rAV/AWAVr?mViTrt5S'r»'AV4fAWi*fla CORNELL UNIVERSITY THE Sflomer Hetenttarg Hibrarg FOUNDED BY ROSWELL P. FLOWER for the use of the N. Y. STATE VETERINARY COLLEGE 1897 Digitized by Microsoft® CORNELL UNIVERSITY UBRARY 3 1924 104 224 856 Digitized by Microsoft® This book was digitized by Microsoft Corporation in cooperation witli Cornell University Libraries, 2007. You may use and print this copy in limited quantity for your personal purposes, but may not distribute or provide access to it (or modified or partial versions of it) for revenue-generating or other commercial purposes. Digitized by Microsoft® Digitized by Microsoft® Digitized by Microsoft® A MANUAL OF CLINICAL LABORATORY METHODS JOHN BENJAMIN NICHOLS, M.D. IQ Cliargp ot Clinical Laboratory, (jarfleld Hospital ; Hsematologist to Columbian University Hospital ; Professor of Normal Histology In Medical Department of Colum- bian University, Wasbington, D. C. ILLUSTRATED NEW YORK WILLIAM WOOD AND COMPANY MDCCCCII p Digitized by Microsoft® Ho. \Zli,a Copyright, 1901, \ By WILLIAM WOOD AND COMPANY. M6/ Digitized by Microsoft® ^0 m^ WLite ANNIE GLEDHILL NICHOLS Digitized by Microsoft® Digitized by Microsoft® PREFACE. It is the purpose of this work to present in practical and systematic form the most important laboratory methods em- ployed in clinical medicine. Covering established methods, it cannot profess to present much that is new or original, or to cover a field not already occupied, but must base its raison d'etre on the collection of numerous technical procedures within small compass and on presentation of the subject in a manner conven- ient and clear to students and practitionere of human medicine, to whom it is hoiked the woi-k may prove of use. John Benjamin Nichoi.s. Wasiilngton, D. C, December, 1901. Digitized by Microsoft® Digitized by Microsoft® CONTENTS. PAGE I. Introduction 1 11. Laboratory equipment and general methods. . 4 III. The blood, ... . 13 Constitution of the blood, . . 13 Technique of blood examination. 43 IV. The stomach, .... 72 Characters of the stomach contents, . . 72 Examination of the stomacli contents, . 79 Examination of motor power, 95 Examination of absorptive power, . . 96 V. The fseces and intestinal discharges. 98 Composition of the fseces, . 98 Physical characters of tlie fsBccs, 114 Examination of the fajces, . . 115 VI. The sputum, ..... . 120 VII. The urine, . . . ItJo Composition of the urine. 133 Physical characters of urine. . 173 Examination of urine, . 178 nil. Miscellaneous secretions and body fluids, 210 IX. Pathological fluids, . . . . ooo X. Calculi, ... ... 331 XI. Parasites, 340 Animal parasites 240 Vegetable parasites, .... 245 XII. Clinical bacteriology, 253 Cultural methods . 254 Microscopical methods, 366 Animal inoculation, .... 270 Characters of pathogenic bacteria, . 373 ail. Autopsies, . 383 Digitized by Microsoft® Digitized by Microsoft® A MANUAL OF CLINICAL LABORATORY METHODS. I. INTRODUCTION. In the clinical practice of medicine there are two distinct and equally important divisions : diagnosis, the determination of the nature of the disease condition present, and therapeutics, the treatment of that condition. The processes and nature of dis- ease are often so obscure, and their recognition is so essential as a guide to proper treatment, that the clinician needs to utilize all information that may throw light on his cases. Various classes of data are available for diagnostic purposes, such as the history of the case, the subjective phenomena experienced and commu- nicable by the patient, and the objective characteristics exhib- ited by the patient and determinable by various methods of in- vestigation. In the latter class of data are included the results derived by examination of fluid and solid materials obtainable from the patient's person. Such materials it is the province of the clinical laboratory to elaborate. The chemical, microscopical, bacteriological, and other methods utilized in this kind of work are very diverse in their nature, principles, and technique ; yet as all these methods have to be applied to the examination of the same material, and the assemblage of the requisite reagents and apparatus naturally results in the equipment of a laboratory convenient for working purposes, it comes about that the worker in the clinical labora- tory combines the functions of the cheniist, the microscopist, and the biologist, and this entire field forms quite a distinct depart- ment of medical work. The field of the clinical laboratory is, then, the investigation by appropriate methods (chiefly chemical, microscopical, and bacteriological) of such fluid and solid materials, secretions, 1 Digitized by Microsoft® 2 A MANUAL OF CLINICAL LABORATORY METHODS. ejecta, tissues, and the like, as are obtainable from the diseased subject, for the purpose of obtaining whatever data may aid in ascertaining the nature and course of the disease processes pres- ent. It is the object of this treatise to present, for the use of the student and worker, the most important practical clinical labora- tory methods at present in general use, together with a sufficient exposition of the principles on which they are based, to enable them to be intelligently and rationally understood and their re- sults to receive the proper clinical interpretations. In the fol- lowing pages, in each branch of the subject the composition and general characters of the material to be examined are first con- sidered, so as to place the technique on a rational basis and per- mit clinical interpretation to be made of the conditions and vari- ations found in normal and abnormal circumstances ; secondly, the technique and methods of examination are given by which' the various facts of diagnostic usefulness are elicited. The practical usefulness of the results obtained by clinical laboratory methods should be estimated at their proper value, and neither overestimated nor underrated. In many cases the laboratory findings are .essential to the making of the diagnosis. In other cases they furnish valuable or even pathognomonic data, which yet may not be essential to the making of a diagnosis with reasonable certainty. In many cases the information elicited may be of no positive or definite significance, or significant only in a negative way. On the one hand, laboratory work should not be regarded with excessive enthusiasm, or with the expectation of pathognomonic results in every case, or to be an invariably short, easy, and sure road to diagnosis. On the other hand, these methods should not be underrated as merely academic and im- practical in character ; nor does the laboratory worker desire to be regarded and treated as a visionary enthusiast who enjoys spending his time and labor, regardless of other compensation, in examining specimens patronizingly handed him. Properly utilized the field of usefulness of clinical laboratory methods is very large, but they must not be expected to take the place of other diagnostic aids. The methods are largely of recent devel- opment, and the field is still growing both in technical proced- ures and clinical usefulness ; a well -equipped clinical laboratory is now an essential adjunct to every modern hospital. Clinical laboratory findings once obtained must be properly Digitized by Microsoft® INTRODUCTION. 3 interpreted in order to utilize them advantageously and to avoid erroneous conclusions. Negative results from single or a few- examinations do not necessarily disprove the existence of the disease in question ; thus, failure to find tubercle bacilli, the mal- arial parasite, or the Widal reaction does not necessarily prove the absence of tuberculosis, malaria, or typhoid fever. Only repeated negative results in such cases under favorable condi- tions can be safely given a negative interpretation. Positive findings are much more definite in their significance. The results should be taken in their clinical connections, and all data obtain- able, subjective symptoms and results of physical examination, as well as microscopical and chemical findings, should be consid- ered together in making diagnosis. Laboratory work is an es- sential part of clinical medicine, precisely corresponding to phys- ical examination, and should be given its place, no more and no less, in the clinical whole. The materials available for examination by clinical laboratory methods during life and in time for useful therapeutic indica- tions are limited to the fiuid and solid substances practically ob- tainable, including most of the secretions and excretions of the body, ejecta, pathological fluids, and small portions of solid tis- sues. Some of these materials are obtainable with ease and in ample quantity, as urine, faeces, sputum ; some are easily obtain- able, but only in small amount, as blood ; some are obtainable in sufficient quantity but with considerable difficulty or discomfort, as cerebro-spinal fluid, stomach contents, portions of tissues. In addition to the substances obtainable during life in time for ther- apeutic usefulness, after death and to a certain extent after oper- ations any of the materials of the body are available for exami- nation for the retrospective light which may be thrown on the case. Histological technique, or the examination of solid tissues, constitutes an important part of clinical laboratory work. It alone is an extensive subject, and from lack of space its consid- eration has necessarily been omitted here. Digitized by Microsoft® II. LABORATORY EQUIPMENT AND GENERAL METHODS. The equipment of the clinical laboratory must be conditioned on the available resources as to space and funds. If necessary, very satisfactory work can be done within small space. For micro- scopical purposes a clear northern exposure, unobstructed by trees, buildings, or other objects, is desirable. Working-tables, shelf room, drawers, sinks, and water and gas supply should be provided. A variety of apparatus, reagents, and supplies is nec- essary. Among the most important items are the microscope, microtome, freezing apparatus, centrifuge, bacterial incubator, hot-air and steam sterilizers, delicate scales. It is not necessar- ily the most expensive and most complicated apparatus that is the most desirable. With ordinary ingenuity much useful ap- paratus can be readily improvised. The microscope should be of the highest grade, provided with iris diaphragm, Abbe condenser, triple nosepiece, two oculars, objectives of about 2 centimetres; 4 or 5 millimetres, and 2 milli- metres (oil-immersion) focal length. A mechanical stage and micrometer outfit are very desirable adjuncts. Cleanliness, order, and system in managing the laboratory are desirable for the sake of appearance and to promote and facili- tate the work. Blank forms are neat and useful to guide and facilitate the examinations, and valuable records may be kept with little labor. Reagents. — Much often depends on the reagents used being exactly right. This is especially the case with aniline stains, minute variations in whose composition or mode of manufacture often make all the difference between success and failure. One commercial make often produces results that another of the same substance does not yield. Hence in some formulae it is neces- sary to specify the maker's name to help insure the desired re- sult. For many purposes, for instance, the stains of Griibler, of Berlin, are the most reliable and satisfactory. Chemically Digitized by Microsoft® LABORATOEY EQUIPMENT AND GENERAL METHODS. 5 pure substances and accurately prepared volumetric solutions should be handled with the same scrupulous care to avoid con- tamination or alteration that is exercised with pure bacterial cultures. In preparing accurate solutions of many chemicals the water of crystallization must be taken into consideration. Perfect and well-formed crystals, dry, are the only uniform standard for direct weighing ; in such crystals as contain a definite proportion of water of crystallization the weight of the active principle is only a part of the full weight of the crystals. In calculating formulse for solutions this factor is taken into account. Sub- stances that deliquesce or effloresce, that is, absorb water or give it off" (in either case losing their crystalline form), can not be weighed with any pretence to accuracy, since the amount of wa- ter present is an unknown and indefinite factor. Exact volu- metric solutions of such substances (sodium hydrate, for in- stance) must be prepared by standardization with solutions of other substances that can be more directly made. Cover-glasses. — Microscopical cover-glasses are sold in four thicknesses, No^. 0, 1, 2, and 3. Of these No. 1, between .12 and .17 millimetre in thickness, is thin enough for oil -immersion lenses, and is the best for general purposes. No. 2 can be used where the oil-immersion lens is not required. No. is too fragile for use and of no advantage over No. 1. Sizes of 18 to 21 milli- metres, square or circular, are the most convenient ; the squares are cheapest. Cleaning Glassware. — It is of the greatest importance that the glassware used in the laboratory, especially cover-glasses and slides for microscopical purposes, should be absolutely clean. There is no better cleaning fluid for glass than the following : Potassium bichromate 10 Sulphuric acid, commercial 10 Water 100 The glassware, just purchased or soiled by use, is left in this fluid for twenty-four hours, or for an indefinite time, and then thoroughly rinsed with water. Slides and cover-glasses should then be kept in alcohol, in large-mouthed jars; as needed for use they are dried with clean gauze. Extemporaneous microscopical work is largely required in the Digitized by Microsoft® 6 A MANUAL OF CLINICAL LABORATORY METHODS. laboratory. Small solid masses may be teased iu a watch-glass or on the slide, or may be crushed aud spread out between the cover-glass and slide, until they are thin or divided enough for microscopical examination. Decinormal (0. 6 per cent) sodium- chloride solution is generally used as a medium for such mani- pulations and for temporarily mounting the specimens for exam- ination ; water or glycerin may also be used. In examining clear colorless unstained specimens in this way the substage illumi- nation should be dimmed to the point of the best effect by con- tracting the iris diaphragm or lowering the coudeusej'. Granular material may be rubbed up with a suitable fluid so as to dilute and scatter the granules sufHcientlv. Fluids are examined by placing a drop or two on the slide and covering with a cover-glass. To preserve fluid mounts for a few hours a rim of vaseline may, with a camel's-hair brush, be applied around the dried edges of the cover-glass so as to prevent evaporation. Undissolved solid elements or particles suspended in liquids, if not sufficiently concentrated, may be collected by allowing them to subside as a sediment after standing a long enough time, or by throwing them down with the centrifuge. When thus concen- trated, one end of a glass tube with the other end closed by the finger is passed to the sediment in the bottom of the liquid, which then on removal of the finger above flows up into the tube; on replacing the finger the material within the tube can be removed and deposited on a slide for examination. The amount of sedi- ment allowed to flow into the tube can be readily controlled by the finger above, the finger and end of the tube being dry. Micro-chemical tests can be made by adding reagents to the material under examination and watching the reaction under the microscope. A drop of each of the materials used may be mixed on the slide and the cover then api^lied ; or with the material being examined already under the cover-glass a drop of the re- agent is placed at the edge of the cover-glass and allowed to flow under. In this manner starch granules may be demonstrated by the addition of iodine solution, fat brought out by Sudan III., acetic acid added to clear up cell granules, etc. A common method of extemporaneous microscopical exami- nation is by means of cover-glass preparations. These are pre- pared by smearing or spreading a small portion of the material Digitized by Microsoft® LABORATORY EQUIPMENT AND GENERAL METHODS. 7 (as pus, etc. ) over the surface of a cover-glass with a platinum loop, or otherwise, and allowing it to dry in a thin film ; or a drop of the material may be placed on one cover-glass, another glass immediately placed over it, and the material pressed out or allowed to spread out in a thin layer between the two cover- glasses; the glasses are then slid (not lifted) apart, leaving a thin film on one side of each cover-glass. When dried, such cover-glass preparations may be stained at once, or kept indefi- nitely before further treatment. The next step after drying the Mms is fixation ; not, as many novices think, to make the film adhere to the glass, but to coagulate the albuminous principles so that the cells will stain well. Various methods of fixation are employed for different purposes. Fixation is most frequently done by "flaming" the cover-glass preparations, that is, by grasping them with forceps by one edge and then passing them three or four times at a moderate speed and at short intervals through the flame of a burner. Specimens may also be iixed with alcohol, alcohol and ether, or in numerous other ^\'ays. After fixing they are stained by appropriate methods. The whole procedure requires only a few minutes for execution. Preservation of Specimens.— For preserving specimens of or- ■ gans and tissues, for exhibition purposes or storage, a number of preservative fluids may be used. Weak solutions of formal- dehyde, 1 or 2 per cent .(= 2 to 5 per cent of formalin), are cheap, effective, and extensively iised. Alcohol of about 70 per cent strength is an excellent preservative medium ; strong- alcohol makes the specimens too hard, an excess of water macer- ates them. Other fluids are used for special purposes. At first the fluid should be changed frequently until it remains free from blood or turbidity. The specimens should not be too crowded in the fluid. Kaiserling's method of preserving gross specimens for exhibi- tion purposes has the advantage of retaining the natural color of the tissues. The method is as follows : 1. Fix for one to five days according to size, in the dark, in the following: Potassium nitrate 15 Potassium acetate 30 Formalin (40 per cent formaldehyde) 200 Water 1,000 Digitized by Microsoft® 8 A MANUAL OF CLINICAL LABORATORY METHODS. 2. Place in 80 per cent alcohol one to six hours, then in 95 per cent alcohol one or two hours. 