLINICAL PATHOLOGY. is, however, most desirable to distinguish between simple and malignant growths of this nature. In dealing with typical warty papillomata of the skin the distinction is easy, but in the case of bladder, intestinal, or laryngeal growths there may be considerable difficulty, and intermediate stages in the conversion of a papilloma into a carcinoma may be encountered. The papilloma removed should have, if possible, an area of the surrounding epithelium and a portion of the subjacent tissues attached to it. Great care should be taken in the " casting " of the paraffin block, so that the sections are cut exactly at right angles to the epithelial surface. If a section is cut in a slanting direction through normal skin an appearance of epithelial thickening and down-growth is obtained, which is confusing to the beginner. The section should be examined first with a hand glass, when the deeply stained line of epithelium is seen projecting towards the surface instead of, as in a carcinoma, dipping down into the subjacent tissues. Under the microscope the epithelial outgrowth is seen to be regular, and to more or less exactly reproduce the character of the adjacent epithelium. The regularity of the outgrowth, the comparatively normal size, shape and appearance of the cells composing it, and the absence of epithelial infiltration deeply into the subjacent tissues, serve to distinguish the simple from the malignant tissue. Adenoma. — Adenomata are somewhat similar tumours to the foregoing, but arise from glandular structures instead of from epidermal or mucous surfaces. They tend to repro- duce the type of glandular tissue from which they develop, and in some instances reproduce it so closely that on purely histological grounds they cannot be distinguished from the normal gland or a general overgrowth of it. An adenoma of the thyroid gland, for example, under the microscope exactly resembles a simple hypertrophy of the gland, but is readily distinguished by its gross appearance, since, like all adenomata, it is a localised and usually encapsulated tumour. Adenomata arising from columnar-celled glandular tissue have a tubular structure lined by columnar-celled epithelium. The tubes may be covered with connective tissue, or the tissue may be thrown into a fold producing a papillary projection lined NEOPLASMS-SIMPLE TUMOURS. 393 by epithelium. Such a tumour is known as a papillary- adenoma- Adenomata arising from spheroidal-celled tissue, such as that of the breast, are composed of similar cells grouped into alveoli. If the interstitial fibrous connective tissue is in much excess the tumours are called fibro- adenomata- Adenomata are met with in the breast, the prostate, the uterus, the ovary, the intestine (where they may form pedunculated growths or "polypi"), the sebaceous glands, the kidney, and the gall bladder. The adenomata '/ ft s G> l % ff 8 V •« 1)1 / / fcb °e •© o Fig. 33. — Adenoma of Breast. Drawn under §-inch Objective. rarely become carcinomata, and have to be distinguished from them. The points of distinction are the following : — The adenomata are capsulated and the carcinomata are very rarely capsulated. The adenoma cells do not infiltrate the surrounding tissues. The cells of an adenoma retain considerable likeness to those of the normal tissues, and tend to reproduce a more or less regular tubular or alveolar structure. They contain as a rule a considerable fibrous tissue framework, which supports the tubular or acinous cell collections, but is not invaded by them. The microscopic appearance of a fibro-adenoma conveys the impression of a 394 CLINICAL PATHOLOGY. fibrous framework supporting a series of glandular processes ; the appearance of a scirrhus or fibro-carcinoma is that of cellular columns invading and eating their way into a fibrous barrier. In distinguishing between an adenoma and a carcinoma of the uterus, it must be remembered that the glands of the endometrium normally penetrate a considerable distance below the surface. The same is true of glands of the gall bladder. Lymphoma. —Lymphoma is the name applied to tumour- like masses composed of lymphoid cells such as occur in the leukaemias and in general lymphoid hyperplasia. They have a very close histological resemblance to round-celled sarco- mata. The term is also applied to a lympho-sarcoma. Fibroma. — This is a connective tissue tumour composed mainly of fibrous tissue, and may occur in any organ contain- ing connective tissue. Fibromata are found commonly in the skin, fasciae, periosteum, tendons, nerves, uterus, ovary, breast, and nose. In the skin the pendulous fibroma may cover a con- siderable area, and is known as molluscum fibrosum. It is, however, more properly described as a neuro -fibroma. The common " fibroid" tumour of the uterus is largely made up of unstriped muscle, and is known as a leiomyoma. The epulis is often a fibroma and less commonly a myeloid sarcoma. The "mucous polyp " of tbe nose consists of a loose cedematous connective tissue framework enclosing a number of inflammatory cells. It is commonly regarded as an cedematous fibroma. Fibromata may be hard or soft, according to the density of the fibrous tissue and the presence or absence of oedema and mucoid degeneration to which these tumours are occasionally liable. Under the microscope the fibroma is readily recognised by its elongated spindle-shaped and wavy cells, which tend to be arranged in whorls. Blood-vessels are usually scanty, but well formed. Many of the fibromata contain other elements than fibrous tissue, and a certain admixture of elastic tissue is usual. Some fibromata contain also unstriped muscle and are known as fibro-myomata, or if the muscle tissue predominates as leiomyomata. Others contain also nerve tissue and are called fibro-neuromata, and the name fibre-adenoma is applied NEOPLASMS— SIMPLE TUMOURS. 395 to an adenoma, such as is commonly met with in the breast, and contains little glandular and much fibrous tissue. Degenerated fibromata and leiomyomata are often difficult to distinguish from spindle-celled sarcoma, and a sarcomatous change may occur in the simple tumour. The regular arrange- ment of mature long fibrous tissue cells arranged in whorls is not seen in sarcoma. Moreover the few vessels of a fibroma are usually thick-walled, while the rather more numerous blood- vessels of a sarcoma are mere clefts in the tissue containing blood, and lined as a rule by a single layer of cells. ~~p/r^^i_z. "" .-::-=-.-_- -^ ~ -W:T"S5: /^^2g|f=^ ^ ^ v^^^r^^pr^ j . ~M ?• ~ z ^P^^~£Sk " ^^ • "\^ N \ i ' ! i 1 , "' 1 4> ' '^sss^ S? 5 ?^^^. Q^^S&^W&S ">s<^ 'f.of v N %S Pi /"^'iS^!^^?:^^^ -" '^ yV ., ~^ £"--.': " : v^^^S^ Fig. 34. — Fibroma of Ovary. Drawn under §-inch Objective. Myoma. — Myomata may be composed of striated or of unstriated muscle fibres. The former class of tissue is called a rhabdomyoma, and is practically unknown. The rhabdo- myoma of the kidney is generally considered to be a sarcoma. Leiomyoma is composed of unstriped muscle fibre, and is the common tumour of the uterus known as the " fibroid." The leiomyomata of the uterus usually contain a varying amount of fibrous tissue, and may properly be called fibro- myomata. Similar tumours are also found in the ovary and less commonly in the prostate. Under the microscope the arrangement of the muscle cells in whorls is very similar to the arrangement of the fibrous tissue in a fibroma, and a similar distinction in the case of cedematous or degenerated growths has to be made between 396 CLINICAL PATHOLOGY. myoniata and sarcomata. The myoniata usually contain few, but well-formed, blood-vessels. The tendency of uterine fibroids to cause bleeding from the uterus results from their situation. The haemorrhages come from the closely-subjacent endo- metrium, and not from the tumour. Myxoma. — The true myxoma is a rare tumour composed of similar tissue to the Whartonian jelly of the umbilical cord. The myxomata may form tumours of considerable size in the subcutaneous tissues, and occur also in the medullary cavities of the long bones. They are frequently mixed tumours, myxo-liioomata, fibro-myxomata, chondro-myxomata, and myxo-sarcomata being met with. The tumour is seen to consist of a mucinous tissue contain- ing spider-like cells with a darkly-staining nucleus and a considerable amount of cytoplasm, the long interlacing pro- cesses of the cells forming the framework of the tissue. The cells are separated by considerable intervals. Lipoma. — The .lipomata are among the commonest and most readily recognised of all the tumours. They are found in almost any situation where fatty tissue is normally abundant, such as the neck, shoulders, axillae, and groins. They may be circumscribed and lobulated, forming more or les3 pedunculated tumours, which may reach a considerable size, or they may be diffuse and consist of a relatively localised overgrowth of the tissue fat. Under the microscope liporoata are seen to consist of fatty tissue with a varying framework of fibrous tissue. In paraffin sections they appear as a loose and empty reticulum of fibrous tissue. Chondroma. — Cartilaginous tumours are of two varieties. The variety which grows from normally situated cartilage is known as an ecchondroma. Those arising in situations where cartilage is normally absent are called enchondromata. Ecchondromata are usually small tumours arising from the cartilage of the ribs, the larynx, or the nasal septum. The enchondromata are usually attached to the long bones, and particularly to the phalanges. Cartilage may also be found in parotid, testis, and kidney tumours. The cartilage found in parotid tumours is supposed to be derived from Meckel's cartilage of the bronchial arches. The enchondromata are often multiple, and may become sarcomatous and form secondary deposits. NEOPLASMS— SIMPLE TUMOURS. 397 Under the microscope chondromata are seen to consist of hyaline cartilage containing a variable number of unevenly distributed cartilage cells. In some cases the matrix is not homogeneous, but fibrillated like that of fibro-cartilage. Osteoma. — By osteoma is meant a tumour composed of bone, and not a bony overgrowth, such as is found in the callus which surrounds a fracture. Osteomata of two kinds are recognised. The cancellous osteoma is composed of bony trabecule including large irregular spaces, a considerable amount of medulla, and a number of blood-vessels. The ivory osteoma is composed of dense bone, with little medulla and few vessels. Osteomata of the teeth composed of dentine are known as odontomata, and those composed of cement as dental osteomata. Osteomata are frequently multiple, and their most usual situations are the skull, the extremities of the long bones, and the pelvis. Ivory osteomata are practi- cally confined to the skull. Angeioma. — The angeiomata are tumours composed of vessels which may be either blood or lymph channels. The former class of tumour is called a hsemangeioma, the latter a lymphang-eioma. The hpemangeiomata are further subdivided into capillary or simple angeiomata, and into venous or cavernous angeiomata. Both classes of haemangeionia are also known as nffivi. Capillary nsevi contain large numbers of tortuous, distended, and thick-walled capillaries lined by a well-marked layer of endothelial cells and lying in ordinary connective tissue. They are congenital tumours for the most part, and often tend to increase in size. They are very obvious during life, and may be much less distinctive in a microscopic section. If the tissue has been fixed with the vessels distended by blood the histological aspect of an area composed of many small vessels containing red cells is unmistakable. If, however, as not infrequently happens, the vessels have contracted their fibrous walls and emptied their lumina of blood, the section has rather the appearance of ordinary connective tissue. Capillary angeiomata may occur on any part of the body, but are particularly common on the face. They may be single or multiple. Venous or cavernous angeiomata consist merely of wide, 398 CLINICAL PATHOLOGY. irregular clefts lined with endothelium and separated by fibrous septa. They occur on the skin, forming the raised purplish patches known as naevi or birth marks. They are found occasionally in the internal organs, but very rarely in any other organ than the liver, where they are not particularly uncommon. Lymphangeiomata are rare tumours consisting of collec- tions of dilated lymph-vessels. Two varieties are described. The cavernous lymphangeiomata occur in the tongue and the lips, and are concerned in the production of macrogiossia and macrocheilia. Cystic lymphangeiomata are rare tumours which occur in the subcutaneous tissues, and particularly those of the neck, forming diffuse, cystic, and fluctuating swellings. The lymphangeioma spaces are recognised under the microscope by their being filled with a homogeneous watery lymph, and by the absence of red cells in them. Moles. — Moles are usually pigmented and often hairy tumours growing in the skin. Some confusion in the nomen- clature is caused owing to the custom of German authors in naming these growths naevi and of English authors for the most part calling the common capillary angeiomata of the skin nsevi. The pigmented and often hairy growths described here as moles may be highly vascular, but are histologically and clinically quite distinct from the capillary angeiomata. Apart from the confusion of nomenclature the classification of these tumours is sufficiently difficult. They are considered by some authors as epithelial, by others as of mesoblastic origin. The simple moles have a tendency to become malignant, and may give rise to the most intensely malignant of all tumours, namely, the melanotic sarcoma. It is by no means easy in all cases to judge on histological grounds whether a mole is simple or malignant, and it is particularly necessary that a mole, if removed, should be taken away with a wide margin and a portion of the deeper structures, both to avoid recurrence, and to enable a proper histological examination to be made. The majority of moles are composed of epithelioid cells in an alveolar arrangement. Prolongations of the overlying epithelium downwards help to form the alveolar network, within the meshes of which are found groups of the somewhat angular epithelioid cells. The cells are divided into groups by fibrous tissue, but the stroma does not NEOPLASMS— SIMPLE TUMOURS. 399 pass between the cells. The great majority of the tumours contain a large number of brownish-black pigment granules. The pigment as a rale is most abundant towards the surface of the growth. Blood-vessels may be numerous, and small cysts may develop within the tumours. Even in the non-malignant moles the extension of the tumour downwards beneath the epithelium may be consider- able, and the margin of the growth is often ill defined and irregular. There is no capsule. The extension of the growth through the subcutaneous loose connective tissue into the fasciae and muscles is evidence of malignancy, as is also the replacement of the compact alveolar arrangement by a looser and more irregular cell growth. Moles are found on almost any part of the skin and on the conjunctiva. They are congenital tumours, and if pig- mented arise only in situations where pigment is normally present. Glioma. — Gliomata are comparatively rare tumours, the nature and classification of which are considerably confused. They can hardly be considered simple tumours, since they infiltrate the surrounding tissues, yet they are usually classified as such. The gliomata are essentially tumours of the central nervous system, and as such are confined to the brain and the spinal cord. The gliomata of the retina are customarily grouped with those of the brain and cord, but are in reality tumours of a very different nature, and are mentioned here only in obedience to the usual custom. The gliomata of the brain and cord are of two kinds. They may form definite and more or less encapsulated tumours, which may be single or multiple, or they may be indefinite infiltra- tions the edges of which cannot be determined by the naked eye. The diffuse gliomata tend to soften and break down and to destroy the neighbouring tissues. The condition of the cord producing the disease syringo-myelia is considered to result from a gliomatous growth. The gliomata are usually slowly-growing tumours, and occur in adult life. Under the microscope the gliomata are seen to consist of glial cells, which contain a relatively small amount of cytoplasm and have delicate branching processes which divide and interlace in all directions. The nuclei are relatively large and stain 400 CLINICAL PATHOLOGY. deeply. The number of blood-vessels in the tumour varies considerably, as also do the nerve fibres and cells which may be incorporated in it. This variety of glioma, consisting almost entirely of mature glial tissue, is not met with in the retina. The retinal glioma differs markedly both on clinical and on histological grounds from those found in the brain and cord. It is met with in babies or very young children and is intensely malignant, growing with great rapidity and killing by invasion of neighbouring structures. It may commence simultaneously in both eyes. Several members of the same family may be affected. Under the microscope the tumour is seen to consist of an embryonic type of tissue in which all the structures of the retina have been traced. Eods and cones may be found. The cells have few or none of the delicate processes seen in the gliomata of the brain. The best-formed cells are seen growing round the vessels in small masses, which are separated by non-vascular areas of necrosed cells. It is evident that a retinal glioma can hardly be described as a simple tumour and with difficulty as a sarcoma, since all the retinal structures may be repeated in it. It might, appropriately, be called a retinal "neuro-blastoma." Neuroma. — Neuromata are tumours partly composed of nerve fibres or cells. The conditions to which the term "neuroma" should be applied are in considerable confusion owing to the disputed character of the growths. Some have been considered to be fibromata which have merely entangled nerve fibres in them, but the general tendency is to call those tumours which contain nerve fibres or cells neuromata or neuro-fibromata. The tumours are rare and can only be referred to here. Molluscum fibrosum has already been briefly considered, and, since the cutaneous growths tend to follow the lines of nerve supply and contain nerve fibres, they are generally classed as neuro-fibromata. Single tumours may also arise in nerve sheaths and may apparently be composed purely of fibrous tissue derived from the nerve sheath, or more commonly contain nerve fibres in them. The rare condition known as plexiform neuroma is a multiple growth affecting one or more systems of nerves, and associated with great thickening of the nerve sheaths and a formation of new and often abnormal nerve fibres. NEOPLASMS- SIMPLE TUMOUES. 401 Endothelioma. — The endothelioma is a comparatively rare tumour of uncertain classification. It is commonly classed among the sarcomata because of its mesoblastic origin, but in its histological characters it resembles rather the carcinomata or papillomata, and from its clinical characters, since it has little tendency to infiltrate the surrounding tissues or to form metastases, it might well be classed as a simple tumour. Numerous other tumours difficult to classify, such as the mixed tumour of the parotid, are for no very good reasons included among the endotheliomata. The endothelioma arises from endothelium, and is mainly composed of endothelial cells. It is found growing from the membranes of the brain and less commonly from the pleura, or peritoneum. Under the microscope the tumours are seen to consist of collections of large endothelial cells arranged in groups, some of which show a whorled arrangement. The individual cells are not separated from each other, as in the sarcomata, by connective tissue, but are in apposition. Tumours composed of cells arranged in columns the centres of which have undergone degeneration are known as cylindromata. Endothelial tumours arising from the dura and containing a considerable amount of fibrous tissue together with calcareous granules are called psammomata. The parotid tumour. — A peculiar form of "mixed" tumour, known as the parotid tumour, is not infrequent in the parotid gland, and may occur in the other salivary glands. The tumour is a very distinctive and remarkable one, which has been variously classified as an endothelioma, a chondroma, a sarcoma, an adenoma, etc. It is perhaps wisely described as a " mixed " tumour. The parotid tumour is localised and encapsulated, and, though it has a distinct tendency to recur after removal, does not disseminate, and is conveniently classed here among the simple tumours. Under the microscope are seen strands, columns, and often whorls of small, somewhat angular and elongated cells with deeply -staining nuclei. The cellular portions are often separated by considerable areas of a hyaline groundwork p. 26 402 CLINICAL PATHOLOGY. containing few and small groups of cells only. Occasional fairly well defined areas of cartilaginous material are met with, which are said to arise from inclusions of Meckel's cartilage. The cells in the cartilage are usually scanty and their arrangement is atypical. Portions of the stroma may be mixed with fibrous tissue, which in other parts forms the hyaline groundwork previously mentioned. The tumours may reach a considerable size, and are not always wholly encapsulated. Very rarely they may grow into neighbouring glands, but dissemination, as previously stated, does not seem to occur. Relationship between the simple and malignant tumours. — Many of the simple tumours may become malig- nant, invade neighbouring tissues, and disseminate. This conversion of a simple into a malignant tumour is a different process, and can often be distinguished histologically from the invasion of a simple tumour by a malignant one. A glandular carcinoma, for example, can be seen to infiltrate the very different structure of an old-standing parotid tumour. If a simple tumour becomes malignant it necessarily can only take on one form of malignant growth structure, namely, the form which arises spontaneously in the tissue of which the simple tumour is made and from which it grows. Thus a papilloma of the skin becomes an epithelioma or squamous- celled carcinoma : an adenoma becomes a glandular carcinoma : a fibroma becomes a spindle-celled sarcoma. The great majority of malignant growths, however, arise from normal tissues and not from simple tumours. CHAPTEE XXVIII. CARCINOMATA — SARCOMATA — OTHER TUMOURS — CYSTS. Malignant tumours differ from simple neoplasms in that they have no well-defined edge and no capsule ; that they grow rapidly and invade neighbouring structures ; that they form multiple secondary growths of the same nature at a distance ; and that they commonly produce cachexia. All malignant growths, however, do not conform to these characteristics. The malignant tumours are divided into two main groups : tumours arising from epiblast or hypoblast are known as carcinomata ; malignant connective tissue tumours arising from mesoblast are known as sarcomata. The division is thus analagous to that made between the simple neoplasms. Carcinomata differ from sarcomata in several respects, and in the great majority of instances are readily distinguished from them. Sarcomata have many thin-walled blood-vessels, but no lymphatics. A delicate connective tissue penetrates between each cell, but may be so delicate as to be discerned only with great difficulty. The tumours are very cellular, and the arrangement of the cells is commonly irregular. Dissemina- tion takes place by the blood-stream rather than by the lymphatics. The tumours may arise at any age, but are most common in young subjects. Carcinomata possess a stroma carrying more or less wel]- formed blood-vessels and lymphatics. The cells of the growth are in apposition to each other. The groups of cells are arranged in columns or in rude alveoli. Dissemination takes place by the lymphatics rather than by the blood-stream. The tumours arise most commonly in middle-aged and elderly subjects. The Carcinomata. Carcinomata tend to reproduce the structure of the tissue in which they arise, but very imperfectly. The cells of which they are composed are evidently similar to the cells of the 26—2 404 CLINICAL PATHOLOGY. tissue of origin, and the secondary deposits, in whatever organ they occur, are composed of cells similar to those of the primary growth. Yery cellular carcinomata are often called " encephaloid," and tumours with comparatively few cells and much fibrous tissue are known as " scirrhous." All forms of carcinoma are vulgarly referred to as cancers. Carcinomata are conveniently divided, according to the type of cell of which they are composed, into squamous, spheroidal or cuboidal, and columnar celled carcinomata. The term " epithelioma " is applied by some to all epithelial tumours, and to avoid confusion is not used here. Squamous-celled carcinoma. — This variety of carcinoma necessarily arises from squamous epithelium, and consequently is found upon any part of the skin, the lips, the tongue and buccal cavity, the tonsil, pharynx, larynx and oesophagus, the anus, the cervix uteri, the glans penis, and the bladder. The commonest situations are the cervix uteri, the lip, and the tongue. Squamous-celled carcinoma is particularly liable to develop in response to chronic irritation. It rarely occurs upon the skin, but may be grafted upon X-ray burns, upon a chronic sore, or notoriously upon the scrotum of a sweep. Carcinoma of the lip is practically confined to pipe smokers, and particularly to those who suck clay or painted horn mouth- pieces, and it arises at the place on the lip where the pipe is habitually held. Carcinoma of the tongue is rare except among patients with syphilitic leukoplakia. Carcinoma of the penis is practically confined to the uncircumcised. This variety of carcinoma is locally very malignant, and spreads rapidly into the neighbouring glands. It rarely disseminates generally over the body. The least malignant form of carcinoma is that affecting the lip. It attracts notice early and remains localised for a