3. Preserve, in the dark, in Potassium acetate 300 grams. Glycerin 400 c.c. Water 3,000 c.c. Weights and Measures. — The metric system is used throughout in this work. The equivalents between the Apothecaries' or Troy and the metric system are as follows : f 15,433.6 grains 1 , ^i- tiounds Trov 1 kilogram = I 2.679 pounds Troy I = approxi- \ ^J ^^^^^^ ^^^^_ exactly] 3.305 pounds avoir- j mately 1 dupois L dupois J 1 gram = exactly 15.4336 grains, or approximately i drachm. 1 centigram = " .1543 " " " i grain. 1 milligram = " .0154 " " " ^ grain. 1 ounce Troy = exactly 31.1009 grams, or approximately 31 grams. 1 drachm = " 3.8876 " " " 4 " 1 grain = " .0648 " " " 6 centigrams. 1 litre = exactly 33.8683 fluid ounces, or 3.1168 pints. 1 millilitre or cubic ) ^ . -^g ggg^ minims, centimetre ) 1 pint = exactly 473.4736 cubic centimetres. 1 fluid ounce = " 39.5296 " 1 minim = " .0615 " 1 metre = exactly 39.37 inches, or approximately 40 inches. 1 centimetre = " .3937 " " " | inch. 1 millimetre = " .0394 " " " -^ " lmicromillimetre(rt=" .000039 " " " tswtt" 1 inch = exactly .0354 metre, or approximately 35 millimetres. Degrees Centigrade (C) may be converted into degrees Fah- renheit (P) and vice versa by the following f ormulse : ^^ _ 5 (F - 33) 9 F = 5-^ + 33. 5 The specific gravity of organic fluids is ordinarily taken by means of special floating instruments on the order of a hydro- meter. For more accurate determinations, or where only a small amount of liquid is available, insufficient to float the instrument, the pyknometric method of direct weighing may be employed. This may be carried out by the use of receptacles in which a Digitized by Microsoft® LABORATORY EQUIPMENT AND GENERAL METHODS. known volume of fluid can be very accurately measured ; or the necessary receptacle may be prepared impromptu. In the latter case a piece of glass tubing of proper capacity is drawn to a fine (but permeable) point at each end. This tube is accurately weighed while empty and dry ; it is then completely filled with the fluid under examination, the outside dried, and weighed; it is then similarly filled with pure water and again weighed. In this manner the weights of an equal volume of water and the fluid tested are obtained, from wh|ch the specific gravity of the latter can be calculated by dividing its weight by that of the water. Ethereal Extracts. — Eeference will be occasionally made in this work to the extraction of substances from their solution in water by means of ether, chloroform, or other fluid not miscible with water. This is done by shaking the watery solution with ether or chloroform, etc. ; the latter will extract or take into solution certain substances from the water, and after standing the two fluids will separate. Either the water or the other fluid may then be re- moved separately with a pipette, and the desired substance thus separated ; or by placing it in a filter paper pre- viously saturated with either water or chloroform (for instance), the watery or chloroform portion respec- tively will filter through alone. Centrifugal Apparatus. — The centri- fugal machine (Fig. 1) is a very great convenience in laboratory work, and for some purposes is a necessity. Its purpose is to concentrate the undis- solved solid materials in any liquid (as blood, urine, etc.), in the bottom of the liquid, either with a view of determining the relative amount or bulk of the solid constituents (as in "centrifugal analysis") or of collecting them for microscopical examina- tion. This is accomplished by the fluid being placed in tubes Fig. 1.— Hand Centrifuge, showing urine tubes attached, lisematokiit or Wood tubes above. (Bauscb & Lomb.) Digitized by Microsoft® 10 A MANUAL OF CLINICAL LABORATORY METHODS. at the euds of a horizontal arm, which is revolved at a high rate of speed by means of hand, electric, or water power acting on a gearing. There are separate attachments for blood, urine, spu- tum, milk, etc. The powerful centrifugal action forces the heavier undissolved solids very quickly, and compactly to the bottom of the fluid. Contact Tests,— Testing by the contact method is frequently practised, as in examining for albumin, hsemoglobin, etc. The method consists in introducing the test fluid and the fluid to be tested together into a test tube or conical glass so that instead of mixing they form separate layers, the lighter fluid overlying the heavier. The reaction takes place at or near the plane of junc- tion of the two fluids. Either fluid may be introduced first, preferably the heavier ; the tube or glass is inclined so that its side forms a slight slope, and the other fluid is then allowed to flow from a pipette slowly and gently down the inclined side so as to overlie or underlie the other fluid, without being mixed ; the glass is then carefully turned upright again. Or the lighter fluid may be first introduced; the other fluid is drawn into a long pipette ; the upper end of the pipette being tightly closed with the finger, the point of the pipette is passed to the bottom of the glass, and the finger carefully raised so as to permit the fluid to flow gently from the pipette and underlie the other fluid. For observing delicate color changes in contact or other tests, the test tube is best held in front of a white background, as a sheet of paper ; the light from the window should fall upon the white background, whence it is reflected through the test tube to the eye of the observer. For observing white precipitates or clouds, as albumin, an unilluminated black background is prefer- able. Before taking up the special lines of laboratory investigation in detail it will be con^'enient to consider a few methods and tests of general application. Fat. — The presence of microscopic particles of fat is best de- termined by treatment with Sudan III. The test solution is pre- pared by first making a saturated solution of Sudan III. in alco- hol ; after standing several days one part of this solution is mixed with one part of alcohol and one part of water ; the mixture is at first turbid, but clears on standing. Sudan III. has an afiinity for fat only, staining fat particles red and leaving everything Digitized by Microsoft® LABORATORY EQUIPMENT AND GENERAL METHODS. 11 else uncolored ; the specimens must not be treated with alcohol either before or after staining, as that dissolves out the fat and stain. Permanent mounts must be made in glycerin-jelly, or similar medium. For making the test a drop or two of the Sudan III. solution is added to a few drops of the fluid or material (as gastric fluid, faeces) to be tested for fat, or is allowed to flow un- der the cover-glass covering the latter ; particles of neutral fat are by this procedure conspicuously differentiated under the micro- scope by their taking a red color. Sections of solid tissues, cut by the freezing method and untreated by alcohol, are stained in the solution and examined or mounted in a watery or glycerin medium. Starch in solution or granular form is tested for by free iodine, with which it strikes a deep-blue color. The stock test solution (Lugol's, Gram's) consists of iodine 1, potassium iodide 2, in a variable amount of water, say 300 parts. For microscopical pur- poses*a drop of this may be mixed with a drop or two of the sediment or fluid under examination, or allowed to flow under the cover-glass; starch granules, if present, are stained deep- . blue. For testing liquids for the presence of starch or erythro- dextrin dilute the iodine solution with water till it is of a light- yellow color, and add a few drops of the suspected fluid ; if a blue color develops starch is present ; if a deep-brown color ap- pears erythrodextrin is present. Achroodextrin gives no reaction. Glycogen granules when treated with the above iodine solution turn a deep mahogany -brown, and may be thus diff'erentiated under the microscope. Iodides. — Occasions at times arise iu clinical work to test the secretions (saliva or urine) for the presence of iodides or othei' iodine combinations experimentally or otherwise administered. The starch method is employed for this purpose ; starch is not affected by iodine in combination, but on setting the iodine free with nitric acid or chlorine the characteristic blue color devel- ops. The test is best applied as follows : A watery solution or thin paste of starch is made with the aid of heat ; filter paper is saturated with it, dried, and cut into pieces of suitable size. These pieces keep until ready for use. To make the test one of the pieces is well moistened with the fluid to be tested, and then touched with a drop of nitric acid. The appearance of a blue color indicates the presence of an iodine compound. Digitized by Microsoft® 12 A MANUAL OP CLINICAL LABORATORY METHODS. Occasions constantly arise in laboratory practice where un- usual or unexpected results, obscure reactions, or perplexing con- ditions are presented. Methods could hardly be presented that would obviate all difSiculties that might possibly arise; these must be met and mastered at the time by the exercise of common sense, judgment, and ingenuity, guided by enlightened experi- ence. Digitized by Microsoft® in. THE BLOOD. The blood is so intimately concerned in the processes of life and disease, and comes into such close contact with all parts of the body, that the conditions manifested by it throw important light on the nature of many vital and abnormal processes that may be in progress, and the clinical examination of the blood affords assistance in diagnosis in many conditions, both in a positive and negative way. Specimens of blood are easily ob- tainable for purposes of examination, though only in minute quantities. A. CONSTITUTION OF THE BLOOD. The blood is composed of a fluid portion, the plasma, in which are suspended large numbers of cells or corpuscles, which are of three kinds, red blood corpuscles, leucocytes, and blood plates. The total quantity of blood in the body (normally about one- thirteenth of the body weight) is doubtless variable, and would be a valuable clinical datum if there were any method of deter- mining it. The relative proportions of the plasma and corpus- cular elements of the blood are easily determinable ; but to ascer- tain if there is any absolute total increase or decrease in the plasma or hsemocytes, and its extent, which is the real criterion of an anaemia or plethora, it would be necessary to know also the total volume of blood. The normal color of arterial blood is bright red, that of venous blood darker ; in abnormal conditions the color may vary, being paler in anaemia and hydrsemia, darker in concentrated blood, cherry -red in carbon-monoxide poisoning, dark and venous in poisoning by hydrocyanic acid and other poisons, somewhat milky in leuksemia. The consistency of blood is normally some- what creamy, but may be thinner and watery in anaemic and hydrsemic conditions, thicker and less fluid where the blood is more concentrated (as cholera). The reaction of the blood is Digitized by Microsoft® 14 A MANUAL OF CLINICAL LABORATORY METHODS. normally alkaline, but the degree of alkalinity may vary, and. the reaction may even be acid (in cholera Asia,tica). The specific gravity of the blood is normally about 1.056 to 1.060, somewhat higher in males than in females. In conditions in which the proportion of solids is reduced, as in anaemia, it may range down to 1.030 or lower; the specific gravity should be a useful index of the degree of concentration or dilution of the blood. To a large extent the specific gravity varies parallel with the haemoglobin. Blood Plasma. This is a clear, homogeneous, colorless fluid normally com- prising about three-fifths of the volume of the entire blood. Its quantity is susceptible of variation in different circumstances. It may be increased in amount from ingestion of fluids, subcuta- neous or intravenous injection of fluid, in cedematous conditions, or as a result of lowered blood pressure or vascular dilatation, which permits the entrance of lymph from the lymphatic ves- sels; this condition, known as "hydrsemia," causes a dilution or thinning of the blood, the corpuscles not being correspondingly increased, and hence comprising a smaller relative proportion of the whole amount of blood. The amount of the plasma may be diminished, after profuse loss of fluid from free purging, vomiting, or sweating, or from insufficient ingestion of water ; the corpuscles not being corre- spondingly reduced, the result of such a diminution of the fluids of the plasma is a concentration of the blood and relative increase in the proportion of the corpuscles ; such losses of serum from the blood are very quickly replaced. Increase or decrease in the volume of the plasma (the corpuscles not being correspondingly affected) is manifested in- hsematologic examination by a relative contrary change (decrease or increase respectively) in the num- ber of corpuscles per cubic millimetre ; and it is necessary to take other conditions into account in order to determine whether abnormalities in the corpuscle-count are due to changes in the amount of the plasma or of the corpuscles. The blood plasma consists of water holding in solutioi; nor- mally about 9 per cent of various substances, as albumins, globu- lins, fibrin factors, chlorides, phosphates, compounds of potas- sium, sodium, and calcium, glucose, glycogen, fat, fatty acids, Digitized by Microsoft® THE BLOOD. 15 urea, uric acid, and other substances, besides adventitious mate- rial derived from food and other ingested substances. Varia- tions in the kind and amounts of the various constituents of the plasma occur according to the substances absorbed, the processes of metabolism, or the action of the excretory organs ; thus there may be a decrease in the amount of proteids, an increase or de- crease of the fibrin factors, an increase of glucose (diabetes mel- litus), an increase of katabolic products or leucomains (as in ursemia, lithtemia), or increase of fat (lipsemia) ; or unusual and abnormal substances may be present, as bile constituents (cholse- mia), toxic agents of extra- or intra-corporeal origin (toxaemia), bacterial toxins, antitoxins, and agglutinins developed in conse- quence of infectious conditions. Qualitative and quantitative determinations of many of the chemical constituents of the blood plasma would afford valuable clinical information in many cases ; but unfortunately, owing to the limited quantities of blood ordi- narily available for examination and the difficulty of identifying many of the substances (as toxins), practicable methods of inves- tigating the blood plasma have as yet been developed only to a ^ery limited degree. There are a few tests, as for glucose (Bre- mer's, Williamson's), glycogen (iodine), agglutination tests, that require only minute quantities of blood ; and other constituents can be estimated if sufficient amounts of blood can be obtained. But there are many sxibstances, especially those having toxic ac- tion, the recognition of which, if it were practicable, would be of great aid in diagnosis and treatment. One important class of substances, the agglutinins, are amen- able to examination. The agglutinins are substances which ap- pear in the blood serum in consequence of bacterial infection, various bacteria producing their own specific agglutinins. These substances when added to cultures of the corresponding germs cause the bacteria to become aggregated or "agglutinated" to- gether in clumps or masses, at the same time arresting motility of the bacteria. Each agglutinin affects only its own specific bacterium. The presence of an agglutinin is tested by adding a small amount of blood serum from the case to a culture of the suspected bacteria, under proper conditions ; if the agglutinating action occurs, the presence of the agglutinin of that germ is demonstrated, and this again shows that the subject furnishing the serum had suffered infection by the corresponding parasite. Digitized by Microsoft® 16 A MANUAL OF CLINICAL LABORATORY METHODS. This method is utilized in the diagnosis of typhoid fever, under the common name of "Widal reaction," which is typical of the whole group of agglutination tests. Although chiefly used in typhoid fever, the method is more or less applicable in a few- other infectious conditions, as cholera, bubonic plague, Malta fever. Small particles of fat occasionally appear in the plasma, as after a fatty diet. Pigment is sometimes present, notably black pigment granules in malaria, in melanosis, and sometimes, it is said, in Addison's disease ("melansemia"). The haemoglobin of the red corpuscles sometimes (in toxic or other conditions) passes into solution in the blood plasma ("hsemoglobinsBmia") ; the serum which separates after clotting is in such cases red instead of straw-colored. Glycogen in granules may occur in the plasma normally ; it is increased in suppurative conditions, pneumonia, leuksemia, and other conditions, not only in the plasma but abun- dant in the polynuclear leucocytes. Granules of a peculiar kind, known as hgemokonia or "blood dust" (Miiller), are frequently to be seen in blood plasma. They are very minute in size (1 or 2 micromillimetres in diame- ter), rounded or irregular in shape, colorless and refractile, and in constant active oscillating or Brownian movement. Their nature is not fully determined, but they are probably minute bits of protoplasm or granules extruded from leucocytes. They occur in both normal and abnormal conditions ; ordinarily they are few in number, occurring singly here and there, but some- times they are present in large numbers. They have no known clinical significance. Coagulation and Fibrin Formation. — Human blood normally co- agulates in about from three to ten minutes. Departures from the normal time of coagulation may occur in consequence of individ- ual peculiarities or pathological processes. In some individuals coagulation occurs almost instantly. In pernicious ansemia, other anaemic conditions, inflammations, and haemophilia, coagulation is retarded. The process of fibrin formation may be observed in micro- scopical preparations of fresh blood, and some judgment as to coagulation or the amount of fibrin may be formed in that way. Under such circumstances numerous delicate, colorless, straight filaments of fibrin appear in the plasma, running in different di- Digitized by Microsoft® THE BLOOD. 