con- siderable time. Operative treatment is far more successful in cases of carcinoma of the lip than in any other form of carcinoma with the exception of rodent ulcer. The histological diagnosis of squamous-celled carcinoma is usually easy. Care should be taken, however, to cut the sections exactly at right angles to the epithelial surface. The only growths which could be mistaken for this form of carci- noma are papillomata and rodent ulcers. CARCINOMATA— SARCOMATA, ETC. 405 Under the microscope the following points can be made out : — An ulcer bare of epithelium is usually present, and at the edges of the ulcer the epithelium is thickened and irregular, and dips down into the subjacent tissues. Beneath the base Fig. 35. — Squamous- Celled Carcinoma of Tongue. Drawn under f-inch Objective. "S7 & ® O flir^wf » s s 9 (V Fig. 36. — CelL Nest. Drawn under £-mch Objective. of the ulcer and at its edges are columns and groups of epi- thelial cells, which, in a single section, do not join directly with each other or with the overlying epithelium. The cells composing the columns are of the same nature as the prickle cells which occupy the central rows of the normal Malpighian 406 CLINICAL PATHOLOGY. layer of the skin. They are flat, faintly- staining cells with a central and usually lightly-stained nucleus, and their prickle processes can often be made out. Lying in the centre of some of the columns, or lying free in the connective tissue, are found in the great majority of sections the so-called "cell nests." These are round bodies, easily recognised under the f-inch objective, staining a bright red with eosin, and more or less structureless. In some cell nests the outlines, and more often the nuclei, of the compressed elongated and curved epithelial cells can still be made out. The cell columns and nests lie in a connective tissue stroma, and, penetrating beneath the sub- epidermal alveolar tissue, pass between the muscle bundles. An inflammatory exudate of cells is usually present in the stroma at the edges and base of the growth. A carcinoma is distinguished from a papilloma by the direction of the growth, which is inwards and not outwards, by the character of the ramifying columns of epithelial cells, which involve the deeper structures, and by the presence of cell nests. The points of distinction between a squamous-celled carci- noma and a rodent ulcer will be given under the description of the latter tumour. Rodent ulcer. — Eodent ulcer is an epithelial neoplasm which spreads and destroys the tissues as it grows. It is, however, only locally malignant and never disseminates, thus differing from all other varieties of carcinoma. Rodent ulcer affects the skin, and particularly the skin of the face. Its most characteristic situations are at the outer canthus of the eye, on the nose, the eyelid, and less frequently the pinna : multiple rodent ulcers are not very uncommon. The histological aspect of a rodent ulcer is very characteristic. The growth arises from the skin or from one of the skin appendages, such as the sebaceous glands or hair follicles, and is evidently a growth of the palisade cells of the Malpighian layer. If a section of the normal skin be examined under the micro- scope the basal row of cells of the Malpighian layer is seen to consist of tall, narrow cells with deeply-staining nuclei. The appearance of these cells is in very strong contrast to the squamous cells of the more superficial layers. The same layer of palisade cells is present also in the skin appendages. CARCINOMATA— SARCOMATA, ETC. 407 A section of a rodent ulcer shows columns of epithelial cells passing beneath the epidermis. The columns tend to be longer and somewhat more regular than those of a squamous- celled carcinoma. They are composed of the narrow elongated Fig. 37. — Rodent Ulcer. Drawn under |-inch Objective. Fig. 38. —Rodent Ulcer. Drawn under ^-inch Objective. cells with deeply-staining nuclei. Few or no prickle cells are present, consequently there is no tendency to keratinisation and no formation of cell nests. An inflammatory exudation into the stroma is common, as also into the neighbouring 408 CLINICAL PATHOLOGY. tissues of the majority of malignant growths, and particularly those of cutaneous surfaces. The distinction between squamous-celled carcinoma and rodent ulcer is rarely difficult, since the squamous growth is composed of squamous-celled columns, with at most an imperfect edging of palisade cells, and of cell nests, while the rodent ulcer is composed of smaller elongated cells with deeply- staining nuclei, very few squamous cells, and no cell nests. Spheroidal-celled carcinoma.— Spheroidal or cuboidal celled carcinoma originates from spheroidal glandular epithelium. It is the common carcinoma of the breast and prostate. It occurs also in the ovary, testis, pancreas, thyroid, salivary glands, and stomach. Very cellular growths of this nature are known as encephaloid carcinomata, as opposed to the fibrous, which are called scirrhous carcinomata. A prognosis based upon the cellular character of the growth is very un- certain; as both forms of growth invade the neighbouring tissues and disseminate freely in the lymph glands. A fibrotic carcinoma of the breast may remain latent for a number of years, and after removal of the main growth dissemination may rapidly occur. It is not really possible to say from the histological findings in any form of carcinoma whether the growth will recur rapidly or not. Carcinoma in elderly patients often shows very little malignancy : carcinoma in young subjects usually terminates fatally in a short time. The histological aspect of the growth in the two cases is identical. Under the microscope the spheroidal-celled carcinoma is seen to consist of a number of columns composed of oval cells ramifying in a more or less dense fibrous stroma. The columns consist of atypical glandular acini, very irregular in shape and size, and with their lumina filled as a rule by the cells. Small groups of spheroidal cells are characteristically present in addition, irregularly placed and lying free in the fibrous stroma. Some of the cells may show mitotic figures, and these may be present in any form of malignant neoplasm, but afford no particular evidence of malignancy, since they may occur also in simple growths and in normal tissue. Areas of cell degeneration are common in these tumours, and particularly in the encephaloid variety of them. The degeneration consists of necrosis as a rule, but colloid change is not infrequent. CARCINOMATA— SARCOMATA, ETC. 409 The spheroidal-celled carcinoma has to be distinguished from an adenoma and from a sarcoma. The acini of an adenoma are far more regular in shape and arrangement than those of a carcinoma, and are commonly composed of one or two layers of cells enclosing a well-defined lumen. In the majority of cases the distinction is easy. The more degenerated portion of a carcinoma, in which the alveolar arrangement is broken up, may bear a considerable resemblance to a sarcoma. The diagnosis is made clear by examining also the undegenerated portions. The areas of J /v; ■ ?^SWSUri^ ' » , VVR':, *\\&P)J ( V^JttfilP mm- £ ; #« \Ai \. %dl) V v /- jHbi!| /;fj L 7% }'/ h ?#V ; ^M U^*^^ ?S^;; Fig. 39. — Spheroidal-Celled Carcinoma of Breast. Drawn under £-inch Objective. degeneration are readily recognised and avoided in the fresh specimen by their colour and soft consistence. If the growth is entirely necrotic it may be impossible to distinguish its nature. In examining breast sections in particular it is essential to have an acquaintance with the histology of the normal gland. Areas of glandular activity in a lactating breast have been frequently mistaken for malignant growths. The secondary deposits in glands have the characters of the primary growth, and considerable fibrosis of the gland is often present in addition to the alveolar columns of spheroidal 410 CLINICAL PATHOLOGY. cells. The secondary deposits of all tumours, however, tend to be more irregular than the primary growth, and it may not be possible to tell their exact nature in all cases. The lymph glands draining a carcinomatous area are frequently enlarged, but not necessarily infected by cells of the neoplasm. Such glands show great increase in their epithelioid cells, and fortunately these cellular areas hear little or no resemblance to areas of spheroidal cells. The epithelioid elements have, however, been oiten mistaken for malignant deposits. The glands show areas in which the lymphoid cells are very scanty or absent, and have been replaced by the angular epithelioid cells of chronic inflammation. Columnar-celled carcinoma.— This variety of carcinoma arises from, and is mainly composed of, columnar epithelial _<*.<■ C Fig. 40. — Columnar- Celled Carcinoma of Colon. Drawn under g-inch Objective. cells. It is met with throughout the alimentary tract from the pylorus to the commencement of the anal canal, in the body of the uterus and in the gall bladder : also in the larger ducts of those glands which have been mentioned as usually affected by spheroidal-celled carcinoma. Columnar-celled carcinomata arising in spheroidal-celled glands provided with columnar- celled ducts are known as duct carcinomata. Under the microscope are seen numbers of alveoli lined by a single layer of columnar cells, and separated from each CAECINOMATA— SAECOMATA, ETC. 411 other by a delicate and scanty connective tissue. In many sections the columnar-celled alveoli differ bat little from those of the normal epithelium, except in the usual absence of a regular arrangement relatively to each other. The diagnosis of malignancy in such cases rests mainly upon the position of the alveoli, for example, beneath the submucous coats and between the muscle bundles of the muscular coats in the case of an intestinal carcinoma. Frequently, however, the alveoli are atypical and of very irregular size, and in rapidly-growing tumours may be so compressed as to resemble rather solid columns of more or less spheroidal cells, the alveolar arrangement being present only at the growing edges. In other cases there may be proliferation of the lining cells of the acini to form papuliferous processes projecting into the lumina, and a few cells may be seen passing out from the lining layers into the adjacent tissues. In secondary deposits the columnar-celled acini may be well formed, but they are frequently most atypical, and difficult to distinguish from similar deposits of a spheroidal- celled growth. The Sarcomata. The sarcomata are mesoblastic tumours, and, with the exception of myeloid sarcoma, are intensely malignant. The sarcomata may very rarely disappear spontaneously or after the injection of Coley's fluid. Cure of a round or spindle- celled sarcoma by purely surgical means is extremely unusual, so rapidly do the secondary growths disseminate. The sarcomata are classified mainly according to the type of cell of which they are composed, and we have to consider round-celled, spindle-celled, and giant-celled sarcomata. The round-celled growths are further subdivided into small and large round-celled sarcomata. In addition are recognised alveolar and melanotic sarcomata. The sarcomata are particularly liable to the various degenerations, and the growths are commonly necrosed in part. In some necrotic growths it may be impossible to obtain for section a portion of the tumour which sufficiently indicates its structure. Mucoid and hyaline degeneration may also occur. Sarcomata, usually of the spindle-celled variety, may 412 CLINICAL PATHOLOGY. be associated with an excessive production of fibrous tissue, and such growths are known as fibro-sarcomata. Other growths may develop cartilage, and are called chondro- sarcomata ; others again may produce bone, and are called osteo-sarcomata. The majority of the sarcomata are very soft and cellular tumours, but the varieties just mentioned are tough, and the osteo-sarcomata may be of stony hardness. The hard sarcomata may disseminate with great rapidity. Small round-celled sarcoma. — The distinction between * ^ © * f> fl ® ®^ » • *#*'^ • @ ©<©© #" © ^ @ ©. © © • ^@ ©|°® ©@i$ %* © ft • •- »* %*t**/ ##*'••$*• 2* • Fig. 41. — Small Bound- Celled Sarcoma. Drawn under J-inch Objective. the two varieties of round-celled sarcoma is not very marked, and cells of varying size are common in the two forms. The round-celled sarcomata are the most generalised of all tumours, and may occur in any part of the body, among other places in the connective tissues, and particularly in the bones, in the ovary, testicles, pharynx, and pancreas. The round and spindle celled sarcomata of bones are especially characteristic, and almost uniformly run a rapid and fatal course. For- tunately they are comparatively rare tumours. Under the microscope the small round-celled sarcoma is seen to consist of a scanty intercellular connective tissue con- taining a large number of cells, which show no particular CARCINOMATA— SARCOMATA, ETC. 413 arrangement in respect to each other or to the tissue in which they lie. The cells have a round and usually reticular nucleus, and a small but definite amount of cytoplasm. An ordinary microscopic field bears a considerable resemblance to an