17 rections, radiating from points or centres, or interlacing to form a network ; blood plates or granular materials are often located at the points of intersection. In general the amount of fibrin formation is increased in infections and inflammatory conditions, running largely parallel with the number of leucocytes and the temperature; it is not increased in the leucocytoses of leukae- mia and malignant disease, and is diminished in pernicious anaemia. In the days of bleeding the character of the blood clot was a matter of clinical significance, but at the present time opportu- nity for taking advantage of such data is only rarely afforded. The firmness of the clot, the occurrence of the "buffy coat" (the pale upper layer caused by subsidence of the corpuscles when coagulation was slow, or the corpuscles were relatively heavy), or the formation of a concave or "cupped" upper surface of the clot, are characters that were once considered significant, but are now obsolete. Bed Blood Corpuscles. Eed blood corpuscles or erythrocytes consist of a soft, pliable, and elastic proteid stroma, in which haemoglobin is suspended in solution. Their form and consistency are due to the stroma, their color to the haemoglobin. The normal number of red corpuscles in given volumes of blood under ordinary conditions is quite constant, being in adult males about 5,000,000 and in adult females about 4,500,000 in each cubic millimetre of blood. Under various conditions, normal and abnormal, the number varies within wide limits (the ex- tremes observed being 143,000 and about 9,000,000 per cubic millimetre), and these deviations constitute important facts for clinical consideration. The number of erythrocytes is increased ("polycythaemia") in various conditions. In vigorous health the number may run up to 6,000,000 or even more. For a few days after birth, the number is high, up even to 8,000,000. With increase of alti- tude above the sea level there is a remarkable increase in the number of red cells, up to 8,000,000 at the altitude of 4,400 me- tres ; the relation of the total volume of the erythrocytes to their number under these circumstances has not been worked out, and the cause of this altitude polycythaemia is very obscure. After 2 Digitized by Microsoft® 18 A MANUAL OF CLINICAL LABORATORY METHODS. recovering from ansemic conditions increased Lteniopoietic activ- ity may cause a marked increase in the number of red cells over the normal. Concentration of the blood by decrease of the plasma, as by loss of fluid from severe sweating, diarrhoea, or vomiting, or by vascular contraction and increased blood press- ure causing an expulsion of blood serum into the lymph channels, increases the number of red corpuscles to the cubic millimetre up to 6,000,000 or more. In acute poisoning by phosphorus or carbon monoxide there may be a great increase in the num- ber of red cells, up to 8,000,000 or over. In localities where there is congestion, stasis, or accumulation of corpuscles there may be a local increase of red cells. There may occasionally be conditions of general cellular plethora or stasis exhibiting poly- cythsemia. The number of erythrocytes is decreased ("oligocythsemia") especially in ansemic conditions of various origin, primary and secondary, most notably in pernicious anasmia, in which the cor- puscles often run as low as 400,000 to 800,000. Diluted or hy- drsemic conditions of the blood caused by access of fluid from ingestion by the mouth or subcutaneous or intravenous injec- tions, oedematous conditions, decreased blood pressure or vascu- lar dilatation (as after administration of amyl nitrite), permit- ting entrance of lymph into the blood, lower the count of red corpuscles. In itself the number of red corpuscles per cubic millimetre simply expresses the quantitative relation between these cells and the plasma, and when the number present is abnormal all the concomitant circumstances must (in the absence of a method of determining the total amount of blood) be taken into account in order to draw a conclusion as to whether the variation is due to changes in the total amount of the corpuscles or of the plasma. Thus in a case of anaemia, a severe sweat or diarrhoea may cause such a loss of fluid as to carry the number of red cells up to or above the normal ; and yet the increase would be only ap- parent and relative, and not real and absolute. The normal volume of red corpuscles relatively to the total vol- ume of the blood is about 40 to 43 per cent ; the ordinary centrif- ugal method of determining the volume of the corpuscles gives a slightly higher proportion, about 50 per cent. The volume ratio varies in different conditions, depending on (a) the number Digitized by Microsoft® THE BLOOD. 19 and (6) the size of the individual corpuscles. If the size of the corpuscles were constant, the volume would vary in direct ratio with the number of cells ; and from a determination of the volume the number could be directly calculated, and vice versa. Varia- tions in the size of the cells in different cases would, however, affect the total relative volume of the corpuscles, though to a less de- gree than deviations in the number. Independent estimations of both the volume and the number afford a means of calculating the average bulk of the individual corpuscles, or the "volume index. " The average volume of the red corpuscles, as calculated from their dimensions, is normally about 85 cubic micromillime- tres each ; as calculated from the findings given by the centrif- ugal apparatus the volume is about 100 cubic micromillimetres, which, while somewhat in excess of the real bulk, is the only prac- ticable method of determination and provides a useful standard figure. The distribution of the red corpuscles is ordinarily probably uniform throughout the circulatory system, so that a specimen taken from the peripheral blood-vessels in the ordinary manner must be regarded as sufficiently typical and representative of the entire blood. In situations where there is a local congestion or oedema, however, there may be a concentration or dilution of the blood,' so that specimens from such localities would not be repre- sentative of the general blood mass. The color of the red corpuscles when seen singly is a peculiar pale greenish-yellow, lighter and paler in shade at the centre, where the corpuscle is thinnest, and gradually deepening toward the periphery. The color is due to hsemoglobin, which makes up about 95 per cent of the solid matter of erythrocytes ; and varia- tions in the proportionate amount of this important substance cause corresponding variations in the color of the blood as a whole and of the individual corpuscles. The amount of haemo- globin necessarily to a certain extent corresponds to variations in the number of red corpuscles ; but the hsemoglobin may and does vary independently of the number of the cells, disturbing influences usually causing the hsemoglobin to be less in amount than the number of corpuscles would account for. Thus, in anaemic conditions the haemoglobin is reduced in amount along with the number of erythrocytes; but it is usually reduced rela- tively more, notably in chlorosis. Great decrease in hsemoglo- Digitized by Microsoft® 20 A MANUAL OF CLINICAL LABORATORY METHODS. bin, without a corresponding reduction in the number of cells, is sometimes called " oligochromfemia, " or "chloro-ansemia." The amount of hsemoglobin may be considered with reference to the blood as a whole and with reference to the individual cor- puscles. As a whole it may be expressed in percentages, being normally about 12.5 to 13.75 per cent of the blood by weight; in hsematological work the hsemoglobin when normal is commonly expressed as being 100 per cent, and variations from the normal are recorded in percentages with this as the standard. The color of the blood varies with changes in the amount and with chemi- cal changes of the hsemoglobin, and affords a colorimetric method of estimating it. The specific gravity of the blood also corre- sponds closely with the amount of hsemoglobin. The hsemoglobin can also be considered with reference to the amount in each red corpuscle. The total percentage of hsemo- globin and the number of corpuscles being normal, each cor- puscle may be regarded as possessing a normal amount of hsemo- globin. If the total proportion of hsemoglobin is relatively less than the number of corpuscles, each corpuscle possesses less than its normal amount of hsemoglobin, and these variations referred to the individual cells have a distinct clinical significance. The corpuscular quantities of haemoglobin are quantitatively ex- pressed by means of ratios, determined by dividing the total percentage of haemoglobin by the percentage to normal of the number of corpuscles present. This ratio is variously termed the "corpuscular hsemoglobin ratio," the "globular value," "color index," etc. When the ratio is 1, each cell possesses its normal quantity of hsemoglobin, even though the number of corpuscles is abnormal. When, as often occurs in ansemic conditions, the ratio is less than 1, each cell possesses less than its normal share of hsemoglobin. The ratio rarely exceeds 1, as in pernicious ansemia and new-born infants. When the red corpuscles are individually deficient in hemo- globin (their ratio being less than 1), their color is correspond- ingly paler, often to a degree distinctly and markedly perceptible in microscopic examination ; in such cases the thin centre of the corpuscles may be entirely colorless, only the thicker periphery exhibiting a pale color. The composition and properties of haemoglobin, and the chemical combinations and transformations to which it is subject Digitized by Microsoft® THE BLOOD. 21 (as oxylisemoglobm, methsemoglobin, hsematin, hsemin, hsematoi- din, liseniatoporpliyrm, carbon monoxide liaBmoglobm, etc.), are interesting and important subjects which cannot be considered here. The most important constituent of haemoglobin is iron, which also occurs in the blood in other combinations. Under pathological circumstances haemoglobin may pass from the corpuscles into solution in the plasma, producing hsemoglobin- aemia. The shape of the red corpuscles is normally circular and disc- shaped, biconcave. The deviations from the normal form may be considered under three classes : (a) Crenation and other non- pathological changes in form; (&) endoglobular changes (vacuo- lation) ; (c) pathological changes in form, or poikilocytosis. Crenation occurs in blood removed for examination, and is caused by increase of density of the plasma, by evaporation from exposure to the air or by the solution of additional substances in it. The corpuscles contract from loss of part of their fluid contents through osmosis ; they become spheroidal or irregular in shape, with rounded or spiny projections. When the density of the plasma is diminished, as by the addition of water, the red cells absorb fluid by osmosis, swell, become spherical, faint, and colorless, the haemoglobin passing into the plasma; these are the "shadow corpuscles." Portions of the corpuscles often become broken off, assuming a rounded form, and appear like minute corpuscles. Sometimes the corpuscles appear bent, twisted, or doubled up irregularly. Familiarity with the changes of form which erythrocytes may manifest under the artificial conditions to which they are exposed during examination is necessary to prevent mistaking them for pathological alterations. Often spaces or vacuoles appear in the interior of the red cor- puscles, from shrinkage of their substance ; the vacuoles may be single or multiple, large or small. Usually they are artefacts and not pathological, but possibly at times they may be of patho- logical nature. In some diseased conditions, especially in anaemias, the red corpuscles, or many of them, may be markedly abnormal or de- formed in shape. Such abnormally shaped corpuscles are called poikilocytes. Sometimes, as often in pernicious anaemia, the cor- puscles generally are elliptical or elongated in form; in other cases the poikilocytes are of various irregular shapes. Digitized by Microsoft® 22 A MANUAL OF CLINICAL LABORATORY METHODS. The size of the red corpuscles is normally 7 to 8 (averaging 7.5) micromillimetres in diameter and about 2 in thickness. Under normal conditions the size of the corpuscles may vary within small limits, either in the same individual or in different individuals. Greater variations in size occur in pathological conditions, especially in ansemias. Abnormally large cells are termed macrocytes, abnormally small cells microcytes. In some cases the cells are generally and almost uniformly, or on an aver- age, undersized or oversized; sometimes in the sanie case the cells exhibit considerable variations in size. Macrocytes are es- pecially frequent in pernicious aufemia ; while in chlorosis micro- cytes are quite characteristic. Nuclei. — Human red blood corpuscles are normally devoid of nuclei, except at an early embryonic period. In certain diseases, especially leukaemia and severe anaemias, nucle- ated red cells may be present either in small numbers or in small pro- portion to the non-nu- cleated cells. Three varieties of nucleated erythrocytes are distin- guished, according to the size of the corpus- cle: (a) normoblasts, nu- cleated corpuscles of the normal size of red cor- puscles; (6) megaloblasts, nucleated cells of ab- normally large size ; (e) microblasts, nucleated cells of \'ery small size. Normoblasts are considered to be closely related to the normal erythroblasts of the bone marrow, from which the red blood cor- puscles are derived; the nucleus is conspicuous, single, and rounded, sometimes divided into segments, or karyokinetic, stains deeply with nuclear and basic stains, and is frequently eccentrically situated. Normoblasts occur regularly in severe anaemic conditions (except chlorosis), and are abundant in sple- %7 Fig. 3.— Normal and Abnormal Bed Blood Corpuscles, o, Normal corpuscles, side and edge view ; 6, vacuole lormatlon ; c, crenated corpuscles ; d, rouleau forma- tion ; e, pale corpuscles, deficient in liaemoglobin ; f, poikilocytes ; ff, maoroeyte ; ?i, microcyte; *, normo- blast ; ft, megaloblast. Digitized by Microsoft® THE BLOOD. 23 nic leukaemia ; they have uo specially evil significance in such cases, and indeed indicate hemopoietic acti^'ity. Megaloblasts are very large erythrocytes, 11 to 20 micromilli- metres in diameter, often somewhat irregular in outline and with a polychromatophilic tendency, and with a large, rounded, pale- staining nucleus. They approach the type of erythroblast occur- ring in early embryonic life. They may be found in most classes of cases in which normoblasts occur, but usually in much smaller number than the latter ; they are especially abundant and char- acteristic in pernicious anaemia. When they are relatively abundant, they are regarded as of grave prognostic significance, representing an embryonic or inadequate process of blood regen- eration. Microblasts are very rarely encountered ; they are abnormally small nucleated red corpuscles. Staining Characteristics, — Eed blood corpuscles are normally oxyphile in their staining affinities, talcing acid stains like eosin or picric acid. In some abnormal conditions, especially severe anaemias, instead of being colored by the ordinary stains in the usual manner some of the corpuscles wholly or in part assume peculiar and unusual colors. This abnormity of the staining re- action is termed polychromatophilia, and the cells exhibiting the change are called polychromatophiles. Polychromatophilia indi- cates a chemical or degenerative alteration in the red corpuscles ; it is one of the less common abnormities of the erythrocytes, and usually occurs only in very severe anaemias. A form of polychromatophilia ("granular degeneration") is rarely observed in which small, strongly basophilic granules are scattered about in red corpuscles, which may themselves be ot normal or polychromatophilic coloration. These granules are probably a peculiar form of degeneration, and appear in lead poisoning and other conditions. In the blood of diabetics the red corpuscles lose their affinity for the acid stains which they ordinarily take with avidity, and, on the contrary, stain with dyes which do not color normal red cells. Rouleau Formation. — When a sufficiently thick layer of fresh blood is placed under the microscope, the red corpuscles normally collect temporarily in rouleaux, becoming arranged or packed to- gether with their sides in contact, like a pile of coins. Patho- Digitized by Microsoft® 24 A MANUAL OP CLINICAL LABORATORY METHODS. logically the tendency to rouleau formation may be absent, as frequently in pernicious anaemia. Many of the abnormities of erythrocytes just considered, as Tariations in size, shape, haemoglobin, and staining aflanities, may be regarded as degenerative conditions of the corpuscles, arising from injurious or toxic alterations of the plasma or from diminished power and inability of the hsemopoietic agencies to produce healthy corpuscles. Other indications of degeneration may occur. The consistency or firmness of the corpuscles may be lessened, so that they change their shape at the slightest dis- turbance ; the cells may become almost semi-fluid, so as to be hardly capable of retaining any definite shape, and almost run together in a gelatinous mass. The power of the erythrocytes to withstand changes in the density of the plasma (their "tonicity" or "isotonicity ") may be lessened so that they more easily suffer osmotic changes, causing loss of haemoglobin, change of shape, or other destructive alterations. The cori)uscles may undergo irregular contractions or movements, or rupture, or fragments may break off. 4 Leucocytes. Human leucocytes are of several varieties, differing in some characteristics, but similar in their general features. They are spherical when quiescent, irregular and variable in form during amoeboid movement. They range in diameter from about 7 to 20 micromillimetres. They are of firmer structure and more vigor- ous vitality than the erythrocytes, not so easily influenced by changes in their environment, and not so subject to degenerative changes. They occur not only in the blood (blood leucocytes), but also in the lymphatic structures (lymphatic leucocytes), in the bone marrow (marrow leucocytes), in the spleen (spleen leucocytes), in the interstices of tissues (wandering cells), iu pus (pus leucocytes), etc. They are typical cells, of active vi- tality. Their nuclei vary in the different varieties of leucocytes as to number, form, size, and staining properties. The cyto- plasm or cell body is typical protoplasm, in which can be distin- guished, especially during amoeboid movement, a clear, hyaline, fluid, homogeneous peripheral portion, the hyaloplasm, and a more central granular portion, the spongioplasm. The cell- bodies of most white blood corpuscles contain protoplasmic gran- Digitized by Microsoft® THE BLOOD. 25 ules, which vary in kind and present important distinguishing characteristics in the different varieties of leucocytes. These granules are distinguished chiefly by their size and staining affini- ties.