area of a lymphatic gland, but there is no glandular arrangement into cortex and medulla, and " germinal " centres are absent. Also the individual cells of a sarcoma are definitely larger than those found in lymph tissue, and are more scattered and more irregularly placed. Thin-walled blood-vessels and hemorrhagic areas are frequent. Mitotic figures are usually numerous. Large round- celled sarcoma. — This variety of sarcoma is distinctly less common than the smaller-celled tumour. The cells have a big, round, deeply-staining nucleus and a large area of cytoplasm. Even in paraffin sections the cells are obviously large, and if unstained specimens teased out in normal salt solution are examined the cells are seen to be very large indeed. Spindle-celled sarcoma. — These sarcomata are met with more frequently than the round-celled varieties. They may be similarly divided into large and small spindle-celled growths, but the relative size of the cells is not materially important. The tumours are met with in much the same situations as the round-celled sarcomata, and in particular in connective tissues, the periosteum of the long bones, and in fasciae. Under the microscope the tumour is seen to consist of elongated cells tapering to a point at each end and containing a central elongated nucleus. The arrangement of the cells may be fairly regular, and whorls of cells similar to but less regular than those met with in fibromata are occasionally present. Such tumours are not often seen, are slowly grow- ing, and tend to recur locally rather than to disseminate. Occasionally tumours are met with in which the spindles are shorter and broader, and the growth is called an " oat-celled " sarcoma. In the more typical sarcoma the long well-formed spindle cells are scanty, and the nature of the sarcoma is more obviously shown by the spindle-shaped nuclei than by the shape of the cells. The arrangement of the cells is irregular, and there are practically no whorls. Often the cells are more or less angular and may show bifid processes. In nearly all spindle-celled sarcomata numerous round cells with round 414 CLINICAL PATHOLOGY. nuclei are seen, and both large and small spindle cells are present. Such tumours are frequently called mixed-celled sarcomata. It must be recognised, however, that the cells do not all lie in the same direction and that many are cut trans- versely and appear as round or oval cells. In this variety of sarcoma, as in other varieties, the great majority of the blood- vessels are mere spaces in the reticulum, and their walls are rarely composed of more than one layer of endothelial cells. *&r U£ 'AnKi '-'WAV »» Q „ 9 t •a * - % , 1 W rf 6 o viWfi e U M »#W l_a_ Pig. 42. — Spindle-Celled Sarcoma. Drawn under jl-inch Objective. The character of the blood-vessels in a sarcoma is of the utmost diagnostic importance. Myeloid sarcoma.— It is doubtful whether this tumour should be classed as a sarcoma. It is an endosteal tumour, which invades and destroys the surrounding tissues, but scarcely ever forms secondary deposits. Local removal, and often the necessarily incomplete local removal by curettage, is usually sufficient for a cure. The growth is called by some a myeloma in preference to a myeloid or giant-celled sarcoma. Myeloid sarcoma is common in the maxilla, forming one variety of epulis, and is frequently met with in the extremities of the long bones, particularly in the upper end of the tibia and in the lower end of the femur. CARCINOMATA— SARCOMATA, ETC. 415 Under the microscope the character of the growth is extremely typical. The essential feature is the presence of abundant myeloid or giant cells. These cells are large, and both variable and irregular in size and shape, the majority being more or less oval. They contain very many nuclei, often 20 or more, scattered irregularly throughout the cytoplasm. Giant cells of a somewhat similar type may be present in ordinary granulation tissue, and exactly similar cells are frequently found in an inflammatory process con- nected with bone. In no other tissue, however, are to be Fig. 4.'3. — Myeloid Sarcoma. Drawn under f-incli Objective. found so many of such cells as to form the predominant feature of a microscopic field. In addition to the giant cells is found tissue precisely similar to that which mainly comprises a spindle-celled sarcoma of the small irregular spindle-celled type. The spindle-celled parts of the tumour penetrate among the myeloid cells, and also form areas free from giant cells. The blood- vessels are very numerous and thin-walled, and hemorrhages into the growth are frequent. The hemorrhagic character of the growth gives to the fresh specimen a characteristic maroon colour. Alveolar sarcoma. — This name is given to sarcomata in which the cells have an irregular arrangement into alveoli 416 CLINICAL PATHOLOGY. separated from each other by strands of fibrous tissue. The alveolar sarcomata are most commonly seen in connection with the skin, and may arise from moles. They are often melanotic. The cells in an alveolar sarcoma are practically always of the small angular spindle-shaped variety. Melanotic sarcoma. — This tumour is confined to situations in which melanin is normally present : consequently it is found in the skin, and particularly in pigmented moles, and in the choroid. It less frequently arises in the nail matrix of the fingers, or in the adjoining skin. Melanotic sarcomata :**- of ^ ^> ^ G— Fig. 44. — Melanotic Alveolar Sarcoma. Drawn under ^-inch Objective. developing in moles always show evidence of an alveolar arrangement. Such moles may remain quiescent for years, suddenly become malignant, and disseminate with great rapidity. The amount of pigment is variable, and it is usual for the secondary growths to be more deeply pigmented than the primary tumours. The secondary deposits occur in the neighbouring lymph glands, and from thence disseminate to the liver, lung, kidney, brain, and other organs. The histological diagnosis of a secondary deposit is simple : that of the primary growth in the skin is often difficult, and CABCINOMATA— SARCOMATA, ETC. 417 has been referred to in the paragraph dealing with pigmented moles. Othee Tumoues. Other malignant growths, difficult to classify or not included in the above account, are referred to here. They include deciduoma malignum, renal tumours, and teratomata. Deciduoma malignum. — Deciduoma malignum, or chorion epithelioma, is a rare malignant growth of the uterus which has been variously classified as a sarcoma and a carcinoma. The growth follows a pregnancy, and in a large number of cases has been associated with a hydatidiform mole. It is an extremely malignant tumour, and rapidly disseminates to the lungs and other parts of the body. Very similar tumours may rarely occur in the testicle. The growth is very vascular, and haemorrhages into it are the rule. Under the microscope the growth is seen to consist as a rule mainly of blood clot enclosing comparatively few and small cellular processes. Some of the processes may show a tubular arrangement, and in the majority of them are to be found cells of two varieties representing the two layers of a chorionic villas. The smaller cells from the investing, or syncytial, layer are seen, together with the large polyhedral cells known as decidual cells, and derived from the inner or Langhan's layer. Renal tumours. — The kidney may be affected by a variety of new growths common to other tissues, but all are rare. One remarkable tumour must be mentioned here, and is apparently peculiar to the kidney. The tumour is known as a hypernephroma, or as a von Grawitz tumour. It is a malignant tumour, probably of carcinomatous nature, and is supposed to originate in adrenal gland inclusions. The tumour may occur in young children or in middle life. Com- mencing in the upper pole of the kidney it gradually extends downwards, compressing the renal substance, and remaining for a considerable period more or less encapsulated. It finally burrows its way down to the renal pelvis and grows into the vessels, whence it disseminates over the body. Under the microscope the tumour is very characteristic. Areas of haemorrhage and degeneration are common, but in the undamaged growth areas is seen a capillary-bearing p. '27 418 CLINICAL PATHOLOGY. stroma enclosing more or less circular collections of cells. The cells are swollen, round or polygonal, contain a central nucleus, and often stain somewhat poorly. The cells may be arranged in columns, the centres of which are often degenerated and thus convey the impression of alveolar processes. The tumours contain a large amount of glycogen and fat, and were originally described by von Grawitz as fatty tumours of the kidney. Teratomata. — These tumours are new growths which contain a variety of tissue elements. The teratomata include the dermoid cysts of the ovary, in which any of the skin appendages, such as hair, nails and teeth, may be found, as well as other cysts, which may contain fragments of numerous internal organs. Such teratomata may occur in the testis as well as in the ovary. The malignant teratomata are remark- able mixed tumours which may contain sarcomatous and carcinomatous tissue. The former are often spindle celled, and the latter columnar. The sarcomatous and epithelial growths may appear side by side in the same section. Areas of well-formed cartilage are common in such tumours, while muscle fibres and almost any variety of tissue may be present in addition. The teratomata have been considered to represent abortive impregnations. It has been suggested that the ova of the embryo have been fertilised by the father of the embryo, and that the teratoma is the imperfect individual resulting from this achievement. Cysts. Cysts are pathological formations enclosed by a membrane, and containing fluid or semi-fluid material. The majority of cysts result from the dilatation of secreting tubules following obstruction to the outflow of the secretion. The tubule may be normal, and the obstruction the result of acquired pathological change outside the secreting tissue, as in the retention cysts of the kidney or breast ; or the cyst may arise in a persistent rudimentary remnant of foetal tissue, as in the case of a branchial or parovarian cyst. All such cysts are known as retention cysts. Other cysts may arise as the result of softening processes in CARCINOMATA— SAECOMATA, ETC. 419 solid tissues, or may be due to the presence of parasites. Only the more important cysts can be mentioned here. Retention cysts. — Retention cysts result from the blocking, and most commonly from the incomplete. blocking, of the duct of a secreting gland. One of the best known examples is the ranula, a cyst arising in connection with the submaxillary gland. Pancreatic cysts may reach a considerable size, as also may the single cysts of the parovarium. The majority of such retention cysts are of little histological interest, since the lining membrane is usually composed of more or less structureless fibrous tissue. The sebaceous cyst, however, has a well-developed epithelial lining, and some of the cysts of vestigial structures are of histological importance. The dermoid cyst, resulting from an epidermal inclusion, has a lining membrane which contains all the layers of the true skin, including the stratum granulosum. The branchial cyst of the neck has an epithelial lining, often composed of columnar, and sometimes of columnar ciliated, epithelium. Cysts of a similar nature may arise in the substance of a secreting organ, and may follow fibrosis of the organ resulting from toxic or inflammatory processes, the fibrosis constricting the duct lumina. Such cysts are common in the kidney and the breast. Cysts may also develop in the adenomata of secreting glands, and are commonly found in the breast and in the thyroid. Such cystic formations are known as cystic adenomata. The cysts in such cases are due to retention of secretion in the abnormal alveoli, and do not always contain the normal secretion of the organ. They are lined with a definite epithelium, usually more or less columnar. The multilocular ovarian cyst is of this nature, and is known as a " cystoma." The epithelial cyst lining of the ovarian cystoma is often composed of flattened and not of columnar cells, the flattening of the cells being ascribed to the pressure of the fluid in the cysts. In fibro-cystic disease of the breast the sections may show considerable areas of fibrosis with little cellular evidence of past inflammation, and numerous cysts of very variable size. Congenital cystic disease of the kidney is almost invariably bilateral, and nearly the entire area of both kidneys may be converted into a number of thin-walled cysts. 27-2 420 CLINICAL PATHOLOGY. Degeneration cysts. — These cysts are not of great importance. They are not infrequent in new growths as the result of necrosis and softening of portions of the growth, and in old haemorrhages. Such collections of fluid are scarcely true cysts in that they have no true limiting membrane. Parasitic cysts. — The occurrence of these cysts has been noticed in the description of the higher parasites. CHAPTER XXIX. HISTOLOGICAL METHODS. The tissues removed at operation are preferably received into the laboratory without the addition of any preserving fluid. The constant handling of tumours gives the pathologist a considerable acquaintance with their various appearances, and the naked-eye observation is of great assistance to him. The addition of alcohol or formalin to the tissues completely alters their appearance, and tends to obscure the relationship of the parts removed. If, for geographical reasons, a preservative is necessary, the tissues may be placed in 50 per cent, methylated spirit or in 10 per cent, formalin. It is also advisable that as much of the material should be obtained as possible in order that the relationship of the normal and abnormal tissues can be noted, and portions removed from the most desirable places. The selection of the portion or portions to be sectioned must be made with considerable care. It is a common error to remove pieces of far greater surface area and thickness than are required for diagnostic purposes. In cases of evident tumour the firmest and least necrotic portion should be chosen from the growth itself, and in the majority of cases a second portion at the junction of growth and normal tissue. If more than one variety of tissue is present, portions should be selected from each variety. Metastatic growths in lymph glands, or elsewhere from the same case, should also be examined. Lymph glands, even if normal to the naked eye, should always be sectioned. Small glands lying in a mass of fat are most readily recognised by their firm and shotty consistence. The direction in which the section is to be cut is frequently of great importance, particularly in the case of tumours or ulcers of the skin and of the intestinal tract. It is often advisable to make a rough sketch of the portion removed for examination, with an arrow indicating the surface to be cut. 422 CLINIC AX PATHOLOGY. Minute fragments of friable tissue which have been removed by the curette are best placed in a layer of gauze tied with a thread in the form of a small sac. The tissues can remain in the gauze sac until the stage of " casting " in the paraffin process. Tissues containing bone must be decalcified by a special process, and it is well, if possible, to examine separately one portion, free of bone, by the ordinary method. Tissues to be examined for the presence and distribution of fat must pass through the gum, and not the paraffin, process, since the latter involves passage through alcohol. The methods of fixing, cutting, and staining sections are very numerous. Only those are given here which are generally suitable for the diagnosis of the great majority of tissues received from the wards and operating theatres. The processes of fixing and cutting tissues described here are those of gum and paraffin. The preparation of celloidin sections is rarely called for in clinical pathology, and is practically only required for sections through the globe of the eye. The preparation of gum sections.— The gum method has numerous advantages, and, since it requires the minimum of apparatus, is to be recommended for diagnostic purposes. Bather more practice is required for the actual cutting of the section, unless one of the more elaborate section cutters, such as the excellent one of Delepine, is available. The apparatus required consists of a hand microtome and a gum-embedding solution. The gum solution is made as follows : — Saturate gum acacia in hot water. Mix 3 parts of the gum solution with 1 part of syrup (B. P.). The most convenient pattern of microtome is Williams' microtome, made by Messrs. Swift & Son. It consists of a circular plate fitted with a clamp, which can be attached to the edge of a firm table, and of an ether spray apparatus, by means of which a spray of ether can be directed against the under surface of a small metal disc let into the circular plate. An ordinary razor blade is fitted into a brass tripod carrier at a fixed angle. The level of the cutting edge is regulated by a screw at the apex of the tripod. An ethyl-chloride spray is an extremely useful adjunct. HISTOLOGICAL METHODS. 423 There are two methods of conducting the process, a rapid and a slow one. The rapid method.— The rapid method can be performed with the majority of tissues, and is particularly useful for the immediate diagnosis of tumours during an operation. The Fig. 45. — Williams' Freezing Microtome. method requires some practice, and the interpretation of the results must in most cases be left to the expert, since the appearance of rapidly-frozen gum sections is very different from that of the more familiar paraffin section. In many instances the tissue can be cut, stained, and reported on within 10 minutes of removal from the body. The method is 424 CLINICAL PATHOLOGY. therefore very useful, but in a proportion of cases sections of tumours requiring long and careful examination are met with, and a certain diagnosis cannot and should not be made on the sj)ot. The diagnosis, unless extremely obvious, should always be confirmed by sections made at leisure. Eapid sections have the great advantage that practically no shrinkage of the tissues takes place, and they are for this reason to be preferred to the paraffin section. On the other hand, the clearing, staining, and flattening out of the section are less satisfactory in the majority of cases. The process is as follows : — With a sharp scalpel remove the most suitable portion of the tissue. Place the portion on the central disc of the microtome stand. Just cover it with gum solution. Do not pour on so much gum solution that it flows over the edge of the disc. Freeze with the ether spray from below, assisted by the ethyl-chloride spray from above. The freezing is complete when the gum is quite white and the tissue firm to the touch. If the freezing process is con- tinued too long the tissue becomes extremely hard and cannot be cut at all, in which case thawing may be hastened by moisten- ing tissue and gum with warm water. If the freezing is insufficient the gum is readily dented with slight pressure of the finger, and the tissue leaves its bed when the razor meets it. The freezing process should be completed in a few minutes. Moisten the stage of the microtome with water to allow the razor carrier to slide easily. Adjust the level of the razor edge exactly to the height of the tissue. Hold the razor carrier in both hands, with the forefinger of the right hand resting on the front adjusting screw. Eapidly sweep the razor across the tissue, keeping the carrier legs pressed against the microtome-stand surface. With the forefinger turn the screw a short distance onwards to depress the cutting edge. The amount of the turn deter- mines the thickness of the section cut. Eepeat the process about a dozen times in rapid succession. " With a small camel-hair brush wipe gently the mixture of gum and tissue from the upper surface of the razor blade into a tall glass beaker filled with warm normal saline. HISTOLOGICAL METHODS. 425 The sections float out on the surface of the fluid, and can be assisted to separate by gently touching with the brush. If complete and thin sections do not appear, cut more, either of different thickness or after altering the consistence of the gum and tissue by further freezing or thawing. Pick up the best section on a clean glass slide by holding the slide vertical and submerged in the saline and drawing it upwards along the section. If the section is curled it may often be straightened out by partially floating it off again in the saline with the aid of a blunt mounted needle or probe. Drain off the fluid as much as possible. Drop on the section 1 drop of Loffler's methylene blue. Carefully let down a cover-slijD in the slain over the section. Press the cover-slip down with 2 mounted needles. Wipe off the excess of stain from around the cover-slip. Examine with a §-inch and ^-inch objective. A differential stain, and a more permanent preparation, can be made by the rapid method as follows : — Take up the section from the salt solution on a section lifter or a blunt needle, and leave it in hsemalum for 2 minutes. Transfer to tap water in a tall beaker for 2 minutes. Pick up on a slide, and flatten out by smoothing with a cigarette paper moistened in water. Peel off the cigarette paper carefully. Cover slide and section with 5 per cent, alcoholic eosin for 1 minute. Cover with methylated spirit for a few seconds. Cover with absolute alcohol for 1 minute. Cover with xylol for 1 minute. Drain off xylol, and mount in Canada balsam. The slow method. — By this method the tissues are first fixed, then cut and stained at leisure. The method is par- ticularly useful for the demonstration of fat in tissues. The method of staining fat in tissues. Place the tissues selected in salt formalin solution of the following composition :— Water 100 c.c. Commercial formalin (i.e., 40 per cent.) . 10 ,, Sodium chloride ..... 1 gramme. Leave in salt formalin for 24 hours. Wash in running water for 24 hours. 426 CLINICAL PATHOLOGY. Transfer to gum solution (see above) for 24 hours. Cut sections with freezing microtome. Place sections in water for 1 hour. Transfer to a small well-stoppered bottle filled with Scharlach R. for 36 hours. Transfer to 75 per cent, spirit for a minute or two. Transfer to water. The sections spin round rapidly and spread out on the surface of the water. Place in filtered hsBmalum for 1 minute. Transfer to tap water in a tall beaker for 3 minutes. Pick up on a slide. Drain off the excess of water, and mount in Farrant's solution. The fat is stained red and the tissues blue. If the demonstration of fat is not required proceed as follows : — Fix in the same manner, and after cutting the sections leave in water for about an hour. Then stain with hsemalum and eosin in the manner described for the rapid method, varying the time of staining according to the nature of the tissue, and mount in Canada balsam. The preparation of paraffin sections. 1. The fixation of the tissues.— This can be done by one of the two following methods. The alcohol method is quite reliable for the majority of tissues, and furnishes very suitable sections for diagnostic purposes. Fixation by Zenker's fluid is suitable for small pieces of tissue, and particularly for soft, friable growths. This method gives better fixation than the simple alcohol process, and should be used when exact cellular details are required. A. The alcohol method. — Place the tissue selected in 50 per cent, alcohol for 24 hours. Transfer to 90 per cent, alcohol or methylated spirit for 24 hours. Transfer to absolute alcohol for 4 hours. Transfer to xylol. If the xylol becomes cloudy return to absolute alcohol for 2 more hours in order to get complete dehydration. Leave in xylol from 12 to 24 hours, or until the tissue becomes transparent. Remove from xylol, and blot off the excess of xylol with blotting paper. HISTOLOGICAL METHODS. 427 Place in melted paraffin in an incubator kept at 125° F. The paraffin should have a melting point of about 120° F. and should not be heated to a higher temperature than is necessary to keep it in a liquid state. After from 8 to 12 hours, during which the paraffin is changed three times, there should be no smell of xylol detected in the paraffin. The tissue is then ready to " cast." Two L embedding blocks are arranged to form a square on any smooth, hard surface, such as a piece of glass, which has previously been lightly smeared with vaseline. The blocks should also have their inner surfaces smeared with vaseline. The tissue is removed from the paraffin bath with a pair of forceps, previously warmed at the tips in a gas flame. The square is then filled with melted paraffin and the tissue care- fully laid in it so that the surface to be cut is lying flat and facing directly downwards against the glass. When the paraffin block has set hard the L blocks are knocked away, and the paraffin block is placed in a labelled chip box until required for sections. The entire process takes from 5 days to a week, but may be considerably hastened if desired. A paraffin section can if necessary be completed in one day. To hasten the process it is essential to select small pieces of tissue for examination. The surface area of the portion removed is less important than its thickness, which should be reduced as much as possible with a sharp knife. The tissue is placed in methylated spirit for about 1 hour, then in absolute alcohol for 2 hours, then in xylol till clear ; then in liquid paraffin, which must be changed every half honr until the smell of xylol is no longer detected, when the block is cast. Very excellent sections can often be obtained in this manner provided a very thin portion of tissue is carried through the fluids. The various strengths of alcohol and the xylol can be used for several specimens. Each specimen must be kept in a separate small bottle securely corked, and the bottle must be emptied out thoroughly before the next fluid is added. The melted paraffin can be kept in small glass pots without a cork ; and a well-regulated oven, or incubator kept at the appropriate temperature, is an 428 CLINICAL PATHOLOGY. advantage. The oven can be dispensed with and the paraffin pots can be kept in a water bath the temperature of which is regulated by the size of the flame under it. Such an appara- tus must be periodically inspected in order to be sure that the paraffin remains liquid and the temperature does not rise too high. B. Fixation in Zenker's fluid. — This fluid has the following composition : — Potassium bichromate . . . 