* The two principal varieties in human blood are fine ueu- trophile granules and coarse oxyphile granules (e and a granules of Ehrlich's classification) ; fine basophile (5) granules occur but are inconspicuous and unimportant, whUe coarse basophile (;') granules characterize mast cells. The granular appearance of fresh leucoc5rtes, which often obscures the nuclei, is cleared away on addition of acetic acid, leaving the nuclei prominent as two or three spherical bodies ; this is a useful means of recognizing and demonstrating leucocytes. Leucocytes exhibit amoeboid movement in a very typical man- ner, and they possess the power of phagocytosis, as may be ob- served especially in connection with the malarial parasite. The number of leucocytes in given volumes of blood in normal conditions ranges from 6, 000 to 10,000 to the cubic millimetre ; 8,000 is about the average standard. The number in the per- ipheral blood varies widely in different pathological conditions, ranging from 419, probably the lowest number on record (Ca- bot), up to 1,500,000 per cubic millimetre, and these variations are of great diagnostic significance. The deviation in number may consist in a parallel variation of all the varieties of leuco- cytes, or in an increase or decrease of single varieties. Increase in the number of the leucocytes is termed "leucocytosis" (also "hyperleucocytosis "), decrease in their number below the normal is called "leucopenia" (also "hypoleucocytosis"). The leucocytes are increased (leucocytosis) in numerous con- ditions. The number is higher in those in vigorous health and well-fed condition. At the height of digestion, as three or four hours after a rich proteid meal, there is ordinarily a temporary Increase in their number of from 30 to 50 per cent ; this is called the "digestion leucocytosis." Digestion leucocytosis is absent in some conditions, as in gastric carcinoma, other gastro-intestinal disorders, and occasionally in other conditions, so that its occur- rence may be a matter of clinical significance. Exercise, mas- sage, and brief cold baths cause a transient increase of leucocytes, * Basophile granules are those which stain with basic stains, like methy- lene blue ; oxyphile granules are those which take acid stains, as eosin, acid fuchsin; neutrophile granules are those which take neutral stains. Digitized by Microsoft® 26 A MANUAL OF CLINICAL LABORATORY METHODS. perhaps from vascular contraction and concentration of the blood. In young infants, especially the first week or two, the number is high (up to 30,000 or more). In the latter part of pregnancy, especially in primiparse, and after delivery, leucocytosis occurs. In the moribund period there is often a leucocytosis. The leuco- cytes may be increased after the administration of certain drugs and poisons, as ether, salicylates, pilocarpin, illuminating gas. After hemorrhage leucocytosis appears very quickly, within an hour or so. In most inflammatory and infectious diseases, and especially in suppurative conditions, marked leucocytosis appears and affords valuable diagnostic information. In pneumonia the leucocytosis may be extreme, the number occasionally reaching 100,000. In cases of infectious disease, when the infection is very mild or when it is overwhelming, without a vigorous bodily reaction, there may be no leucocytosis, but where the infection is moderate, so as to permit a vigorous body reaction, the leuco- cytosis is marked. In uncomplicated tuberculosis, typhoid fever, malaria, measles, and influenza, leucocytosis is conspicuously absent, — a negative point that is sometimes of diagnostic significance. In malignant disease, carcinoma and sarcoma, leucocytosis is usu- ally present, and may be extreme (up to 100,000). Leucocytosis may be present also in various chronic affections. The greatest increase in the number of leucocytes occurs in leukaemia, in which they may exceed 1,000,000 cells per cubic millimetre. The leucocytes are diminished (leucopenia) in certain cases. In conditions of lowered health, debility, malnutrition, insuffi- cient food, and starvation the leucocytes are decreased, some- times below 1,000. Short hot baths and prolonged cold baths decrease the leucocytes, probably from dilatation of vessels and dilution of the blood. Pernicious anaemia usually exhibits marked leucopenia, the corpuscles sometimes falling below 1,000. Infectious diseases in which leucocytosis is absent, as tuberculo- sis and typhoid fever, may show a diminution in the number of leucocytes. In leukaemia complicated by infectious disease leu- copenia may result. The proportion of white to red corpuscles is ordinarily in the neighborhood of 1 to 600. When the two kind of cells increase or decrease proportionately, this ratio continues ; but when they do not vary in correspondence, the ratio is altered accordingly. Digitized by Microsoft® THE BLOOD. 27 In leukaemia tlie ratio of white to red cells may go as high as 1 to 4, or the white may even exceed the reds in number. As to the distribution of the leucocytes in the blood, many au- thorities believe that they do not always occur in equal quanti- ties in all parts of the circulation. In some forms of leucocyto- sis, for instance, it is believed that the leucocytes are aggregated in the peripheral circulation, the internal structures being corre- spondingly depleted, so that there is no alteration in the total number of leucocytes in the entire body. In many cases, how- ever, there is doubtless an actual increase in the total number of leucocytes. Whether the peripheral blood, which alone is avail- able for examination, is representative of the entire blood or not, the fact remains that marked and characteristic changes occur in the numbers of leucocytes there present, and that these changes are of valuable diagnostic significance. Varieties of Leucocytes. — The leucocytes that occur normally or abnormally in human blood are of a number of different kinds, varying as to size, nuclei, granules, and staining properties. These varieties are small mononuclear leucocytes, large mononu- clear leucocytes, transitional leucocytes, polynuclear leucocytes, eosinophile leucocytes, neutrophile myelocytes, eosinophile mye- locytes, and mast cells, besides occasional atypical and as yet unclassified leucocytes. Small Mononuclear Leucocytes (or small lymphocytes). — ^These are small spherical corpuscles about 6 to 8 micromillimetres in diameter. They are made up chiefly of a nucleus, which is sur- rounded by a narrow shell of body protoplasm. The nucleus is spherical and usually stains deeply with nuclear and basic stains. The body protoplasm stains but faintly, and is usually non- granular, though sometimes basophile granules can be demon- strated. Large Mononuclear Leucocytes (or large lymphocytes).— These are large corpuscles, 12 to 15 micromillimetres in diameter. They contain each a single, large, rounded, pale-staining nucleus, which is surrounded by a large cell -body of faintly staining pro- toplasm that is non-granular or contains only inconspicuous basophile granules. Transitional leucocytes are like the large mononuclear leuco- cytes in all respects, except that the nucleus instead of having a circular outline is indented at one side, giving it a horseshoe or Digitized by Microsoft® 28 A MANUAL OP CLINICAL LABORATORY METHODS. TJ shape. The cytoplasm also sometimes contains a few neutro- phile granules. The small and large mononuclear and transitional leucocytes are closely related to one another, forming together a veil- defined group, the "lymphocytes," and are regarded by some as younger stages of leucocytes. Intermediate forms, as to size and nuclei, may at times be observed between all three varieties, so V,^./ r si f M Fig. 3.— Human Leucocytes, a. Small mononuclear leucocyte, stained appearance ; 6, large mononuclear leucocyte, stained ; e, transitional leucocyte, stained ; d, polynuclear leu- cocytes, living amoeboid appearance : e, polynuclear leucocytes, stained ; /, eosinopliile leucocyte, stained; g, myelocyte (neutropWle), stained; ft,, eosinopliile myelocyte, stained. that sometimes sharp dividing lines between the three can hardly be drawn. Transitional leucocytes especially are often to be re- garded as simply a variety of the large mononuclear group. Polynuclear (polymorphonuclear or neutrophile) Leucocytes. — This corpuscle is about 10 micromillimetres in diameter. It has a conspicuous body of vitally active protoplasm containing large numbers of fine (e) neutrophile (or rather, perhaps, faintly oxy- phile) granules. The nucleus stains deeply, and is very variable and irregular in shape, because of which the term "polymorpho- nuclear " is often applied to this form of leucocyte. On treating the fresh leucocyte with acetic acid, the cell-body clears up and the nucleus stands out as about three separate spherical bodies, whence the name polynuclear applied to this variety. Instead of possessing three separate nuclei, however, it is generally be- lieved that this leucocyte has only a single nucleus, very irregu- Digitized by Microsoft® THE BLOOD. 29 lar, twisted or lobed in form, often having the appearance of being divided into separate parts, which, are, however, united by- strands of the nuclear substance. This is the most abundant leucocyte of normal hmnan blood, and exhibits very active amoe- boid movements and phagocytosis. The corpuscles of pus are identical with polynuclear leucocytes. Eosinophile Leucocytes. — These are about 10 micromillimetres in diameter. They have a rather large irregular and polymor- phous nucleus, or perhaps two or three separate rounded nuclei, staining less deeply with basic stains than the nucleus of the preceding variety. The cell-body is crowded with conspicuous coarse oxyphile («) granules, which stain intensely with acid stains such as eosin (whence the name eosinophile) ; the charac- teristic granules enable this variety of leucocyte to be very dis- tinctly recognizable, both in the fresh and stained condition. Neutrophile myelocytes are found in the blood only in abnor- mal conditions; they (or corpuscles very similar) occur nor- mally in the bone marrow. They are usually larger than other varieties of leucocytes, ranging from 20 down to 12 or 13 micro- millimetres in diameter. They possess each a single large rounded pale-staining nucleus eccentrically placed, and the ample cell body is crowded with fine neutrophile granules. Myelocytes are perhaps intermediate forms between large mononuclear and polynuclear leucocytes. They occur in the blood in small num- ber in pernicious anaemia, chlorosis, and other anaemic condi- tions, and in some leucocytoses ; but they are especially abun- dant and characteristic in splenic leukaemia, in which they form a large percentage of the leucocytes present. Eosinophile Myelocytes. — These may occur in the blood in any condition in which the neutrophile myelocytes appear (chiefly in splenic leukaemia and occasionally in pernicious anaemia) but in much smaller number than the latter. They resemble the ordi- nary neutrophile myelocytes in every respect except that their cell bodies are crowded with coarse oxyphilic granules instead of neutrophile granules. Mast Cells. — These cells ordinarily occur in the body tissues, but occasionally they find their way into the normal circulating blood. In splenic leukaemia they may be present in the blood in large numbers. They are usually large cells, ranging up to 20 or 30 micromillimetres in diameter, each with an irregular or lobu- Digitized by Microsoft® 30 A MANUAL OP CLINICAL LABORATORY METHODS. lar pale-staining nucleus, and coarse basophile (r) graniiles in tlie body protoplasm. Atypical Leucocytes.— Nearly all leucocytes observable in the blood are distinctly referable to one or another of the classes described. Occasionally, however, leucocytes may be seen with atypical characters, as to size, nuclei, granulation, etc., which do not permit them to be placed with any of the generally recog- nized classes, ^ — such as mononuclear leucocytes with neutrophile granules, leucocytes with basophile granules, etc. Degenerating forms, as leucocytes with vacuoles, are also sometimes seen. Basophile Granules. — It will be observed that the most abun- dant and important granules of human leucocytes are the fine neutrophile or oxyphile (e) and the coarse oxyphile (a) gran- ules. Basophile granules can often be demonstrated by proper methods, but they are ordinarily not very abundant or conspicu- ous, and little or no clinical importance is attached to them. They may be demonstrated sparingly in lymphocytes and myelo- cytes, are marked in mast cells, and may occur in atypical leu- cocytes. One class of basophile granules that has been specially differ- entiated are the periimdear basophile granules. These are baso- philic granules that can sometimes be demonstrated immediately surrounding the nuclei of mononuclear and polynuclear leuco- cytes. Their clinical significance, if they ha^^e any, has not yet been settled. Relative Proportions of Different Varieties of Leucocytes. — In normal blood the different varieties of leucocytes are present in tolerably constant proportions to one another, which are, in the adult, in the neighborhood of the following figures: Small mononuclear leucocytes Large mononuclear and transitional leucocytes. Polynuclear leucocytes Eosinophile leucocytes 100 No. per cubic millimetre. 24 1,900 6 450 68 5,500 2 1.50 8,000 Mast cells may also be present normally up to the proportion of about 0.5 per cent. In abnormal conditions these proportions may undergo great Digitized by Microsoft® THE BLOOD. 31 fluctuations. In leucocytosis and leucopenia all the varieties of leucocytes may be increased or diminished together, without any change in their relative proportions ; or the change in the total number may be due to an increase or decrease in some particu- lar variety of leucocyte, which would correspondingly modify the relative proportions of the different kinds. Also, the total number of leucocytes may be normal, and yet their relative pro- portions may be modified. All these variations are in many cases of highly important diagnostic significance. The mononuclear leucocytes, ■ large or small, or both, are in- creased ("lymphocytosis ") relatively to the other varieties, either without or with an increase in the total number of leucocytes, in various conditions. In infants the lymphocytes normally are much more numerous (up to 50 or 60 per cent) than in the adult, at the expense of the polynuclears ; this lymphocytosis subsides parallel with the development of the child. Often debilitated conditions in the adult present relative lymphocytosis. Lympho- cytosis may also occur in chlorosis, pernicious ajisemia, syphilis, thyroid disease, and in various other conditions. In lymphatic leuksemia the greatest increase in the mononuclear leucocytes occurs, both in relative proportion (over 90 per cent of all the leucocytes) and absolute numbers. The polynuclear leucocytes may be relatively decreased where there is an increase in the proportions of other varieties. In infancy the polynuclears are relatively less than in the adult, ranging as low as 20 per cent. In most of the ordinary leucocy- toses, especially those accompanying inflammatory and infectious processes, hemorrhages, and malignant disease, there is a relative and absolute increase of the polynuclears, these corpuscles being the ones chiefly concerned in the ordinary vital processes and reaction to disease. Even when the total number of leucocytes is not above normal, the relative proportion of polynuclears may be increased ; such an increase has a similar significance to that of an absolute polynuclear leucocytosis. The eosinophUe leucocytes are diminished in relative number in many of the conditions presenting polynuclear leucocytosis. They are relatively and absolutely increased (an increase of these cells being called "eosinophilia") in infancy, trichinosis, asthma, certain skin diseases, some bone diseases (osteo-sarcoma, osteo- malacia, etc.), in some cases of leukaemia, syphilis, nervous and Digitized by Microsoft® 32 A MANUAL OF CLINICAL LABORATORY METHODS. mental disease, chlorosis and pernicious anaemia, and in various other conditions without known cause. Their relative number may vary from a slight increase up to 6, 10, or 20 per cent, or even more; in one case of trichinosis 68.2 per cent of eosino- philes have been observed, and in another case 77.3 per cent. Myelocytes are absent from normal blood. They occur occa- sionally in small numbers in many cases of severe anaemia and ordinary leucocytosis without any special evil significance. They are present in large proportion (averaging about 35 per cent) in splenic leukaemia ; and their presence in large numbers is pathog- nomonic of this disease. Mast cells and basopMle leucocytes are occasional stray visitors to normal blood. In splenic leukaemia they may comprise up to 10 per cent of the leucocytes. The percentages of the different kinds of leucocytes express only their relative number, and do not in themselves alone give any indication as to whether there is an absolute change in the number of the individual varieties of leucocytes per cubic milli- metre. To determine the absolute variations of leucocytes it is necessary to calculate the number of the different kinds in each cubic millimetre, and compare this with the normal standard (such as that given on page 30). Thus, in a case of leukaemia exhibiting 100, 000 leucocytes to the cubic millimetre, of which 50 per cent (50,000) were myelocytes, 35 per cent (35,000) poly- nuclears, 10 per cent (10,000) mononuclears, 2 per cent (2,000) eosinophiles, and 3 per cent (3,000) basophiles, all the varieties would be absolutely increased over their normal number per cubic millimetre, while in relative proportions the polynuclears and mononuclears would be diminished and the eosinophiles un- changed. Blood Plates. The blood plates are small, colorless, homogeneous, clear, hy- aline, spherical bodies, about 3 or 4 micromillimetres in diameter. They very readily disintegrate and disappear when the blood drop is exposed to the air, so that they are not usually to be seen in specimens of blood unless special precautions are taken to preserve them. They occur singly, or aggregated in clusters, often surrounded by or in the neighborhood of granular matter, probably the debris of disintegrated blood plates. They number Digitized by Microsoft® THE BLOOD. 