2'5 grammes. Sodium sulphate Corrosive sublimate Glacial acetic acid Water to . 1 gramme. 5 grammes. . 5 c.c. 100 „ The corrosive sublimate and the potassium bichromate are dissolved in the water by heating. If a considerable amount of stock solution is made up it is advisable to omit the acetic acid, adding it in the proper proportion as required. The tissue to be fixed should be cut moderately thin and placed direct in the Zenker's fluid and left for from 8 to 12 hours. The tissue is then washed in running water for 24 hours; then placed in 30 per cent, spirit for about 4 hours, then in 60 per cent, spirit for the same time, then in methylated spirit containing sufficient tincture of iodine to give it a pale port wine colour, then in absolute alcohol, with two changes, for about 4 hours. The remainder of the process is the same as in the alcohol fixation method. The object of the tincture of iodine in the methylated spirit is to dissolve out of the tissue the mercury which tends to pre- cipitate from the Zenker's fluid. The period in absolute alcohol and the change of alcohol must be sufficient to discharge the iodine from the tissue. C. Decalcification of tissues.— If much bone is present in the tissue it is necessary to decalcify. Before decalcifying the tissue must be fixed and treated by method A or B until the end of the alcohol stage. The tissue is transferred from alcohol to the decalcifying fluid. The fluid should be made as follows : — Add carefully in the fume closet 1 gramme of phloroglucin to 10 c.c. of nitric acid. Keep for at least 24 hours, or until the reddish mixture has become light yellow. HISTOLOGICAL METHODS. 429 Dilute with 100 c.c. of 10 per cent, nitric acid in water. The extent of decalcification can be tested by probing the tissue with a needle. The process takes 3 or 4 hours as a rule in the above solution, but may take longer. After decalcification remove the tissue to running water for 24 hours. Return to methylated spirit for 24 hours, then to absolute alcohol, and continue as in A and B. 2. The cutting- of the sections. — A special microtome is required, and the apparatus most generally used is the Cambridge rocking microtome. The procedure is as follows : — Pare down the paraffin block, leaving a small margin of wax round the tissue. Heat a metal spatula in the flame and melt with the hot spatula the base of the paraffin block. Press down the block on to the metal carrier so that the surface of the tissue to be cut is horizontal. Adjust the carrier so that the block surface just touches the razor blade. Begin by cutting the thickest possible sections until the entire surface area of the tissue is exposed : then adjust the pointer to the required mark on the scale. The majority of tissues should be cut at from 5 to 7m thick. In cutting, move the handle of the microtome with a rapid and even movement. Provided the razor is really sharp, very little practice is required. Discard all broken and imperfect sections. It is best at first to cut one section at a time, and not a ribbon of sections. Each section is removed and discarded until a perfect section is obtained. Difficulty in cutting a tissue which should be reasonably soft usually means that the xylol was imperfectly removed in the paraffin bath. The xylol is readily detected in the block by its smell and gritty consistence, and the block must be returned to the paraffin bath and subsequently re-cast. Remove from the razor blade the thinnest sections with the aid of a small piece of paper and a mounted needle. Float the sections selected in a tall beaker filled with warm water. The water should feel distinctly warm to the touch and should be sufficiently hot to spontaneously flatten out any wrinkles in the section, but not so hot as to melt the paraffin. 430 CLINICAL PATHOLOGY. As soon as the section is quite flat it is picked up by drawing a clean slide, which has been thoroughly rinsed in hot running water, along the section, in such a way that the section clings to the centre of the slide. The slide is held vertically with the majority of its length in the water and drawn out slowly in a vertical direction when the section floats against it. The excess of water is wiped off the slide. A strip of stout clean paper is soaked in methylated spirit and laid along the slide over the section, which is then firmly smoothed down with the forefinger moistened in spirit. The paper is then peeled off and the slide wiped. After the sections are mounted the slides are put in a warm place, such as an incubator at 37° C, until perfectly dry. They are then ready to be cleared and stained, or can be kept indefinitely until required. 3. The staining of the sections.— In order to prepare the section for staining and to stain it a series of simple reagents are required. These may be poured out into special staining tanks if a number of sections are being treated and the sections can be completely immersed in the tanks. If few sections only are being prepared it is sufficient to pour the reagents over each slide, but care must be taken to cover the slide completely and to renew the reagent if there is danger of evaporation. To prepare the section for staining proceed as follows : — Cover with xylol, and leave till the paraffin is completely dissolved and the section is perfectly clear and transparent. Drain off the xylol. Cover with absolute alcohol for 2 minutes. (The section becomes opaque.) Cover with methylated spirit for 2 minutes. Wash in water for 2 minutes and leave in water until required to stain. Should the section come off the slide at any period of the process, it can be transferred with a section lifter through the intermediate reagents to water, floated in a beaker of water, and picked up again on a clean slide. The water on the slide is drained off so far as possible, and the section is blotted very firmly on the slide with clean, dry filter paper. The preparation of the various staining reagents is given in Chapter XIII. HISTOLOGICAL METHODS. 431 A. To stain with Hsemalum and Eosin. — This is the most useful routine, stain and is sufficient for all ordinary purposes. Eemove from water and drain off excess of water. Filter the haemalum on to the section and control the depth of stain by examining the section, after washing it in tap water, under the low power of the microscope. The period of staining differs for different samples of haemalum and for different tissues. Three minutes is the time for the majority of stains and tissues. Very cellular tissues, such as a lymph gland, require a shorter time and necrotic tissues a longer time. If in doubt it is preferable to understain the section and then to return it to the haemalum for another period. Leave in tap water till the section is quite blue. Cover with 2 per cent, watery eosin for 20 seconds. Dip in tap water. Drain off water and cover with methylated spirit for 1 minute. Drain off methylated spirit and cover with absolute alcohol for 3 minutes. Drain off alcohol and cover with xylol for 3 minutes. Wipe off the xylol from the slide in the neighbourhood of the section, but do not allow the section to dry. Mount in Canada balsam. The cells and nuclei are stained blue and the connective tissue pink. For diagnostic purposes it is often advantageous to stain one section with haemalum only. A single stain frequently gives a better idea of the nature of a doubtful tumour than a brightly differentiated one. B. To stain with Hsemalum and van Gieson :— After staining in haemalum, soak in water. Cover with van Gieson' s stain for 20 seconds. Wash very rapidly in water. Wash very rapidly in methylated spirit. Place in absolute alcohol for 3 minutes. Place in xylol. Mount in Canada balsam. Fibrous tissue is stained pink, muscle brown, and elastic tissue yellow. The haemalum staining should be rather deep, otherwise the picric acid will stain the cell protoplasm yellow. 432 CLINICAL PATHOLOGY. C. To stain with carbol-thionin :— Cover the slide with carbol-thionin for 5 minutes. Wash rapidly in water. Wash in methylated spirit for about 1 minute, observing the section from time to time under the microscope, and stopping as soon as any pink colour appears. Place in absolute alcohol for 2 minutes. Place in xylol, and mount in Canada balsam. Fibrous tissue is stained red and cells purple. Micro- organisms are well shown and stain purple. Fibrin can be stained in this way. The method is not recommended for permanent preparations, since the stain is liable to gradually dissolve out into the mountant, but is useful as a rapid and simple stain for organisms in inflammatory tissues. D. To stain for tubercle bacilli in section :— Take the slide from xylol through spirit to water. Filter boiling carbol-fuchsin on to the slide. Stain for 7 minutes with 3 changes of fuchsin. Dip in water. Wash in 25 per cent, sulphuric acid till decolorised. Wash in water. If the red colour comes back return to acid. Eepeat until the tissue after 3 minutes' washing in fresh water is colourless, or at the most of a very pale pink colour. Stain with hsemalum. Soak in water till blue. Take back through alcohol and xylol, and mount in Canada balsam. The tubercle bacilli are stained red and the tissues blue. Leprosy bacilli are stained in the same manner, but 12 per cent, acid is used to decolorise and a distinct pink is left in the tissue. E. To stain by Gram's method in section :— Take the slide from xylol through spirit to water. Filter freshly-prepared aniline gentian-violet (see page 150), on to the slide for 10 minutes. Dip in water. Cover with Gram's iodine for 1 minute. Wash in methylated spirit until the colour ceases to come out freely and only a faint blue is left in the section. Wash in water. Stain in J per cent, safranin (filtered) for 20 seconds. HISTOLOGICAL METHODS. 433 Wash in water. Dip in methylated spirit. Place in absolute alcohol for 2 minutes. Clear in xylol, and mount in Canada balsam. Gram-positive organisms are stained blue and Gram- negative red. F. To stain with Leishman's stain in section :— Clear in xylol. Place in absolute alcohol for 2 minutes. Wash in distilled water. Cover with Leishman's stain, and add immediately double the volume of distilled water. Stain for 7 minutes. Wash in distilled water. Wash in 1 in 1500 acetic acid in distilled water, observing the section from time to time under the microscope until the connective tissue appears pink. Wash in distilled water. Stain in a saturated solution of eosin in absolute alcohol for 20 seconds. Wash in absolute alcohol. Clear in xylol, and mount in Canada balsam. The method is to be used as a differential stain for cells. G. To stain for free iron in section : — Take sections through to water. Place in 2 per cent, potassium ferrocyanide in distilled water for 1 hour. Place in 1 per cent, acid alcohol for 30 minutes. The acid alcohol has the composition — 1 c.c. strong HC1. 70 ,, absolute alcohol. 29 „ distilled water. Eeturn through methylated spirit to xylol, and mount in Canada balsam. Free iron pigment is stained a greenish blue. The tissue can be lightly counterstained with eosin or safranin if desired. P. 