33 about 300,000 or 400,000 to the cubic miUimetre of blood. Their number, nature, origin, and purpose are not positively known. They fluctuate in various disease conditions, but as their exami- nation is somewhat difficult and unreliable, and as their patho- logical significance has not been definitely determined, they are not generally regarded or utilized in clinical work. Parasites. The parasites that may occur in the blood are both vegetable and animal. The vegetable blood parasites are certain of the com- mon pathogenic and especially septiceemic bacteria (such as ty- phoid, anthrax, and influenza bacilli, streptococci, staphylococci, pneumococci), and the spirillum of relapsing fever. The blood is an unfavorable and antagonistic medium for ordinary bacte- ria, and while they must be present in the blood stream to some extent, their numbers are relatively so small that the chances of finding them in specimens of blood taken for examination are ordinarily very slight. Ocular examination of the blood for these bacteria is therefore usually negative; they can be ade- quately searched for only by cultural methods, employing a considerable quantity of blood for the purpose. The spirillum of relapsing fever (spirochseta Obermeieri) may be easily found in the circulating blood, sometimes in large num- bers, during and for a day or two before the febrile paroxysms of this disease. They are long, slender, wavy, or spiral fila- ments, 30 or 40 micromillimetres long, actively motile. They are best observed in fresh blood, but may also be stained. The animal parasites of human blood are the parasite of mal- aria, the filarise sanguinis hominis, and the schistosoma hsema- tobium. Parasite of Malaria. — The malarial parasite is a protozoan liv- ing in human blood. The full life history of the parasite has not been ascertained ; its period of existence in the human blood is only a portion of its life cycle. A prominent if not the only agency by which the parasite is introduced into the human body appears to be by means of certain species of mosquitoes (of the genus Anopheles) ; some observations have been made as to the life of the parasite in the mosquito, but nothing else is known as to the extra-human existence of the organism. 3 Digitized by Microsoft® 34 A MANUAL OF CLINICAL LABORATORY METHODS. The parasite under consideration is associated specifically and exclusively with malarial disease, and its detection in the blood is absolutely pathognomonic of malaria. Three distinct varie- ties of the malarial parasite and three corresponding types of malarial disease are distinguished, namely, the tertian, quartan, and sestivo -autumnal forms. These three are practically distinct varieties, not merging into one another (except possibly in ex- ceptional instances). 1. The tertian parasite in the human blood goes through a life cycle of about forty-eight hours. Its earliest forms appear during the latter part of or soon after the chill or paroxysm as small, color- less hyaline bodies within and occupying a small part of the red blood corpuscles, but not showing the complete lack of color and sharp outlines characteristic of vacuoles in these corpuscles. These intracorpuscular bodies usually exhibit active amoeboid movements, a feature distinguishing them from vacuoles. As time passes they increase in size, and in a few hours numerous small pigment granules appear within the parasites, dark in color, variable in size and shape, sometimes appearing as minute rods, sometimes as irregular fragments. The granules are dis- tributed irregularly through the parasite, and are generally in very active dancing motion. With the growth of the organism, the infected red corpuscle becomes paler and increased in size. As the parasite approaches maturity its amoeboid movements de- crease, and the pigment, which may be still in active motion or may become quiescent, tends toward a peripheral arrangement. Toward the end of forty-eight hours the organism attains full growth, has become about the size of a normal red corpuscle, and about it may be traced only the pale, narrow margin of the expanded red disc. After full size is reached the parasite in its further evolution may pass through a number of phases ; it may (a) undergo seg- mentation, (&) become a free extracorpuscular form, exhibit (c) fragmentation or (d) vacuolization, or (e) become flagellated. Segmentation is a process by which each parasite divides into a number of spores from which a new generation of the proto- zoan is developed, and is perhaps the normal course of the ma- tured parasites. It occupies a comparatively brief time; the chill or paroxysm occurs simultaneously with the process of segmentation. The process begins by the pigment becoming col- Digitized by Microsoft® THE BLOOD. 35 lected in tlie centre of tlie parasite in coarse, motionless granules. The protoplasm of the organism, which was previously clear, glistening, and hyaline, loses its lustre, becomes less refractile, and acquires a finely granular appearance ; in it radiating lines gradually develop, dividing the organism into segments. The fully developed segmenting body consists of a central collection of pigment surrounded by twelve to twenty separate radiating protoplasmic segments in a rosette or daisy -like form, each ray Fig. 4.— Tertian Malarial Parasite, a. Young hyaline intracorpuscular form ; 6, young pig- mented actively amoeboid form ; c, half-grown form ; d, maturing form ; e, segmenting form ; /, segments and pigment free in the plasma, after segmentation ; g, free extracor- puscular form ; h, fragmenting form ; i, flagellated form. exhibiting a faint punctate marking in its centre. The remains of the red corpuscle may or may not be visible about the para- site. The segmenting bodies sometimes, however, do not present quite such regular figures. A little later the segments become entirely separated from one another and appear as small spheri- cal hyaline bodies free in the plasma, with the collection of pig- ment also free in their midst or in their vicinity. The hyaline bodies are then supposed to make their way into red corpuscles and begin a new cycle of intracorpuscular existence. Sometimes, instead of segmenting, the parasites, having es- caped from or completely destroyed the surrounding red corpus- cles, appear in the plasma as free or extracorpuscular forms. These are spherical or rounded in shape, clear, hyaline, and glis- tening in appearance, and studded with pigment granules, which are usually quiet, though sometimes in motion ; they are of about Digitized by Microsoft® 36 A MANUAL OF CLINICAL LABORATORY METHODS. the size of red corpuscles, and do not exhibit amoeboid move ments. Sometimes half -grown intracorpuscular organisms leav their hosts and become free. The extracorpuscular parasite sometimes slowly disintegrate and disappear ("cadaveri forms ") ; sometimes they exhibit fragmentation, vacuolation or flagellation. Fragmentation is a division by the process of budding not in frequently manifested by the free extracorpuscular forms. A rounded prominence grows out from the margin of one of thes forms, increases in size (at the expense of the parent cell), am is finally severed from its parent as a smaller spherical separat pigmented body. Four or five smaller forms may be thus de rived from one large one, sometimes connected for a time bj filaments. Earely the large free parasites become vacuolated, a numbe: of round vacuoles appearing within them. The flagellated forms of the parasite are very striking and in teresting objects. In different countries they are observed witl different degrees of frequency, but in this vicinity they are seei in only about four or five per cent of the cases. They do not ap pear immediately after withdrawing the blood from the body, bu develop after the lapse of some time, ten minutes or more. Th( large, free, extracorpuscular bodies are the forms which becomi flagellated. Their pigment becomes exceedingly active and col lects in the centre of the parasite, while from the periphery ar( protruded from one to five thread-like processes or flagella, ii length several times the diameter of the organism, and usually possessing slightly bulbous extremities or slight bulbous expan sions in their course. These flagella whip about with vigoroui undulating motions and often cause a violent commotion amonj the neighboring red corpuscles. Undulating movements of th( periphery of the organism are also observable, and frequently thi flagella break off and, still undulating, make their way about ii the blood. The flagellate movements cease and the flagella dis appear after a few minutes. The significance of flagellation ii these parasites is not well understood ; the process may be a stagi in the extracorporeal existence of the parasite, or the flagella an perhaps a form of spermatozoon and associated with reproduc tive processes. Infection by the tertian parasite may consist in the presenc Digitized by Microsoft® THE BLOOD. 37 of one set of organisms ("single tertian") or of two sets ("dou- ble tertian "). All the parasites in each set begin their develop- ment at the same time, pass simultaneously through all the stages of their 48 -hour life cycle, and arrive at maturity and undergo segmentation together. The parasites do not exhibit all stages of growth at one time, but all the individuals of each group are always in like stages of development ; only in double infections two stages of growth are visible at the same time, the individuals of one set being full grown, while those of the other set are half grown. In single tertian infections a paroxysm occurs every other day ; in double t«rtian one set of parasites segments each day, and consequently a chill occurs daily. The tertian parasite produces typical intermittents and milder forms of malarial fe- ver, and is very susceptible to the action of quinine. It is a common form of malarial infection, and the only form indige- nous in the northern parts of the United States. 2. The quartan parasite is rare in this country, occurring in about one per cent of the cases of malaria in the vicinity of Baltimore; it is commoner in some other countries. Its life cycle is about seventy-two hours. Its appearance and course of development are similar 'to those of the tertian parasite, but there are certain well-marked points of difference. The protoplasm of the growing forms of the quartan parasite has a sharper, more refractive appearance, the amoeboid movements are less marked, the pigment is coarser and less actively motile, the infected red corpuscles are not enlarged or decolorized. The mature parasites are somewhat smaller than the red corpuscles. Segmentation is often preceded by a radiating or star-shaped arrangement of the pigment, which seems to pass from the periphery to the centre by definite channels. The segmenting forms divide into six to twelve segments or rays, frequently exhibiting exquisite sym- metrical forms. Instead of segmenting, the quartan organisms may become free in the plasma or undergo fragmentation, vacuo- lization, or flagellation in a manner similar to the tertian organ- ism. One, two, or three sets of quartan parasites may be pres- ent, each ripening on a separate day and forming a single, double, or triple quartan infection, with a chill every three days, two chills in three days, or a chill daily. 3. The sestivo-autumnal parasite is associated with the more se- vere, the more irregular and protracted, the continued and remit- DigKized by Microsoft® 38 A MANUAL OF CLINICAL LABORATORY METHODS. tent, the chronic and cachectic, and the more malignant forms of malarial disease, and is the most resistant to quinine. This form of malaria is abundant toward the tropical regions, gradually diminishes northward, and does not extend so far north as the tertian form. The earliest visible forms of the sestivo-autumnal parasite are small non-pigmented hyaline bodies in the red cor- puscles, rounded, irregular, or often characteristically ring- shaped, and usually showing active amoeboid movement. These forms are usually smaller and more hyaline than the correspond- ing tertian forms. These hyaline bodies gradually enlarge and fine pigment granules develop in them, not so numerous as in the tertian variety, and showing but little motion. In this stage the diameter of the organism usually does not exceed one -third that of the red corpuscles. The infected corpuscles are frequently Fig. 5.— iEstivo-autumnal Malarial Parasite, a, 6, c, d, Young Intracorpuscular forms ; e, seg- menting lorm ; /, crescent form ; g, elliptical form ; ft, crescent form within red corpuscle ; i,Ti, ovoid forms. brassy or deepened in color, shrunken, or their substance is re- tracted about the parasite. As the organism approaches matu- rity the pigment collects in the centre in solid clumps, and the protoplasm becomes peculiarly refractive ; these are the preseg- menting forms and occur only just before or during the parox- ysm. The segmenting forms are closely similar to those of the tertian variety, except that they are much smaller. Segmenta- tion of the aestivo -autumnal parasite takes place in the spleen or other internal organs, and during this process the parasites are retained within the viscera. Before and during sestivo-autumnal paroxysms, therefore, no parasites at all may be visible in the peripheral blood, and segmenting forms especially are seen only in very severe cases. Extracorpuscular free forms of this parasite are common, Digitized by Microsoft® THE BLOOD. 39 round, ovoid, or crescentic in shape. The crescentic and related ovoid organisms are very striking and distinctive forms of the sestivo-autumnal parasite, not occurring in other varieties of mal- aria. They are crescentic or elliptical-shaped masses of clear hyaline protoplasm, containing near their centre a conspicuous collection of coarse, dark, motionless pigment granules or rods ; a faint curved line can often be distinguished on the concave side of the crescents joining the two extremities, or on one side of the long elliptical forms. The length of the crescents is slightly greater than the diameter of the red corpuscles. The crescents develop from intracorpuscular forms, and may occa- sionally be distinctly seen within red corpuscles; often, espe- cially in stained specimens, the remains of a red corpuscle may be seen attached to the crescents ; and the faint curved line is a last vestige of the red corpuscle. Further than that they are a phase of the sestivo-autumnal parasite, little is really known as to the nature and significance of the crescents. They appear in aestivo-autumnal cases only after the lapse of several days from the onset of the disease; they are very persistent, resistant to treatment, and may be present alone in the blood for a long time without giving rise to paroxysms or definite symptoms, though relapses may occur attended with the advent of the hyaline forms. The rounded extracorpuscular free sestivo-autumnal parasites may undergo fragmentation and flagellation in a manner pre- cisely similar to the tertian f oz-ms, and both round and crescentic forms may become vacuolated. The life cycle of this variety of malarial parasite is not definite, but ranges from twenty-four to forty-eight hours. Several sets of the parasite may be pres- ent. The overlapping of the periods of segmentation of the sev- eral sets may cause continuous febrile manifestations without regular periodicity ; and the clinical manifestations of this infec- tion are very various. Mixed infections occasionally occur, in which two varieties of the parasite, as the tertian and sestivo-autumnal, are present in the blood at the same time. Phagocytosis is frequently observable in connection with the malarial parasite, in a very striking and typical way. The leu- cocytes can be seen to approach free parasites and engulf them within their substance, where the parasite after a time disinte- grates and disappears. Digitized by Microsoft® 40 A MANUAL OF CLINICAL LABORATORY METHODS. The pigment left after breaking up of the segmenting form remains free in the plasma for a time as conspicuous coarse blacl particles, constituting the condition known as "melansemia.- Leucocytes may also be seen containing similar pigment particle which they have taken up as such or which remain from disinte grated engulfed parasites; these are called "melaniferous leuco cytes. " The presence of pigment in the plasma or leucocytes ha an intimate association with malaria, that was recognized year before the parasite was discovered. A point that has not been much studied in connection witl the parasites is that relating to the septenary periodicity of mal aria, the tendency of the paroxysms to recur after intervals o: twenty-one, fourteen, or other multiple of seven days. The examination of blood for the malarial parasite is a simple and satisfactory procedure. The blood may be examined eithe: fresh or stained, but the former is much the easier and more sat isf actory, as the parasites are as conspicuous when fresh as whei stained, and in the preparation of stained specimens many para sites disappear. The tertian parasites are most conspicuous dur ing the chill, the sestivo-autumnal forms during the intermission but the examination may be made at any time (except that ses tivo-autumnal forms are apt to disappear during the paroxysm) The examination should be made before quinine is administered as that drug often very quickly clears the parasites from thi blood. The objects to be searched for are the parasites, als( pigment ia the plasma or leucocytes. All the pigmented formi of the parasite are conspicuous and can be distinguished witl ease and absolute certainty; the variety present, the stage o: growth, and usually the number of infecting sets are recogniz able, and usually the day when the next chill is to be expecte( can be foretold. The small, non-pigmented, intracorpuscularhy aline forms are much less easy to recognize with certainty or t( distinguish from vacuoles ; the most reliable differential point ii the occurrence of amoeboid movement in the parasite, though evei vacuoles may undergo changes of form. Filaria Sanguinis Hominis. — The filariee found in the circulat ing blood of man are embryonic forms of a parental nematodi worm which is lodged at some point in the lymphatics or tissues Pour varieties are known — filaria diurna, filaria perstans, filarij nocturna, and filaria Demarquaii, similar in their general feat Digitized by Microsoft® THE BLOOD. 41 ures, but with specific differences. The parental form of the filaria nocturna is the filaria Bancrof ti ; that of filaria diurna is supposed, to be the filaria loa ; those of filaria perstans and filaria Demarquaii are unknown. The filaria nocturna is the only va- riety which has been found in the United States. The filaria diurna is present ta the peripheral blood chiefly during the daytime, the filaria perstans continuously ; both occur in Western Africa. These filarise may affect the body for years, often without giving rise to appreciable symptoms, but in some cases producing marked disturbances. The filaria diurna is not known to produce pathological consequences ; filaria perstans in many individuals causes no morbid symp- toms, but in others is perhaps etiologically associated wdth the Congo sleeping sickness. Filaria Demarquaii has been differentiated in blood' (both nocturnal and diurnal) from a few individuals of Saint Vincent, an island in the West Indies, and possibly of the Niger region also ; little is known about this parasite. In one case rhabditis Mellyi, an immature nematode related to filaria, has been seen in the blood. The filaria nocturna is common in the tropics, and numerous cases indigenous in the southern half of the United States have been reported. The parent form of this worm, called the filaria Bancrofti, is about 94 millimetres long by .185 millimetre in diameter ; it is lodged in the lymphatic ves- sels about the genito-urinary organs and lower extremities, and causes obstruction of the lymph circulation, manifested by chy- luria, lymph scrotum, filarial abscesses, ele- phantiasis, and other conditions. The pa- rent female discharges large numbers of small embryonic forms, the filaria nocturna, which enter the circulating blood, and are also frequently found in the urine and pus from abscesses. The filaria nocturna is found in the blood only or chiefly in the nighttime, although it may be found in the daytime after prolonged rest in bed. They are about .3 millimetre long and Fig. 6. —Filaria Sanguinis Hominis Nocturna, in the blood, magnified 400 dia^ meters. From photograph ol living specimen. (F. P. Henry.) Digitized by Microsoft® 42 A MANUAL OP CLINICAL LABORATORY METHODS. .0075 millimetre in diameter, and therefore small enough, to pass through the capillaries. They appear in the blood in compara- tively small numbers, so that it may be necessary to examine a large quantity of blood in order to find them. They have a slen- der, worm-like shape ; the posterior extremity is tapering and pointed, the anterior end rounded and blunt. The embryo is enveloped in a delicate sheath. The live worm exhibits active wriggling motions, but without much progressive locomotive movement. In microscopical specimens it retains its vitality and motility for a long period, and is remarkably resistant to cold. The mosquito is supposed to be concerned in the life history of filarise, taking them from man and ultimately- depositing them in water, from which they are ingested by the human subj^t. Schistosoma lisematobium. — This is a trematode worm occurring chiefly in the veins of the pelvic viscera and giving rise to the disease called " bilhar- ziosis. " As the worms and their ova do not occur in the peripheral blood, their detection is not a matter of hsematological examination. In variola and vaccinia small actively amoeboid bodies appear in the blood in small numbers, about 3 or 4 micromillimetres in diameter, some with granular protoplasm, some pale and containing a few dark pigment-like gran- ules in their centre. In vaccinated children they appear about the sixth to the fourteenth day. They have been also observed in vaccinated calves and monkeys, and the granular body has been seen in the blood of normal children, and monkeys. Whether these bodies are hsemocytes or protozoa has not been settled; but if they occur in normal blood they are doubtless hsemocytes. B. TECHNiaUE OF BliOOD EXAMINATION. The object of clinical hsematological work is to determine the various features of pathological significance that have just been reviewed. As an aid to systematic examination and for record- ing and reporting the results, a printed blank form presenting the points searched for is useful, such as that on the following page. To determine these features the following are the principal procedures in general use, besides which there are a number of unusual, difficult, or unsatisfactory methods rarely employed : Obtaining the specimen of blood. Macroscopic examination. Estimation of haemoglobin. Enumeration of red corpuscles. Digitized by Microsoft® THE BLOOD. 43 Date, , 190 . Examination of Blood. Name of patient Macroscopic appearance Bed blood corpuscles : Number of red corpuscles per cubic millimetre ( . . per cent of normal). Haemoglobin per cent. Corpuscular haemoglobin ratio Macrocytes Microblasts Microcytes Normoblasts Polychromatophilia. . , Megaloblasts Poikilocytes Rouleau formation Volume of red corpuscles Leucocytes: Number of leucocytes per cubic millimetre Proportion of white to red corpuscles, 1 to Relative proportions of leucocytes : Small mononuclear Polynuclear. Large mononuclear Eosinophile. Transitional Myelocytes. . Blood plates '. Granules Pigment Fat Specific gravity Coagulation Widal reaction: result ; dilution, 1 to ; time. Parasites Malarial parasite Remarks (Signature) Digitized by Microsoft® 4:4 A MANUAL OF CLINICAL LABOEATOBY METHODS. Determination of corpuscular haemoglobin-ratio. Enumeration of leucocytes. Determination of proportion of white and red corpuscles. Microscopical examination of fresh specimens. Microscopical examination of stained specimens, differential counting of leucocytes, etc. Widal test for typhoid fever. Determination of volume of red corpuscles. Determination of specific gravity. Bacteriological examination. Chemical examination. Micrometry. Determination of coagulation time. Study of blood plates. Tests for blood. The various points to be determined, as outlined in the blank form presented, are ascertained by the technical procedures men- tioned, as follows : Macroscopic appearance, by macroscopic inspection. Number of red corpuscles per cubic millimetre, by enumerating tliem. Amount of lisemoglobin, by special tests therefor. Corpuscular hgamoglobin-ratio, by calculation from number of red corpus- cles and amount of haemoglobin. Macrocytes, microcytes, polychromatophilia, poikilocytes, microblasts, normoblasts, megaloblasts, and rouleau formation, by microscopic examina- tion of fresh and stained specimens. Volume of red corpuscles, by centrifugal method. Number of leucocytes per cubic millimetre, by enumerating them. Proportion of white to red corpuscles, by calculation from the numbers of these cells. Relative proportions of leucocytes, and number of nucleated red corpus- cles, by differential count of stained specimens. Blood plates, by examination of fresh or stained specimens, or by special methods. Granules, pigment, and fat, by examination of fresh or stained specimens. Specific gravity, by special methods. Coagulation, by macroscopic examination, microscopic observation of fibrin formation in fresh specimens, or special methods. Widal reaction, by special "test. Parasites, by examination of fresh or stained specimens, or (in the case of bacteria) by cultural methods. In making a hsematological examination all the apparatus and materials required for the tests proposed to be made should Digitized by Microsoft® THE BLOOD. 45 be in readiness and convenient, so that the work necessary in the presence of the patient can be completed with despatch and with but a single puncture of the skin, which with nervous patients is quite an ordeal. The needle for puncturing the skin, a mixture of equal parts of alcohol and ether for cleaning the skin, sterile or clean gauze, water, the hsemoglobinometer and its capillary tubes, the pipettes for counting the red and white corpuscles, the dilating fluids, cleaned cover-glasses and slides, thumb forceps, or what- ever is needed at the bedside in the tests to be made, should be at hand,' ready for instant use. After duly procuring the specimens of blood, the outfit is taken to the laboratory and the remainder of the work done there. Obtaining Specimens of Blood. — For ordinary purposes only a drop or two of blood is necessary or available. This is usually taken from the lobe of the ear, which is conveniently accessible and comparatively insensible lo pain. To make the puncture a tiny lancet or a surgical needle with a cutting edge is employed ; this shoiild of course be aseptic when used. A straight Hagedorn needle about 5 centimetres long is very satisfactory for the purpose ; this may be run through the cork of a vial of about 4 cubic centimetres capacity, which, filled with alcohol to keep the needle sterile, makes a very convenient receptacle or case for the needle, while the cork serves as a handle for the instru- ment (Fig. 7). Before making the puncture the skin should be cleaned and sterilized by rubbing it with gauze saturated with alcohol, or equal parts of alcohol and ether, and then dried. The needle is then plunged with a quick, firm motion into the skin, and on withdrawing it the slight puncture can be enlarged if necessary by means of the sharp cutting edge of the needle. The puncture should be large enough so that a drop or two of blood will emerge from it spon- taneously, as if too much pressure is required to press the blood drop out lymph will also be forced out and affect the result. The needle prick causes very little pain, though some individuals will be nervous about it. Macroscopic Examination.— The appearance of the blood drop, IG. 7.— Hage- dorn Needle in Vial of Alcohol, for use in ob- taining speci- mens of blood. Digitized by Microsoft® 46 A MANUAL OF CLINICAL LABORATORY METHODS, as it oozes out on the skin, can be noted. Usually it will appear normal ; but marked variations of color may be perceptible to the naked eye, as undue pallor in severe anaemias, etc., or marked changes in consistency may be noted, as a thin and watery or a thick and tarry condition. The time required for coagulation to occur may be roughly estimated and any tendency to haemo- philia be detected. Estimation of Haemoglobin. — Most of the methods proposed for the determination of the haemoglobin are colorimetric in char- acter, carried out by \ / „ matching the tint of a L .^9*1 — »^ - definite dilution of the blood with a standard color scale. According to another method, the haemoglobin is estimat- ed indirectly from the specific gravity. Of several instru- ments introduced for the purpose the haemo- globinometer of Fleischl is the one in most general use. Fleischl 's instrument (Pig. 8) possesses a sliding frame gradu- ated along one side (PP), to which is attached a wedge-shaped red glass slide (KK), thin atone end and increasing in thickness to the other end, so that all shades of red are presented along the slide from a deep red at the thick end to a very pale red at the thin end. By means of a sort of rack-and-pinion arrangement (E), worked by a milled disc (T), the sliding frame can be moved one way or the other and the scale can be read off at the mark M. On the stage of the instrument is a cylindrical metal receptacle G, with a glass bottom, divided into two compart- ments (a, a') by a vertical partition. Beneath the stage is a white reflector (S) by which light can be thrown upward through the receptacle G. The latter is so situated that the colored glass scale KK lies directly underneath the compartment a', while noth- Fiu. «.— Fleiscm's HsemogloMnometer. (Leitz.) Digitized by Microsoft® THE BLOOD. 47 ing is interposed between compartment a and tlie reflector S. Accompanying the instrument is a small capillary glass tube of definite capacity, fastened in a metal handle (Fig. 9). To make the estimation of haemoglobin one end of this capil- lary tube is touched to the blood drop, when the blood by capil- lary action should promptly and immediately flow into and com- pletely fill the tube. The cap- l|piIZ:Jl»- illary tube when used should be dry, as a trace of moisture will ^"*- ^■~;9^Pi;,i*^y ^'p^"? accompanying •' ' Flelschl's Haemoglobinometer. dilute the blood and impair the test ; it should also be perfectly clean and free from oil (as by the use of equal parts of alcohol and ether), or the capillary ac- tion will be interfered with and the blood will not flow into it. The tube being completely filled, any superfluous blood on the outside should be removed, and the blood within it is entirely washed, by means of a jet of water from a pipette or medicine dropper, into the compartment a of the haemoglobinometer. This compartment iS then flUed with water to the brim, the blood at the same time being thoroughly mixed with it by stirring with the handle of the capillary tube ; the upper level of the fluid should form a convex surface rising slightly above the upper edge of the metal receptacle. The other compartment (a') is then filled with water to the same height as compartment a, care being taken that the fluids on the two sides of the partition do not mix. The estimation must be made by artificial light, such as a candle placed a short distance in front of the hseraoglobinometer, in a darkened room from which all daylight is excluded. This is necessary because white daylight gives a wrong value to the color scale. The refiector S is adjusted so as to reflect the light upward through the liquids in receptacle G. A tube of paper or other material, preferably black inside, about two and a half centimetres in diameter and twenty -five or thirty centimetres long, is then placed upright over the receptacle G, to exclude extraneous light during the examination. The eje is then ap- plied to the upper end of the tube ; on looking down this there is seen a definite dilution of blood, pale red in color, in com- partment a, while through compartment a' is seen a portion of the red color scale KK, both illuminated by the light entering Digitized by Microsoft® 48 A MANUAL OF CLINICAL LABORATORY METHODS. from below. On moving the scale by means of the disc T, the color seen through compartment a' changes in its intensity. The scale should be moved back and forth untU the color in the two compartments is made to match exactly. Then the reading of the graduated scale PP at the line M represents the percentage of hsemoglobin present, the normal amount of haemoglobin being taken as 100 per cent. This method is only approximate, as an exact matching of the tints is difficult, and the eyes of different individuals, or even the two eyes of the same individual, will vary in their judgment of colors. In order to promote accuracy in the matching of the colors the eye should be used only for a few seconds at a time, and then rested, in order to avoid fatigue ; it is said also to be better while using the instrument to face one end of the sliding scale than to face the light, as the two lateral halves of the eye are more sensitive to color differences than the upper and lower halves. The paler tints are especially difficult to match closely ; so that when the amount- of haemoglobin is very low it is better to use two capillary tubes of blood in making the dilution, rather than one, and divide the result by two. The method is probably practically correct iu its findings within about five per cent, and is sufficiently close to afford clinical information of great value. A possible defect is that the readings of the Pleischl apparatus are rather low, as often blood from normal and robust individu- als registers several points below 100 per cent. As the proportion of hsemoglobin in most conditions is supposed to vary- closely according to the specific gravity of the blood, tables such as the fol- lowing have been prepared showing the approximate percentage of hssmo- globin corresponding to the various specific gravities (Hammerschlag's method). Specific gravity. Hajmoglobin. 1033-1035 35-30 per cent, 1035-1038 30-35 1038-1040 35^0 1040-1045 40^5 1045-1048 45-55 Specific gravity. Hsemoglobin. 1048-1050 55-65 per cent. 1050-1053 65-70 1053-1055 70-75 1055-1057 75-85 1057-1060 85-95 The specific gravity being determined, this table shovrs the correspond- ing percentage of hsemoglobin; but these figures are not applicable in cases of dropsy, leuksemia, and perhaps certain anaemias. Enumeration of the Red Corpuscles. — The number of red and white corpuscles to the cubic millimetre of blood is determined Digitized by Microsoft® THE BLOOD. 49 by means of the Thoma-Zeiss htemaeytometer (Fig. 10), an ap- paratus wliich yields VL'vy .satisfaetory results, reliable probably within two or tlii-ee per cent. Tliis outfit consists of (a) two pipettes for diluting the blood to a detiuite degree, one for the red and one for the white coi-puscles, and (b) a glass side with a ruled ehanibcr for counting the coi'puscles under the microscope. In counting the I'ed corpuscles it is tiivst necessary to dilute the blood to a. sufticient and known degree (one or two hundred Fi(i. 10.— Tlioina-Zpiss Ha^'maeytonietpr. (Leitz.') times) to bring the coi-puscles within cduntable numbers. For this purpose a number of dilution fluids are available, about the best of wliicli is that of Gowers, the formula for which is as follows : Sodiiuii sulpliate 7.5 Acetic acid 20 Water, distilled 12."i This and all other diluting fluids should lie kept perfectly clear and free from solid itarticles l)y filtering frequently, or whenever sediment collects fi-om long standing; the presence of foreign particles seriou.sly interferes willi the process of count- ing the corpu.scles. Another diluting fluid for the red corpuscles is that of Toi.sson, which stains tlie leucocytes violet, and is there- fore especially useful when the leucocytes are relati\cl\' numer- ous and if. is convenient to have them inadi- conspicuiius so as to 4 Digitized by Microsoft® 60 A MANUAL OF CLINICAL LABORATORY METHODS. omit them in counting. The composition of Toisson's fluid is as follows : Methyl violet 5 B 0.025 Sodium chloride 1- Sodium sulphate 8. Neutral glycei in 30. Water 160. Tor counting the red corpuscles the blood is measured and diluted by a special pipette for these corpuscles (Fig. 11). This has a bulbous expansion or chamber (E) in the course of a cap- illary tube. The longest portion of the capillary tube is grad- uated into ten equal parts from the point (S) up to the mark 1. Fig. U.— Pipette for Diluting Red Blood Corpuscles. (Spencer Lens Company.) The bulbous portion is of such size that from the mark 1 to mark 101 its capacity is exactly 100 times that of the ten divisions of the capillary tube. The bulb contains a small piece of glass to aid in mixing the blood. To the blunt end of the pipette can be attached a rubber tube (G), provided with a mouthpiece M. To use the pipette, the mouthpiece is taken in the lips or ■ teeth, the point of the tube is immersed in the drop of blood flowing from the ear, and by gentle s action blood in a continuous and unbroken column is drawn into the capillary tube exactly to one of the divisions. Superfluous blood being removed from the outside, the point of the pipette is then quickly plunged into the diluting fluid, which is drawn up into the instrument exactly to mark 101. Twirling the tube while drawing in the dilution fluid aids in breaking up clots and mixing the corpuscles. With the blood drawn up to mark 1, and the diluting fluid to mark 101, the blood is diluted exactly 100 times. The blood may be drawn to any of the divisions of the capillary tube, not neces- sarily to mark 1, but the corresponding dilution must be noted to be used in the subsequent calculations. Usually it is most convenient for counting to draw the blood to mark 0.5, which Digitized by Microsoft® THE BLOOD. 