'AS INDEX. Abscess, 116, 165 pulmonary, 367 tropical, blood changes in, 19 Acarus scabiei, 352 Aceto-acetic acid, 244 Acetone, 244 Acholuric family jaundice, 28 Achorion Schonleinii, 146, 355 Achylia gastrica, 295 Acid, aceto-acetic, 244 " active " hydrochloric, 294 ammonium urate, 257 B-hydroxybutyric, 244 -fast bacillus, 123 free hydrochloric, 293 glycuronic, 247 lactic, 297 Acidosis, 228 Acne bacillus, 130 Acne vulgaris, 165, 359 Acquired syphilis. Wassermann re- action in, 68 Actinomyces, 144 Actinomycosis, 371, 384 Acute inflammation, 376 blood changes in, 17 Adenoma, 392 papillary, 393 fibro-, 393 Mrogenes capsulalus bacillus, 132 Agar-agar, 106, 182 Agar-oleic acid, 183 plate cultures, 106 slope cultures, 106 stab cultures, 106 Agglutinins, 48 as a test for organisms, 57 as evidence of infection, 57 in diagnosis, 49 in dysentery, 56 in Malta fever, 56 in paratyphoid infections, 55 in typhoid fever, 49 Ague, 86 Albumin in faeces, 321 tests for, 215 Albuminometer, Esbach's, 238 Albuminuria, 239 Albus staphylococcus, ¥15'' Alkalinity of the blood, 97 Wright's method, 97 Alkaptonuria, 224 Alveolar sarcoma, 415 Ammonia nitrogen, 228 Amceba coli, 340 Amoebic dysentery, 340 Amorphous phosphates, 258 urates, 256 Amyloid casts, 255 disease, 220 Anaemia, aplastic, 11 infantum pseudoleukaemica, 27 pernicious, 7, 8, 99 secondary, 24 splenic, 22 splenic of infants, 27 Von Jaksch's, 27 Anaerobic cultures, 162 Angeioma, 397 Anginosus streptococcus, 119 Angular conjunctivitis, 133 Animal inoculation, 109 parasites, 329 blood changes with, 19 Ankylostoma americanum, 336 duodenale, 336 Anophelinae, 86 Anthrax bacillus, 130 Anti-colon bacillus serum, 176 Anti-diphtheritic serum, 175 Antiformin, 156 Antigen, 66 syphilitic, 72 Anti-meningococcus serum, 176 Anti-plague serum, 178 Anti-pneumococcus serum, 176 Anti-sera, 174 Anti-tetantic serum, 175 Appendicitis, 213 blood changes in, 18 Arabinose, 181 Arthritis, 117 gonorrhceal, 164 rheumatoid, 208 Ascaridae, 338 Ascaris lumbricoides, 338 Aspergillosis, 350 28—2 436 INDEX. Aspergillus niger, 146 Asthma, 367 blood changes in, 20 Aureus staphylococcus, 115 Autoclave, 178 Avian tubercle bacillus, 125 Bacilltjbia, 282 Bacillus, acid-fast, 123 acne, 120 cerogenes capsulatus, 131 anthrax, 130 avian tubercle, 125 Bordet-Gengou, 80 bovine tubercle, 124 butter, 125 coli communis, 137 comma, 140 diphtheria, 127 Ducrey's, 278 dysentery, 137 enteritidis (Gaertner), 136 enteritidis sporogenes, 107 fish tubercle, 125 Flexner's, 137 Friedlander's pneumo-, 370 Gaertner's, 136 glanders, 110 Hofmann's, 128, 130 influenza, 132, 198 Kitasato, 133 Koch-Weeks, 133, 348 lepra, 126 malignant oedema, 140 mallei, 133 Moeller's timothy-grass, 125 Morax-Axenfeld, 133, 348 Morgan's No. 1, 343 paratyphoid A, 136 paratyphoid B, 136 pestis, 134 Pfeiffer's, 132 plague, 372 proteus, 139 proteus in urine, 282 pyocyaneus, 139 Rabinowitch's butter, 125 saprophytic, 198 Shiga, 137 smegma, 125 subtilis, 123 suipestifer, 136 tetanus, 130 tubercle, 123 typhosus, 133 Vincent's fusiform, 143 whooping-cough, 131 xerosis, 128, 347 Bacteriology of the bile, 305 urine, 276 Barber's rash, 358 Basophil, coarsely granular, 5 Basophil, finely granular, 5 Bed bug, 354 Bence-Jones protein, 241 B-hydroxybutyric acid, 230, 344 Bile, 304, 319 bacteriology of, 305 Bilharzia hsematobia, 255, 330 Biuret reaction, 325 Blastomyces, 355 Blepharoblast, 89 Blood, alkalinity of, 97 Wright's method, 97 Blood changes in — acute inflammation, 17 animal parasites, 19 appendicitis, 18 cachexia, 26 carcinoma, 19, 21 cardiac failure, 23 chicken-pox, 20 children, 26 chronic inflammation, 21 congenital morbus cordis, 24 congenital syphilis, 29 gonorrhoea, 21 haemorrhage, 25 infantile scurvy, 30 influenza, 22 lead poisoning, 26 malaria, 22 measles, 22 metallic poisons, 26 pneumonia, 17 purpura, 30 rickets, 29 scarlet fever, 20 skin lesions, 20 small-pox, 20 spasmodic asthma, 20 spring catarrh, 20 syphilis, 21 tropical abscess, 19 tuberculosis, 19 typhoid fever, 22 Blood cultures, 81 in rheumatic fever, 81 in typhoid fever, 83 Blood films, to make, 44 fresh, 3 in test meal, 299 in urine, 249 normal, 3 oxygen content of, 98 parasitology of, 81 platelets, 3, 7 primary diseases of, 7 secondary diseases of, 17 serum, Loffler's, 185 serum medium, 184 specific gravity of, 96 spectroscopic examination of, 92 to obtg.jn, 33 INDEX. 437 Blood, unstained, 34 viscosity of, 47 Boils, 165, 358 Bordet-Gengou reaction, 67 Bothriocephalus latus, 331 Branchial cyst, 419 Brevis streptococcus, 119 Bronchial fluke, 330 Bronchiectasis, 165 Broth, 105 glucose, 181 glycerine, 181 litmus carbohydrate, 107 media, 180 neutral red, 108, 181 Buchner's tube, 162 Cachexia, blood changes in, 26 Calcareous degeneration, 389 Calcium carbonate, 259 oxalate, 259 Calculous anuria, 220 Callosity, 391 Calmette's reaction, 173 Cambridge microtome, 429 Cammidge's method of estimating fats, 321 pancreatic reaction, 273 Capsules, 103 to stain, 162 Carbol fuchsin, 187 -thionin tissue staining, 432 -thionin, to stain with, 149 Carboluria, 225 Carbonic oxide poisoning, 92 Carboxyhaemoglobin, 92 Carbuncles, 358 Carcinoma, 403 blood changes in, 19 columnar-celled, 410 encephaloid, 404 of stomach, 295 scirrhous, 404 spheroidal-celled, 408 squamous-celled, 404 Cardiac failure, blood changes in, 23 Casts, 253 amyloid, 255 cellular, 254 granular, 254 hyaline, 255 prostatic, 250 uratic, 253 Cell, endothelial, 192 endothehoid; 377 epithelioid, 378 giant, 380 giant, of Virchow, 386 lymphoid, 191, 377 malignant, 193 plasma, 378 Cell-nest, 405 Cellular casts, 253 CeUulitis, 118 Centrifugal machine, 63 Cerebro-spinal fluid, 203 Wassermann reaction in, 69, 77 Cestoda, 329, 331 Chancre, 142 soft, 278 Charcot-Leyden crystals, 366 Chicken-pox, blood changes in, 20 Children, blood changes in, 26 Chlorides, estimation of, 272 Chloroma, 15 Chlorosis, 8, 98 Cholsemia, 95 congenital family, 28 Cholelithiasis, 296 Cholera, 141 red reaction, 141 vibrio, 105, 140, 343 Cholesterin crystals, 307 Chondroma, 396 Chorion epithelioma, 417 Chronic gastritis, 296 inflammation, blood changes in, 21 Chylous fluid, 203 Gitreus staphylococcus, 115 Clonorchis sinensis, 329 Cloudy swelling, 387 Coagulation time, 46 Coffin-lid crystal, 258 Coli communis bacillus, 137 Colitis, ulcerative, 137 Colloid degeneration, 388 Colon bacillus in urine, 137, 279 Colorimeter, Duboscq, 270 Colour index, 4 Columnar-celled carcinoma, 410 Comma bacillus, 140 Complement, 66 fixation test in — ■ gonorrhoea, 65 hydatid disease, 65 tuberculosis, 65 Condenser, paraboloid, 161 Congenital family cholsemia, 28 syphilis, Wassermann re- action in, 69 Coniferin, 181 Conjunctival sac, 347 Conjunctivitis, angular, 133 diplo - bacillary, 133, 348 membranous, 349 Cornea, 350 Corneal ulcer, 350 Cornet's forceps, 147 Corpuscles, red, 3 white, 4 Cover-glasses, to clean, 42 Crab louse, 353 438 INDEX. Creatine, 269 Creatinine, 269 Gulex fatigans, 91 Cultural characters, 104 Culture, blood, 81 plate, 106 slope, 106 stab, 106 Cultures, anaerobic, 162 Curschmann's spirals, 365 Cylindroma, 401 Cysticerus bovis, 333 cellulosce, 333 Cystine, 259 Cystoma, 419 Cysts, 209, 418 branchial, 419 degeneration, 420 dermoid, 211, 410 hydatid, 209, 193 mesenteric, 211 other abdominal, 211 ovarian, 211 pancreatic, 210 ranula, 419 renal, 211 retention, 419 retroperitoneal, 211 sebaceous, 419 Cyto-diagnosis, 191 Decalcification of tissue, 428 Decalcifying fluid, 428 Deciduoma malignum, 417 Degeneration, 387 calcareous, 389 colloid, 388 fatty, 389 granular, 10 hyaline, 388 lardaceous, 388 polychromatophilic, 4 Dental osteoma, 397 Dentium, spirochceta, 143 Dermoid cyst, 211, 419 Desmoid test, 299 Dextrose, 181 Diabetes, 220 insipidus, 220 Diacetic acid, 230 Diagnosis, agglutinins in, 49 Wassermann reaction in, 68 Diastase, 210 Dibothriocephaloidea, 331 Differential count, 45 Diphtheria, 129, 288 bacillus, 109, 127 nasal, 363 Diphtheroid bacillus, 109, 128 Diplo-bacillary conjunctivitis, 348 Diplococcus intracellularis meningi- tidis, 121 Frankel's, 116 lanceolatus, 116 rheumaticus, 119 Diplogonoporus grandis, 332 Dorset's egg medium, 157, 184 Dracunculus, 354 Duboscq colorimeter, 270 Ducrey's bacillus, 278 Duodenal ulcer, 31, 296 Dysentery, 137 amoebic, 340 bacillus, 137 Widal reaction in, 49 Dyspepsia, 289 ECCHONDROMA, 396 Edestin method, 297 Emphysematous gangrene, 131 Empyema, 117, 196 Encephaloid carcinoma, 404 Enchondroma, 396 Endemic haemoptysis, 368 Endocarditis, infective, 81, 119, 164 Endothelial cell, 192 Endothelioid cell, 377 Endothelioma, 401 Endotoxins, 167 Entamoeba coli, 340 histolytica, 340 Enteritidis bacillus, 136 sporogenes bacillus, 107 Enteritis, infantile, 137 Envelope crystals, 259 Eosin, 188 Eosinophil cell, 5, 193 Eosinophilia, 19 Epithelial cells in urine, 251 Epithelioid cell, 378 Epithelioma, 404 chorion, 417 Epulis, 394 Erysipelas, 118, 357 Erythremia, 23, 24 Erythrasma, 355 Esbach's albuminometer, 238 reagent, 238 Estimation of chlorides, 272 phosphates, 271 purines, 268 sulphates, 272 uric acid, 266 Ewald's test meal, 290 Examination of throat, 129 Exudates, 199 tuberculous, 198 Eyre's scale, 181 Fcecalis, streptococcus, 118, 119 Faeces, abnormal ingredients, 317 albumin in, 321 INDEX. 439 Faeces, amount of, 316 bacteriology of, 341 banana fibres in, 317 bile in, 318 chemical examination of, 318 colour of, 316 consistency of, 317 crystals in, 327 elastic fibres in, 326 epithelium in, 327 fat in, 321 gall-stones in, 318 intestinal sand in, 318 microscopical investigation of, 325 mucin in, 324 mucous casts in, 317 muscle fibres in, 326 naked- eye examination of, 316 occult blood in, 318 odour of, 317 peptone in, 324 tubercle bacilli in, 157 Fasciolopsis buski, 329 Eat, estimation of, Cammidge's method, 321 in fseces, 321 Fatty degeneration, 389 Favus, 146 Fehling's solution, 217 Fever, relapsing, 90 Fibrin, 3, 265 Fibro-adenoma, 393 Fibroblast, 378 " Fibroids." 394 Fibroma, 394 Fibro-myoma, 394 Fibro-neuroma, 394 Filaria diurna, 91 noclurna, 90 perstans, 91 Filarise, 255 Filariasis, 90 FilariidEe, 335 Finkler-Prior vibrio, 141, 344 Fish tuberculosis, 125 Fixation of tissues, 426 Flagella, to stain, 163 Flea, 354 Flexner's bacillus, 137 serum, 176 Fluid, cerebro-spinal, 203 chylous, 203 decalcifying, 428 opalescent, 202 pericardial, 201 pleural, 196 pseudochylous, 203 spermatocele, 208 synovial, 207 Zenker's, 428 Follicular impetigo, 357 Follicular tonsilitis, 288 Forceps, 147 Cornet's, 147 Frankel's diplococcus, 116 Free hydrochloric acid, 293 iron in section, 433 Friedlander's pneumo-bacillus, 370 Fungus, ray, 144 Fusiform bacillus of Vincent, 143 Gaertner's bacillus, 136 Gall-stones, 306 Gangrene, emphysematous, 131 pulmonary, 367 Gastric juice, 289 ulcer, 296, 311 Gastritis, chronic, 296 Gelatin, 106, 183 Gentian violet, 187 Gerhardt's reaction, 244 Gerrard's method, 242 solution, 243 ureometer, 232 Giant cell, 380 of Virchow, 386 Giemsa's stain, 160, 187 Glanders, 133, 384 bacillus, 110 Glioma, 399 Globulin, 206 _ Glossina morsitans, 88 palpalis, 88 Glucose broth, 181 in urine, 241 tests for, 216 Glycerine broth, 183 Glycogensemia, 94 Glycosuria, 243 Glvcuronic acid, 247 Gmelin's test, 96, 223 Goat's milk, 120 Gonococcus, 82, 121, 276 Gonorrhoea, 122, 278 blood changes in, 21 complement fixation test, 65 Gonorrhoeal arthritis, 164 Gracilis spirochceta, 143 Gram's iodine, 187 method, 103, 150 in section, 432 Granular bodies, 141 casts, 254 degeneration, 10 vaginitis, 114 Granuloma, 375 Gravel, 257 Griinbaum-Widal reaction, 49 Guiac test, 222 Guinea-worm, 354 Gum sections, 422 440 INDEX. Gum solution, 422 Giinzburg's test, 291 Hemagglutinins, 30, 48, 58 Hsemalum, 188 and eosin stain, 431 Hsemangeioma, 397 Hsematoporphyrinuria, 224 Hsemochromatosis, 352 Hsemocytometer, 39 Haemoglobin, 4, 34 reduced, 92 Haemoglobinometer, 34 Haldane's, 34 Oliver's, 36 Tallqvist's, 34 Hemoglobinuria, 222 Haemolysis, 65 Haemophilia, 28 Haemoptysis, endemic, 368 Haemorenal index, 237 Haemorrhage, blood changes in, 25 Hanging drops, 5 Harvest bug, 354 Hay fever, 363 Hay's test, 223 Hecht-Fleming modification, 75 Heller's test, 216 Histological methods, 421 Hodgkin's disease, 22, 384 Hofmann's bacillus, 128, 130 Hot-air steriliser, 177 Hyaline casts, 255 cell, 5 degeneration, 388 Hydatid cyst, 193, 209 disease, complement fixation test, 65 Hydrocele, 208 Hydrochloric acid, " active," 294 free, 294 Hyperchlorbydria, 296 Hypernephroma, 417 Hyphomycetes, 145 Idiopathic peritonitis, 314 Immunity unit, 174 Impetigo contagiosa, 357 follicular, 357 Incubator, 148 Index, opsonic, 165 Indian-ink method, 160 Indole reaction, 105 Infantile enteritis, 137 scurvy, blood changes in, 11 Infection, agglutinins as evidence of, 57 Infective endocarditis, 81, 119, 164 vaccine treatment in, 83 Inflammation, 375 acute, 376 blood changes in, 17 Inflammation, chronic, blood changes in, 21 Influenza bacillus, 132, 198 blood changes in, 22 Inoculation, prophylactic, 136 Inspissator, 178 Intestinal obstruction, 314 toxaemia, 286 Intracellular toxins, 167 Inulin, 181 Involution forms, 127 Iodine solution, 84 test, 223 Iodophilia, 94 Iodopin test, 300 Itch, 352 Ivory osteoma, 397 Jat/ndice, acholuric family, 28 Jenner's stain, 43, 45 Kala-azae, 89 Keratoma, 391 Kitasato bacillus, 133 Kjeldahl's method, 230 Klebs-Loffler baciUus, 127 Koch's old tuberculin, 173 vibrio, 344 Koch-Week's bacillus, 133 Lachrymal sac, 350 Lactic acid, 297 Lactose, 181, 246 Lardaceous degeneration, 288 Latent syphilis, Wassermann reaction in, 69 Lead poisoning, blood changes in, 26 Leiomyoma, 394, 395 Leishman's stain, 43, 45 in section, 433 Leishman- Donovan body, 89 Leishmania, 89 Lepra bacillus, 126 Leprosy, 126, 363, 383 Leptus autumnalis, 353 Leucine, 260 Leucocytes, 5 enumeration of, 40 origin of, 6 Strong's method for, 40 Thoma-Zeiss method for, 41 Leucocytosis, 4, .17 Leucopenia, 21 Leukaemia, lymphoid, 13, 15 myeloid, 7, 13 Lipaemia, 93 Lipase, 210 Lipoids, 67 Lipoma, 396 Lipuria, 227 INDEX. 