51 gives a dilution of .5 to 100, or 1 to 200. After filling the pip- ette, the rubber tube is removed, the two ends of the pipette are closed by the thumb and a finger, and the pipette is shaken vig- orously a few times to mix the corpuscles thoroughly with the diluting fluid; the pipette may be also twirled for the same pur- pose. The pipette is then laid aside in a horizontal position until the count is made ; or if it is to be transported some dis- tance it may be placed lengthwise in a rubber band which tightly closes the two ends. The pipette must be perfectly clean and dry at the time of use. No time should be lost in the manipulations, otherwise the ( fi bed a bod Fig. 12.— Euled Slide and Chamber tor Counting Blood Corpuscles, top and side plans. blood will clot in the tube and cause trouble, vexation, and delay in cleaning. If the blood is once drawn up above the mark at which the dilution is to be made, the pipette must be cleaned and dried before further use, as the corpuscles adhering to the tube will falsify the result. The next step is counting the corpuscles, which is done by means of a ruled counting chamber (Fig. 12). This consists of a glass slide (a) to which is cemented a square glass plate (&) with its center cut out in circular form. In the middle of this circular opening is cemented to the slide a smaller glass disc ( a ■ a c d Fio. 20. — Ova ol Intestinal Worms (X 275). a. Taenia saginata, with and without albuminous covering; 6, ascaris lumbricoldes ; c, trlohurls trlcMura (trichocephalus dlspar); d, uncl- naria duodenalis (ancbylostoma duodenale). up to 2.5 centimetres in length. Sometimes the shape of the segments is atypical, as where they are very short (a few milli- metres), but of full width, where they are bead-like, or where the segments are not distinctly marked off from one another. The most conspicuous and distinctive feature of the anatomy of the segments is the uterus, which consists of a central longitudinal portion giving off 17 to 30 branches on each side. When filled with ova the uterus is very conspicuous. The ova (Fig. 20) are short elliptical or spherical in form, about 30 by 35 micromillimetres in size. Each is enclosed in a conspicuous, thick, translucent, double-contoured vitelline mem- brane exhibiting fine radial markings ; outside of this is some- times a clear, granular, albuminous envelope. The interior of the ovum is a brown, opaque, granular material containing the embryo in various stages of development. Digitized by Microsoft® 112 A MANUAL OP CLINICAL LABORATORY METHODS. The ova and segments are passed in the faeces, on which the diagnosis is made. If a large length of the worm is passed, it is important to search for the head to see if it is entirely expelled. Trematodes. — These are flat or conical worms, common in ani- mals, but rare in man. They inhabit the liver, the cavity of the intestine, or the intestinal wall, from which the ova or worms may pass into the faeces. The following trematodes or ova have been found in the faeces of man : schistosoma haematobium, fas- ciola hepatica, dicroccelium lanceatum, opisthorchis felineus, opisthorchis sinensis, distoma conjunctum, fasciolopsis Buskii, heterophyes heterophyes, amphistoma hominis, and paragonimus Westermanii. Nematodes, or round worms. — The worms of this class which have been found in the human intestine are: ascaris lumbri- coides; ascaris canis; ascaris maritima; oxyuris vermicularis ; unciuaria duodenalis; strongyloides inestinalis; trichuris tri- chiura ; trichinella spiralis. Ascaris Lumhricoides. — This common parasite is a slender round worm about 4 to 6 millimetres in maximum diameter, tapering to both ends. The head is tri-lobed, the tail pointed and in the male bent at an angle and provided with two spicules. The female is 30 to 40 centimetres long, the male about two-thirds as large. The ova (Fig. 20) are of an oval form, about 60 by 75 micro- millimetres in size. The interior of the egg is a brown, granular material surrounded by a vitelline membrane, and outside this is an irregular, nodular, hyaline, albuminous envelope. Both the worms and the ova may be present in the faeces, es- tablishing the diagnosis. Oxyuris Vermicularis. —This common worm somewhat resem- bles the ascaris, but is much smaller, the males being about 4, the females about 10 millimetres long. The worms may be pres- ent in the intestine (especially the caecum) and in the faeces in large numbers, appearing like bits of thread. The ova are about 50 by 25 micromillimetres in size, coarsely granular, and sur- rounded by a well-marked, doubly-contoured vitelline envelope. The eggs are not ordinarily voided by the worms within the intes- tine, hence they do not usually occur by themselves in the faeces. Uncinaria duodenalis (anchylostoma duodenale) causes a very severe anaemia, and occurs especially in underground workers. Digitized by Microsoft® THE F^CES AND INTESTINAL DISCHAEGES. 113 While commonest in Egypt, Italy, the West Indies, and else- where, cases originate in the United States, and in severe anae- mias the faeces should always be examined for the ova of this worm. The diagnosis of pernicious anaemia cannot be considered established until such an examination has been made with nega- tive result. The ova are abundant in the faeces, and the worms appear after the use of proper vermifuges. The ova (Fig. 20) are elliptical, 30 by 50 micromillimetres or more in size, and consist of a colorless, thin, distinct vitelline envelope enclosing from one or two to numerous rapidly dividing cells, which are brown, granular, and prominently nucleated. The male adult worm is 6 to 12 millimetres long, with an expanded copulatory pouch and slender penile organ at its posterior end. The female is 10 to 18 millimetres long. The head is turned dorsally and has a hollowed mouth armed with six hooks. Strongyloides intestinalis is a small worm 1 or 2 millimetres long, which causes diarrhoea. It is common' in Cochin China, but has been found in other countries and even in the United States. The female is viviparous, so that the immature worms and not the ova occur in the faeces. Triohuris triohiura (trichocephalus dispar) is a common intes- tinal parasite and its ova are frequently seen in the faeces, but it causes no troublesome symptoms. The worms are about 4 or 5 centimetres long, and being attached to the intestinal mucosa do not often themselves appear in the faeces. The ova (Fig. 20) are brown and finely granular, covered with a thick, hyaline, doubly contoured vitelline envelope ; they are elliptical in shape, with a small, clear, rounded protuberance at each end (a sort of plug fill- ing openings in the shell), and measure about 50 by 25 micro- millimetres in size. Trichinella Spiralis. — The adult forms of the trichina, 1.5 to 4 millimetres long, occur in the intestine. They are viviparous, and the discharged larval forms penetrate the intestinal wall and are carried to the muscles, where they become encysted. In cases of trichinosis the parasites may be found in the stools. Insect larvae, as those of lucilia macellaria and others, are at times passed with the faeces. Meconium is the dark, tarry material in the intestine of the foetus. It is composed entirely of materials secreted ' into or Digitized by Microsoft® 114 A MANUAL OF CLINICAL LABORATORY METHODS. given off from the alimentary tract, such as fat, cholesterin, bili- rubin, bile acids, varous salts, epithelium, etc. Its examination is rarely of diagnostic service. B. PHYSICAL CHARACTERS OF THE F^CES. Amount. — The daily quantity of faeces passed varies with the diet and condition of the intestine, being greater with vegetable food possessing abundant indigestible material, and less with a more completely digestible proteid diet. The normal amount ranges from about 60 to 250 grams per twenty-four hours. As the water of the faeces is such a variable factor in normal and abnormal conditions, in a precise determination of the daily amount of faeces passed it would be necessary to determine the amount of solids and liquid of the faeces separately. Consistency. — The consistency of the faeces depends upon the relative amount of solids and fluids present. The consistency is ordinarily sufficiently firm to enable the faeces to assume and maintain definite form. When the water is increased, as in di- arrhoeal conditions, the faeces become softer, semi-solid, creamy, or watery. When the water is diminished, as from reabsorption by prolonged stay in the intestine in constipation, the faeces be- come hard and dry. In some instances the stools are pasty or tarry. Form. — Soft or fluid faeces are incapable of retaining perma- nent form. Normal faeces possess the well-known cylindrical shape. Faeces that have been a long time in the intestine may be passed in hard, dry, scybalous balls or masses. Stenosis of the rectum may result in flattened and ribbon-like faeces. Homogeneity. — Ordinarily the faeces are fairly macroscopically homogeneous, all the elements being evenly mixed together. Large undigested vegetable masses may even normally be present, destroying the homogeneous character of the stools. When the faecal masses are passed along with a quantity of fluid, or with unmixed mucus, casein coagula, calculi, foreign bodies, worms and the like, the ensemble is heterogeneous. Color.— The color normally ranges from light yellow to dark yellow, brown, or almost black, depending chiefly on the pres- ence of stercobilin. The greater the amount of water present, the lighter usually is the color. In young infants a green color Digitized by Microsoft® THE F^CES AND INTESTINAL DISCHARGES. 115 from biliverdin is not abnormal ; or stools colored yellow with bilirubin may turn green on standing. Milk diet, as in infants, gives the fseces a light yellow color ; meat diet produces a darker color, huckleberries and claret a blackish brown. Chlorophyll may give a greenish tinge. Iron, manganese, and bismuth dark- en or blacken the faeces ; calomel may turn them greenish ; rheum, senna, and other drugs give a bright yellow. In pathological conditions the color may have much diagnos- tic significance. Acholic, putty-colored, or colorless stools are due to deficiency of bile, or excess of fat, or both. Blood mixed with the stools gives a blackish color. In infants a green color may be generated by some of the bacteria causing the diarrhoea. Odor. — The characteristic repulsive odor of the f feces is due chiefly to indol and skatol, also to volatile fatty acids, gases (methane, hydrogen sulphide), etc. This odor is increased after meat diet, in proteid indigestion, in constipated conditions, or from any similar causes giving rise to excessive proteid decom- position and indol formation. The faeculent odor is less in lien- teric and diarrhceal conditions, on vegetable diet, and especially in infants on milk diet, proteid decomposition being less under these circumstances. Excessive acid fermentations produce a sour odor of the f seces, especially in infants. A fetid odor may arise from necrotic processes, as in rectal cancer. Reaction. — ^The reaction of the faeces is normally neutral, weakly acid, or weakly alkaline. The acidity is due to the or- ganic acids, the alkalinity to ammonia and other principles. The reaction of the surface of the f secal masses may be different from that of the interior. Abnormally the reaction may be strongly acid or strongly alkaline, as from excessive acid or ammoniacal fermentations. C. EXAMINATION OF THE E^ffiCES AND INTESTINAL DISCHABGES. The chief methods of examination of the faeces for the deter- mination of the points above considered are macroscopical, mi- croscopical, chemical, and bacteriological. Macroscopical examination of faeces gives information as to their form, consistency, homogeneity, color, etc., with all that these imply, and enables a sufficiently close estimate to be made of th& Digitized by Microsoft® 116 A MANUAL OP CLINICAL LABORATORY METHODS. relative amount of water present. It i-eveals the presence of macroscopic masses or fragments of undigested vegetable tissue, fat, coagulated casein, mucus, blood, pus, pieces of tissue, cal- culi, foreign bodies, and parasites. The presence or deficiency of stercobilin, and the presence of blood or other abnormal col- oring matters are also manifested. The odor may afford some information as to the extent and nature of fermentation processes in progress (proteid, acid, am- moniacal, etc.). To examine segments of tapeworms as to the morphology of their uterine organs the segments may be flattened out by being pressed between two glass slides, so as to make their internal or- gans more visible. To concentrate small parasites, ova, etc., so as to render their detection more easy, the faeces may be thoroughly stirred up with a large quantity of water in a tall cylindrical vessel, and the mixture allowed to settle for a few minutes. The ova and para- sites settle very quickly, and the supernatant material can be poured off without loss of parasites. More water is added and stirred up with the residue, the mixture allowed to settle for a minute or two, and the upper portion poured off. This process is repeated a number of times, gradually diminishing the amount of water added, until the faecal material is largely washed away. The residue containing the parasites may then be spread in a shallow flat dish and the parasites picked out ; or it may be ex- amined microscopically. Microscopical Examination of Faeces. — If the faeces are fluid a representative drop is placed on a slide and covered with a cover- glass ; or if hard a minute portion is taken (the bacteriological platinum loop is convenient for the purpose) and mixed with a drop of water on the slide, and the cover applied. The specimen thus made reveals the following under the microscope so far as they are present: small fragments of vegetable tissue, chloro- phyll and starch granules, particles of muscle or fibrous tissue remaining from the food, a large amount of amorphous and gran- ular detritus, fat, fatty-acid crystals, bismuth crystals, choles- terin crystals, mucus, ammonio-magnesic phosphate crystals, calcium and magnesium phosphate, calcium oxalate, calcium carbonate, spermin-phosphate crystals, haematoidin, red blood corpuscles, leucocytes, epithelium cells, fragments of intestinal Digitized by Microsoft® THE F^CES AND INTESTINAL DISCHARGES. 117 tissue, saccharomycetes, bacteria, small animal parasites or their ova. Special treatment is necessary to aid in the recognition or demonstration of some of the microscopic bodies. The addition of Sudan III. solution (page 10) stains particles of fat red and so makes them obvious. The addition of iodine solution (page 11) demonstrates any starch granules that may be present by coloring them blue ; iodine also gives cells a yellow color, and stains saccharomycetes and some bacteria brown, others Wue. The addition of acetic acid may make leucocytes and epithelium cells more distinct by clearing the granules from their cytoplasm and making the nuclei more prominent ; acetic acid also makes mucin more distinct, dissolves any phosphatic crystals present, and clears up carbonate crystals with the evolution of bubbles of gas. Cells may also sometimes be demonstrated by the use of the ordinary nuclear stains, dried cover -glass films being pre- pared, fixed, and stained. Bacteria may be brought out by the usual staining methods ; the presence of the tubercle bacillus can sometimes be demon- strated by the use of the specific stains for this bacillus, as Gab- bet's method. To examine for amoeba coli the stool should be fresh, passed in a warmed receptacle, and examined on a warm slide so as not to check the amoeboid movements. Fragments of tissue may be examined extemporaneously by teasing or other- wise, or by hardening and sectioning in the usual manner. For- eign bodies, seeds, vegetable fragments, and the like may be crushed or teased until fine enough for microscopical examina- tion. Pulpy vegetable masses may be distinguished from mucus by microscopical examination, the former exhibiting vegetable structure. Mucin may sometimes be demonstrated by its staining reac- tions, which are of a modified basophilic nature; after proper fixation (alcohol, mercury bichloride, or heat) it stains blue with methylene blue, green with the triple stain, reddish with toluidin blue (1-per-cent solution in 5-per-cent phenol). Fibrin stains red with the triple stain, blue by Weigert's gen- tian-violet method. Cellulose stains yellow with iodine solution, and then turns blue after being quickly washed and allowing pure sulphuric acid to flow under the cover-glass. Digitized by Microsoft® 118 A MANUAL OF CLINICAL LABORATORY METHODS. Chemical Examination of Faeces is only occasionally required to determine as to the presence of special substances, not having as yet been extensively elaborated for clinical application. A number of micro -chemical tests have been already given. The reaction may be taken by litmus paper. If the faeces are hard and dry, the paper should be moistened, thrust into the fae- cal mass for a time, and the adherent faeces then washed off. A quantitative estimation of the degree of alkalinity or acidity may be made by titrating a watery extract of a known amount of the faeces with decinormal acid or alkali. The amount of water and solids may be determined by weigh- ing before and after drying. In exact investigations of metabolism the total nitrogen of the faeces may be determined, as by Kjeldahl's method. Fat may be recognized qualitatively by its solubility in ether or its reaction with Sudan III. Fat and fatty acids can be ex- tracted with ether and thus estimated. Insoluble soaps may be decomposed by digestion with dUute sulphuric acid, and the separated fatty acids extracted with ether. Proteids may be extracted by digesting the faeces in water acidulated with acetic acid, and then filtering. The ordinary tests for albumin, albumose, and peptone are then applied to the filtrate. To test for carbohydrates, boil the faeces with water, filter, concentrate the filtrate by evaporation, and test it for starch and erythrodextrin with iodine, and for sugar by the usual tests. The ferments, proteolytic, amylolytic, or milk-curdling, may be tested for by treating coagulated proteid, starch solution, or milk with acid or alkaline watery extracts of the faeces. Stercobilin is ordinarily indicated by the color of the faeces. To determine if the lack of color of colorless stools is due to ex- cess of fat or lack of stercobilin, extract the fat with ether ; if stercobilin is present the residue will assume a yellow color. Bilirubin may be tested for by adding a drop of nitroso- nitric acid to the faeces, or by testing a watery or chloroform ex- tract in the regular way, a green color indicating the presence of bilirubin. Mucus particles may be tested by hardening them in alcohol or mercuric bichloride and then staining with the triple stain ; mucin stains green, fibrin red. Digitized by Microsoft® THE F^CES AND INTESTINAL DISCHARGES. 