441 Litmus carbohydrate broth, 107, 181 milk, 107, 183 solution, 181 Liver abscess, 269, 304 flukes, 329 Lobar pneumonia, 117 Loffler's blood serum, 185 methylene blue, to stain, 186 Lumbar puncture, 203 Lung puncture, 201, 372 Lymphangeioma, 397 Lymphangitis, 116, 358 Lymphocytes, large, 5 small, 5, 191 Lymphoid cell, 191, 377 leukaemia, 13 chronic, 15 Lymphoma, 394 MacConkey's medium, 108, 182 Madura foot, 145, 355 Malaria, 86 blood changes in, 22 Malarial parasites, 86 Malignant cells, 193 endocarditis, 81 oedema bacillus, 149 pustule, 130 Mallei bacillus, 133 Mallein, 173 Malta fever, 120 agglutinins in, 56 Maltose agar medium, 145 Mannite, 181 Mast cell, 5 Mayhew's ureometer, 234 " Measled " pork, 332 Measles, blood changes in, 22 Meckel's cartilage, 396 Media, standardisation of, 181 sterilisation of, 176 Medium, blood serum, 184 broth, 180 Dorset's egg, 157, 184 MacConkey's, 182 maltose agar, 145 ox bile, 185 potato, 184 Megaloblasts, 4, 10 Melaninuria, 225 Melanotic sarcoma, 416 Meningitis, 117, 206 Meningococcus, 121, 205 Metallic poisons, blood changes in, 26 Metchnikoff's vibrio, 141 MethsemoglobinEemia, 92 Methylene blue, 186 Loffler's, 187 Micrococcus catarrhalis, 120 melitensis, 120 Microscope, 32 ultra, 161 Microsporon Audouini, 145, 354 furfur, 146, 355 minutissimum, 355 Microtome, Cambridge, 429 Williams's, 422 Miliary tubercle, 379 Milk, goat's, 183 litmus, 183 Millon's reagent, 241 Minimum lethal dose, 174 Moeller's timothy-grass bacillus, 125 Mole, 398, 416 Molluscum contagiosum, 387 fibrosum, 386, 394 Morax-Axenfeld bacillus, 133 Morbus cordis, congenital, blood changes in, 24 Morgan's No. 1 bacillus, 343 Mucin, 241 Mucoid degeneration, 387 Mucous polyp, 394 Mulberry calculi, 262 Murexide test, 264 Myeloblast, 6, 13 Myelocytes, 6 Myeloid leuksemia, 4, 7 acute, 13 sarcoma, 414 Myoma, 395 fibro-, 394 Myxoma, 396 Myxo-sarcoma, 396 NiEVi, 397 Nagana, 88 Nasal catarrh, 362 diphtheria, 363 Nasgar, 109, 183 Neisser's method of staining, 159 Nematodes, 334 Nephritis, acute, 231 chronic interstitial, 231 Neuro-fibroma, 394 Neuroma, 400 plexiform, 400 Neutral red broth, 108, 181 Neutrophils, polynuclear, 192 Nits, 353 Nonne and Apelt's method, 206 Normoblasts, 4 Nylander's reagent, 217 test, 217 Oat- celled sarcoma, 413 (Edema, malignant, bacillus of, 140 Oidium albicans, 146 Oleic acid agar, 183 Oligocythsemia, 24 Oncosphere, 331 Opalescent fluids, 203 Ophthalmia neonatorum, 123 Oppler-Boas bacillus, 302 442 INDEX. Opsonic index, 59, 167 modification of, 63 value of, 62 Opsonins, nature of, 58 Oral sepsis, 286 Orcinol reaction, 247 Organisms, agglutinins as a test for, 57 Oriental sore, 90 Osazone reaction, 246 Osier's disease, 24 Osteo-arthritis, 286 Osteoma, 397 dental, 397 ivory, 397 Osteomyelitis, 116 Osteosarcoma, 412 Ovarian cysts, 211 Ox bile medium, 185 Oxygen content of blood, 98 Oxyhaemoglobin, 92 Oxyphil, coarsely granular, 5 finely granular, 5 Oxyuris vermicularis, 339 Pallida, spirochoeta, 141 Pallidum, treponema, 141 Pancreatic efficiency, 303 reaction, 273 Papillary adenoma, 393 Papilloma, 391 Paraboloid condenser, 161 Paraffin sections, 426 Paragonimus westermani, 330, 368 Parasitology of the blood, 81 Parasyphilis, Wassermann reaction in, 69 Paratyphoid bacillus, 136 infections, agglutinins in, 55 Parotid tumour, 401 Pediculi, 353 Pemphigus neonatorum, 357 Pentose, 246 Pepsin, 297 Pericardial fluid, 201 Peritoneal fluid, 202, 311 Peritonitis, 117 idiopathic, 314 post-operative, 314 tuberculous, 315 Pernicious anaemia, 7, 8, 99 Pertenuis, spirochceta, 143 Pertussis, 132 Pestis, bacillus, 134 Petri dish, 154, 185 Pettenkofer's test, 224, 319 Pfeiffer's bacillus, 132 reaction, 110 Phloroglucinol reaction, 246 Phosphates, amorphous, 258 Phosphates, estimation of, 271 stellar, 258 triple, 258 Pigmentation, 389 Pipettes, Strong's, 38 to clean, 42 Pityriasis versicolor, 146 Plague, 134 bacillus, 372 Plasma cell, 378 Plate cultures, 106 Platinum wire, 147 Pleural fluid, 196 Plexif orm neuroma, 400 Pneumococcus, 82, 116, 196, 370 Pneumoconiosis, 368 Pneumonia, blood changes in, 17 lobar, 117 Poikylocytosis, 9 Poisoning, carbonic oxide, 92 Poisons, lead, blood changes in, 26 metallic, blood changes in, 26 Polar staining, 134 Polychromatophilic degeneration, 4 Polycythemia, 4, 23 splenic, 11, 24, 99 Polvnuclear neutrophils, 192 Polypi, 393 Post-operative peritonitis, 314 Potato medium, 184 Prior vibrio, 141 Proglottis, 331 Prophylactic inoculation, 136 use of vaccines, 172 Prostatic casts, 250 Prostatitis, 278 Proteids in urine, 237 Protein, Bence-Jones, 241 Proteoses, 240 Proteus, bacillus, 139 Psammoma, 401 Pseudochylous fluid, 203 Pseudo-glioma, 350 Puerperal septicaemia, 119, 164 Pulmonary abscess, 367 gangrene, 367 Puncture lumbar, 203 lung, 201 Purines, estimation of, 268 Purinometer, Walker-Hall, 269 Purpura, blood changes in, 30 Pus in urine, 250 tubercle bacilli in, 157 Pyaemia, 116 Pyocyaneus, bacillus, 139 Pyogenes, streptococcus, 118 Pyorrhoea alveolaris, 119, 165, 287 Pyosalpinx, 123, 313 Rabin owitch's butter bacillus, 125 Raffinose, 181 Ranula, 419 INDEX. 443 Ray fungus, 144 Reaction, biuret, 325 Calmette, 173 cholera red, 105, 141 Gerhardt's, 244 indole, 105 orcinol, 247 osazone, 246 pancreatic, Cammidge's, 275 Pfeiffer's, 110 phloroglucinol, 246 Salkowski's, 307 Von Pirquet, 173 Reagent, Millon's, 241 Nylander's, 217 Reagents, staining, to prepare, 186 Recurrentis, spirochceta, 144 Red cells, 3 enumeration of, 37 fragility of, 46 sensitised, 66 Strong's method, 37 Thoma-Zeiss method, 38 Reduced haemoglobin, 92 Eefringens, spirochwta, 143 Relapsing fever, 90 Renal cysts, 210 tumours, 417 hypernephroma, 417 von Grawitz, 417 Rennin, 298 Retention cyst, 419 Retinal glioma, 400 Rhabdomyoma, 395 Rheumatic fever, 208 blood cultures in, 81 Rheumatoid arthritis, 208 Rickets, blood changes in, 29 Ringworm, 145, 354 Rodent ulcer, 406 Rothera's test, 245 Round-celled sarcoma, 412 SAP.OTTitA'rJD's maltose agar medium, 145 Saccharose, 181 Safranin, 187 Sahli's desmoid test, 299 Salicin, 181 Salivarius, streptococcus, 118, 119 Salkowski's reaction, 307 Salpingitis, 117 Salt formalin solution, 425 Saprophytes, 111 Saprophytic bacillus, 198 SarcinEe, 123 Sarcoma, 411 alveolar, 415 melanotic, 416 myeloid, 414 oat-celled, 413 osteo-, 412 Sarcoma, round-celled, 412 spindle-celled, 413 Scale, Eyre's, 181 Scarlet fever, blood changes in. 20 Scharlach R., 188 Schistosomum japonicmn, 331 Schmidt-Werner tube, 322 Scirrhous carcinoma, 404 Sclerosis, posterior lateral, 11 Scolex, 331 Scurvy, infantile, blood changes in, 29 Sebaceous cyst, 419 Sections, examination of, 374 free iron in, 433 Gram's method, 432 gum, 422 Leishman's stain in, 433 paraffin, 426 staining of, 430 tubercle bacilli in, 432 Sensitised red cells, 66 vaccine of Besredka, 167 Septicaemia, 82 puerperal, 119, 164 Serum, albumin, 238 anti-colon bacillus, 175 anti-diphtheritic, 175 anti-meningococcus, 176 anti -plague, 176 anti-pneumococcus, 176 anti-streptococcal, 175 anti-tetanic, 175 Flexner's, 51 globulin, 238 to obtain, 51 tube, 96 uric acid in, 96 Shiga bacillus, 137 Skin, 350 bacteriology of, 356 lesions, blood changes in, 20 Slope cultures, 106 Small lymphocytes, 191 Small-pox, blood changes in, 20 Smegma bacillus, 125 Soft chancre, 278 Solution, gum, 422 salt formalin, 425 Sorbit, 181 Spasmodic asthma, blood changes in, 20 Specific gravity of blood, 96 Spectroscope test, 223 Spectroscopic examination of blood, 92 Spermatocele fluid, 208, 209 Spermatozoa, 209, 255 Spheroidal-celled carcinoma, 408 Spindle-celled sarcoma, 413 Spirilla, 140 Spirillum obermeieri, 90 of Vincent, 143 444 INDEX. Spirochceta dentium, 143 gracilis, 143 obermeieri, 144 pallida, 141, 278 pertenuis, 143 recurrentis, 90, 144 ref ring ens, 143 Spirochetosis, 90 Spleen, 308 puncture, 89 Splenic anaemia, 11 of infants, 27 polycythsemia, 11, 24 Spores, 103 to stain, 162, 163 Sporotrichia, 355 Spring catarrh, blood changes in, 20 Sputum, 363 bacteriology of, 368 elastic fibres in, 364 fibrinous casts in, 366 tubercle bacilli in, 155, 369 Squamous-celled carcinoma, 404 Stab cultures, 106 Stain, Giemsa's, 160, 187 hsemalum and eosin, 431 Jenner's, 43, 45 Leishman's, 43, 45 Van Gieson's, 188, 431 Staining capsules, 162 fat in tissues, 425 flagella, 163 polar, 134 reagents, to prepare, 186 sections, 430 spores, 162 with carbol-thionin, 149 with Gram's method; 150 with Loffler's methylene blue, 159 with Neisser's method, 159 Standardisation of media, 181 Staphylococci, 115 Staphylococcus albus, 115 aureus, 115 citreus, 115 Steam steriliser, 178 Stellar phosphates, 258 Stercobilin, 316, 319 Sterilisation of media, 177 Steriliser, hot-air, 177 steam, 178 Stock vaccines, 165 Stomach, carcinoma of, 295 Streptococci, 118 anginosus, 119 brevis, 119 fcecalis, 118, 119 pyogenes, 82, 118 salivarius, 118, 119 Streptococcus, 118 Strong's method for red cells, 37 Strong's pipettes, 38 Strongylidse, 336 Strongyloides intestinalis, 334 Sub-cultures, to make, 153 Subtilis, bacillus, 123 Suipestifer, bacillus, 136 Sulph-hsemoglobinsemia, 93 Sulphates, estimation of, 272 Surra, 88 Sycosis, 358 Synovial fluid, 207 Syphilis, 142, 382 blood changes in, 21 congenital, blood changes in, 29 Syphilitic antigen, 72 Syringo-myelia, 399 Taenia echinococcus, 333 saginata, 333 solium, 332 Tseniidse, 331 Teratoma, 418 Test, albumin, 215 complement fixation, in gonor- rhoea, hydatid disease, tuber- culosis, 65 desmoid, 299 Sahli's, 299 Gmelin's, 96, 223 guiac, 222 Hay's, 223 iodine, 223 iodopin, 300 murexide, 264 Nvlander's, 217 Pettenkofer's, 224, 319 Rothera's, 245 Sahli's desmoid, 299 Uffelmann's, 297 Test meal, 289 blood in, 299 Ewald's, 290 Tetanus bacillus, 110, 130 toxin, 130 Thermos flask, 148 Thoma-Zeiss method for red cells, 38 Thread worms, 255 Throat, examination of, 129 Thrush, 146, 287 Tissues, decalcification of, 428 fixation of, 426 staining fat in, 425 Toison's fluid, 40 Tonsilitis, 288 Topfer's solution, 291 Total acidity, 293, 294 Toxins, intracellular, 167 Trachoma, 349 Transudates, 199 Trematoda, 329 Treponema pallidum, 141 INDEX. 445 Trichineha spiralis, 336 Trichocephalus dispar, 335 Trichophyton megalosporon ectothrix, 355 endothrix, 145, 354 Trichotrachelidae, 335 Triple phosphates, 258 Tropical abscess, blood changes in, 19 Trypanosoma, brucei, 88 evansi, 88 gambiense, 88 lewisi, 88 Trypsin, 210 Tube, Buchner's, 162 Wright's, 51 Tubercle bacillus, 123 in fseces, 157 in pus, 157 in section, 432 in sputum, 155 in urine, 157, 279 Tuberculides, 360 Tuberculin, 167 old, Koch's, 173 Tuberculosis, blood changes in, 19, 21 complement fixation test, 65 histology of, 379 Tuberculous exudates, 198 peritonitis, 315 Tumours, malignant — Alveolar sarcoma, 415 Carcinoma, 401 Chorion epithelioma, 417 Columnar-celled carcinoma, 410 Deciduoma malignum, 417 Encephaloid carcinoma, 404 Endothelioma, 401 Epithelioma, 404 Hypernephroma, 417 Melanotic sarcoma, 416 Myeloid sarcoma, 414 Oat-celled sarcoma, 413 Osteo-sarcoma, 412 Rodent ulcer, 406 Round-celled sarcoma, 412 Sarcoma, 411 Scirrhous carcinoma, 404 Spheroidal-celled carcinoma, 408 Spindle-celled sarcoma, 413 Squamous-celled carcinoma, 404 Teratoma, 418 von Grawitz tumour, 417 Tumours, simple — Adenoma, 392 Angeioma, 397 Branchial cyst, 419 Callosity, 391 Chondroma, 396 Ecchondroma, 396 Enchondroma, 396 Tumours, simple — Epulis, 394 Fibro-adenoma, 393, 394 Fibro-myoma, 394 Fibro-neuroma, 394 " Fibroid," 394 Fibroma, 394 Glioma, 399 Hsemangeioma, 397 Ivory osteoma, 397 Keratoma, 391 Leiomyoma, 394, 395 Lipoma, 396 Lymphangeioma, 397 Lymphoma, 394 MoUuscum fibrosum, 394 Mole, 398 Mucous polyp, 394 Myoma, 395 Myxoma, 396 Myxo-sarcoma, 396 Nsevus, 397 Neuro -fibroma, 394 Neuroma, 399 Osteoma, 397 Papilloma, 391 Parotid tumour, 401 Plexiform neuroma, 399 Polypus, 393 Retinal ghoma, 399 Rhabdomyoma, 395 Wart, 391 Typhoid bacillus in urine, 282 carriers, 135, 306 fever, 135, 161, 172 agglutinins in, 49 blood changes in, 22 blood cultures in, 83 ulcer, 312 Typhosus, bacillus, 135 Tyrosine, 260 Ubtelmann's test, 297 Ulcer, duodenal, 296, 312 gastric, 296, 311 typhoid, 312 Ulcerative endocarditis, 81 Ultra-microscope, 161 Uncinaria, 336 Urates, acid ammonium, 257 amorphous, 256 Uratic casts, 253 Urea, 231 Ureometer, Gerrard's, 232 Mayhew's, 234 Ureteric catheterisation, 213 Urethritis, 122 Uric acid, 257 estimation of, 266 in serum, 96 Urinary calculi, 262 casts, 253 446 INDEX. Urinary deposits, 248 Urine, albumin in, 215 amount of, 220 bacillus proteus in, 282 bacteriology of, 276 bile in, 223 blood in, 221, 249 casts in, 253 colon bacillus in, 279 colour of, 221 deposits in, 218 eosin in, 226 epithelial cells in, 251 glucose in, 216, 241 measurement of, 214 methylene blue in, 226 naked-eye appearance of, 214 phosphates in, 226 proteids in, 237 pus in, 250 reaction of, 214 routine examination of, 214 specific gravity of, 214 tests for albumin, 215 tubercle bacilli in, 157, 279 typhoid bacilli in, 282 urates in, 226 Urinometer, 214 Urobilin, 224 Urostealith, 265 Vaccines, 164 diagnostic use of, 172 method of preparation, 168 of Besredka, 167 prophylactic use of, 172 sensitised, 167 stock, 165 treatment in infective en- docarditis, 83 Vaginitis, 123 granular, 114 Van Gieson's stain, 188, 431 Vaquez's disease, 24 Vibrio, cholera, 140, 343 Finkler-Prior, 141, 344 Koch's, 344 Metchnikoff, 141 Vincent's angina, 144, 288 fusiform bacillus, 143 spirillum, 144 Viscera, bacterial investigation of, 161 Volhard process, 273 Vomit, 301 Von Grawitz tumour, 417 Von Jaksch's anaemia, 27 Von Pirquet's reaction, 173 Walker-Hall purinometer, 269 Wart, 391 Wassermann's reaction, 65 in acquired syphilis, 68 in cerebro-spinal fluid, 69, 77 in congenital syphilis, 69 in diagnosis, 68 in latent syphilis, 69 in parasyphilis, 69 in response to treatment, 70 technique of, 71 Whetstone crystal, 257 Whip-worm, 335 Whitlow, 358 White corpuscles, 4 Whooping-cough bacillus, 132 Widal's reaction, 49 Williams's microtome, 422 Wool-sorters' disease, 131 Xanthine, 265 Xerosis bacillus, 347 Yaws, 360 Zenker's fluid, 428 Ziehl-Neelsen method, 104, 156 — BRADBURY, AGNEW, & CO. LD., PRINTERS, LONDON AND TONBRIDGE. COLUMBIA UNIVERSITY LIBRARIES This book is due on the date indicated below, or at the expiration of a definite period after the date of borrowing, as provided by the library rules or by special arrangement with the Librarian in charge. DATE BORROWED DATE DUE DATE BORROWED DATE DUE m 1 71950 C28(842)MSC Tva fed (o\ P\<\