119 Blood may be tested for by drying a small portion of the sus- pected material and applying the hsemin test, or by applying the guaiacum test to a watery extract. Calculi: In searching for suspected biliary or other calculi, the faeces should be stirred with water and washed through a sieve. The material thus obtained (in the form of small parti- cles or large concretions) may then be examined chemically as to its nature. Bacteriological Examination of the Faeces. — Such a large num- ber of bacteria of so many different kinds occur in the faeces that it is difficult to isolate and determine those present, and in most cases is utterly impracticable with the time and labor available for clinical purposes, although their recognition would be of great service clinically. Tubercle bacilli can sometimes be demonstrated by their spe- cial staining reactions ; care should be taken to exclude smegma bacilli. Methods have been presented for the detection of typhoid bacilli in the faeces, as those of Eisner and Piorkowski, but have not come into general use. In cases of cholera Asiatica the comma bacilli may be so predominant as to be readily sepa- rated. Digitized by Microsoft® VI. THE SPUTUM. The sputum is an abnormal product altogether, and indicative of disease of the respiratory system or adjoining organs. The object of its examination is to aid in determining the nature and seat of the disease. The amount of sputum expectorated is very variable in differ- ent cases, ranging from a few cubic centimetres up to 500 or more in twenty-four hours. Aside from temporary conditions like pulmonary oedema and hemorrhage, or rupture of abscesses, continuous profuse expectoration, serous or purulent, may occur in bronchorrhcea, bronchiectasis, chronic bronchitis, phthisis, etc., and causes a severe drain on the vital energies. Composition. — The chief elements entering into the formation of sputum are: Serum, mucus, pus, blood, air, pigment, putre- factive products, parasites (vegetable and animal), leucocytes, red blood corpuscles, epithelium, fragments of tissue, Cursch- mann's spirals, Dittrich's plugs, fibrin, crystals, calculi, foreign bodies. The serous or watery constituents of the sputum originate from oedematous transudation, inflammatory exudation, or hypersecre- tion, thus being expectorated in large amount in pulmonary oedema and bronchorrhcea. The mucus of the sputum originates from catarrhal inflamma- tion, and contributes to the expectoration its viscid tenacious consistency. It is recognizable macroscopically and (by its form and staining reactions) microscopically. It is usually light and buoyant, and floats in water. The purulent constituents of the sputum originate from sup- purative processes. Sometimes sputum has the creamy consist- ency of pure pus ; but in the suppurative catarrhal conditions common in the respiratory passages the pus is intimately mixed with mucus, giving the sputum a mingling of the characters of the two. The proportion of pus present is measured by the abundance of leucocytes observed microscopically, their number being less the less is the pus and the greater the amount of mucus Digitized by Microsoft® THE SPUTUM. 121 and other materials mingled with the pus. Pure pus usually sinks in water. Blood in the sputum arises from congestive and hemorrhagic conditions. If present it may range in amount from slight streaks of blood in the sputum, through mixtures of blood with serum, mucus, or pus, to pure blood. It is recognized by its color, by the presence of red blood corpuscles microscopically, and if necessary can be tested for chemically. When expecto- rated freshly shed it is bright red in color, venous blood being darker than arterial ; but after remaining in the lungs for a time it turns dark, or the hsemoglobin may undergo transformation to dark or brown pigments (as in the rusty and prune-juice ex- pectoration of croupous pneumonia). Air may or may not be mixed with the sputum, forming a frothy, foamy, or bubbly mass ; and sputum when put into water sinks or floats according to its air content. It is more abundant the smaller the bronchi in which the sputum is generated ; and is especially scanty in sputum originating from phthisical cavities or large bronchi. The main substance of the sputum is ordinarily formed of serum, mucus, pus, and blood; any one of these may predomi- nate, or two or more may be mixed in varying proportions, and air may be present or absent. Sputum owes its consistency and form to these ingredients, and according to the predominating constituents specimens of sputa can be described as serous, muco- serous, mucous, muco-purulent, etc. The relative proportions of the various ingredients indicate the relative intensity of the various processes (catarrhal, suppurative, etc.) that generate mucus, pus, serum, or blood respectively. The consistency of sputum depends on its predominating in- gredients; if serous, it is thin and fluid; if mucous or muco- purulent, it is tough, viscid, and tenacious ; if purely purulent it is creamy. A muco-serous variety of sputum is frequently seen, containing large numbers of bacteria and abundant squa- mous epithelium, but very few leucocytes, that is of creamy con- sistency. Form. — The chief types of sputum are serous, mucous, muco- serous, purulent, muco-purulent, and bloody. The purely serous, mucous, and purulent sputa, sputum of pure blood, many muco-purulent sputa, or any sputum in which Digitized by Microsoft® 122 A MANUAL OP CLINICAL LABORATORY METHODS. the ingredients are thoroughly commingled, are homogeneous. Combinations of mucous or purulent elements with serum are usually heterogeneous, the masses of mucus or pus being sus- pended separate in watery fluid. The so-called nummular spu- tum consists of small rounded or caseous masses sinking in serum ; this is ordinarily stated to be characteristic of phthisis. The rusty sputum of croupous pneumonia is a viscid muco-pus col- ored brown by altered blood pigment. The prune- juice sputum of the same disease is a serous sputum stained dark by trans- formed hsemoglobin. Lamination, — When abundant and thin, sputum on standing often separates into layers, which may be worthy of note. Three layers usually form ; the upper stratum is formed of the buoyant air- containing frothy portions (mostly mucus) ; the middle stra- tum consists of the serous fluid elements ; the lowest stratum is a sediment of cellular elements, tissue fragments, and granular debris. Color and Pigment. — Purely serous and mucous sputa are or- dinarily colorless and hyaline. Other sputa are colored from various pigments. Purulent sputum and sputum containing sulficient purulent elements are yellow or green. A green color may be imparted to the sputum by pigments generated by bac- teria, by biliverdin in cases of jaundice and rupture of the liver into the lung, and sometimes in cases of pulmonary sarcoma and carcinoma. Dark-colored sputum, containing melanin, is some- times expectorated in cases of melanotic tumors of the lungs. Blue and bright-yellow sputa have been observed, due to bacterial pigments, or to altered bile and blood pigments. Peculiar colors sometimes develop (from bacterial growth) after the sputum has stood some time after being expectorated. The presence of blood or the derivatives of hsemoglobin, in hemorrhagic and congestive conditions of the lungs, gives sputum a color varying from bright-red to brown, according to the time elapsing between the extravasation of the blood and its final ex- pectoration. When expectorated freshly shed the color is bright red ; when longer retained before being spit out, it turns first darker and then brown or rusty, as in pneumonia, infarction, brown induration. In pneumoconiosis, or the free inhalation of extraneous parti- cles of dust of various kinds in certain occupations, the sputum Digitized by Microsoft® THE SPUTUM. 123 is colored corresponding to tlie dust inhaled. The particles most frequently inhaled are of carbon, giving the sputum a black color Particles of iron oxide give a dark or reddish color ; ultramarine produces a blue sputum in workers in this substance ; other dusts similarly inhaled are silicious, calcareous, metallic, etc. Putrefactive products are generated in necrotic or foul sup- purative conditions or where the sputum is retained a long time in the cavities or pulmonary passages before being expectorated, and so undergoes decomposition. The odor of fresh sputum often is slight and not markedly disagreeable ; but in connection with putrefactive conditions it is intensely foul, fetid, putrid, and repulsive. The reaction of fresh sputum is usually alkaline. Parasites. — The following animal parasite forms have b'een ob- served in the sputum, but all are rare in this country : Amoeba coli occurs in cases of amoebic hepatic abscesses ex- tending or rupturing into the lungs. Trichomonas, Paramecium, and other protozoa have been observed, especially in putrid pus or gangrene. Booklets, portions of cyst membranes, or even entire hydatid cysts may occur in the sputum derived from the larval cysts (hydatids) of the tcenia echinococcus developed in the lungs or liver. The larval cysts of the tcenia solium (cysticercus cellulosse) at times develop in the lungs. Paragonimus Wester- manii is a trematode infecting the lungs and causing haemoptysis ; it is common in eastern Asia, but a few cases in animals have been observed in the United States ; ova in great numbers and occasionally the entire worm may occur in the sputum, establish- ing the diagnosis. The ova of schistosoma hcematobium have been known to appear in the sputum. Vegetable Parasites. — Fungi exceptionally occur in the sputum, as actinomyces, streptothrix, aspergillus, mucor, oidium albicans, saccharomyces, leptothrix, and others, some pathogenic (causing pneumomycosis), others innocuous or secondary to tuberculous or other lesions. In pulmonary affections caused by fungi, but simalating tuberculosis, instead of tubercle bacilli themycelia of aspergillus, mucor, or streptothrix, etc., are found in the sputum. The most numerous and important of the micro-organisms of the sputum are the bacteria ; many of these are non-pathogenic and harmless, while others are pathogenic and of the utmost diag- nostic import. Digitized by Microsoft® 124 A MANUAL OF CLINICAL LABORATOEY METHODS. The most important pathogenic bacteria are the tubercle bacillus, micrococcus lanceolatus, bacillus influenzae, streptococ- cus pyogenes, staphylococcus pyogenes, micrococcus tetragenus, Friedlander's bacillus. The tubercle bacillus appears in numbers varying from a few to many in pulmonary tuberculosis, and its detection is of the highest importance in the diagnosis of that disease. Failure to find it does not, however, necessarily negative the diag- nosis of tuberculosis unless after many repeated examinations. It is usually associated with a relatively purulent type of spu- tum, containing many leucocytes, few squamous epithelium cells, and few other bacteria. The tubercle bacillus is a long, slender, rod-shaped bacterium, usually with a beaded appear- ance. Micrococcus lanceolatus, the diplococcus of croupous pneumo- nia and other diseases, is observed in the sputum in large numbers in that disease, though it may be present in healthy individuals. It is an oval micro-organism, broader at one end than the other, arranged in pairs, end to end, the broad ends toward each other ; sometimes it forms short chains, and in the sputum and blood is usually surrounded by a capsule. Its presence in the sputum is significant of pneumonia only in connection with the clinical symptoms. The bacillus of influenza, a very small, slender bacillus, single or in pairs, is observed in the sputum in that disease. Other bacteria, pathogenic or innocuous, of numerous varie- ties, and in large numbers or dense aggregations, are frequently present in the sputum, such as streptococci, staphylococci, micro- coccus tetragenus, sarcinse, etc. Leucocytes are present in sputum in numbers proportional to the amount of pus present. The greater the amount of mucus or serum in the sputum, the fewer and more scattered are the leuco- cytes ; while thieir number increases and they may be exceedingly numerous as the sputum approaches a purulent type. Their number is therefore an index of the extent and degree of the suppurative process present. In ordinary suppurative cases the leucocytes are almost en- tirely of the polynuclear variety. In some cases, especially asthma, eosinophile leucocytes may be abundant and even pre- ponderate. Digitized by Microsoft® THE SPUTUM. 125 Red blood corpuscles, either fresh or more or less altered, are observable in bloody sputum. Epithelium cells detached from the respiratory passages fre- quently appear in the sputum in moderate numbers, in their normal or a more or more or less altered condition. There are three types of epithelial cells along the normal respiratory pas- sages, large squamous cells in the mouth and pharynx, ciliated columnar cells in the nose, larynx, trachea, and bronchi, and "alveolar cells " in the air vesicles. In catarrhal conditions and after remaining a time in the sputum these cells may undergo changes so that the site of their origin cannot be determined. Squamous cells, large and flat, are the form most frequently present in sputum. They come from the mouth, pharynx, or upper part of the larynx, and are often numerous in the mucoid sputa, associated with relatively few leucocytes and Avith large numbers of bacteria. In more purulent sputa they are less nu- merous. Ciliated columnar cells, so little altered as to be recognizable as such, are rarely seen in the sputum ; they may come from the bronchi or trachea, but are more apt to come from the nose. Bounded or Oval Epithelium. — Swollen, rounded, or otherwise altered epithelium cells are frequently present mingled in small numbers with the leucocytes, sometimes showing granular or fatty degeneration. They originate from catarrhal surfaces, being im- mature or germinal cells prematurely detached, or from columnar or flat cells that have become swollen or altered by the action of the sputum. Such cells, which are termed "mucous corpuscles" or "mucocytes," are usually spherical in form, larger than leuco- cytes, with a single large, round nucleus and granular cytoplasm. Alveolar Cells. — The pulmonary alveoli are lined with flat squamous cells intermingled with spheroidal cells. The flat squames do not appear in the sputum as such, but if present at all swell to a spheroidal form. The nature of the so-called "al- veolar cells " of the sputum is not definitely settled, but they are supposed to originate in the alveoli of the lungs, perhaps from the spheroidal cells. The alveolar cells are 20 to 50 micromilli- metres in diameter, oval in form, with one or sometimes more than one rounded nucleus ; the body protoplasm is finely granu- lar, and is often studded with granules either of extraneous matter, fat, myelin, or pigment derived from haemoglobin. Digitized by Microsoft® 126 A MANUAL OF CLINICAL LABORATORY METHODS. Sometimes one and the same cell may contain more than one kind of these granules. The epithelial cells from other parts of the respiratory tract rarely show the pigmentation or the fatty or myelin degeneration exhibited by the alveolar cells. The extraneous granules that may infiltrate the bodies of the alveolar cells are particles of dust, most frequently consisting of carbon, inhaled in pneumoconiosis and taken up by the cells. The globules of fat that may be present originate from fatty degenera- tion of the cells. Often the cells exhibit, from degenerative changes, irregular, often large particles of a clear, fat-like, refrac- tile, pale material, sometimes concentrically marked; this sub- stance is regarded as a form of myelin. The cells are sometimes almost broken down into this material, and rounded masses of myelin may appear free in the sputum. Bodies similar in ap- pearance to corpora amylacea, and also resembling myelin drops, are said to appear sometimes in the sputum, turning blue with iodine. In cases of prolonged congestion or brown induration of the lungs, especially from mitral disease, but also in infarctions and pneumonia, the alveolar cells may contain numerous particles of brown or yellow pigment, derived from haemoglobin. Such cells are called heart-disease cells, and are of diagnostic significance. The alveolar cells are of common, almost constant, occurrence in the sputum, being present in small numbers in the slightest respiratory affections, as well as in large numbers in more serious conditions like phthisis. They have little or no diagnostic sig- nificance except in the case of those containing altered blood pig- ment (heart-disease cells), which indicate stasis or extravasation of blood. Carcinoma Cells. — Epithelial cells of various forms derived from pulmonary carcinoma may appear in the sputum, and in two or three instances the diagnosis of cancer of the lung has been so made. It is, however, difficult to identify isolated cells, not in the typical alveolar arrangement, as with certainty coming from carcinomatous growths, except perhaps when abundant and constant and associated with corresponding clini- cal symptoms. Fragments of tissue are at times expectorated, such as macro- scopic pieces of lung substance, pieces of cartilage, and especially elastic fibres (separate or in an alveolar network), in necrotic Digitized by Microsoft® THE SPUTUM. 127 conditions like pulmonary gangrene and phthisis. In two or three instances malignant disease (sarcoma) of the lung has been diagnosticated from fragments of the tumor expectorated ; but this is only rarely possible. Curschmann's spirals are small, elongated, rather firm bodies up to about 1 or 2 centimetres in length and 1 millimetre in diameter, composed of a sort of mucinous, fibrillar material twisted spirally around a central, often sinuous filament. They are more or less opaque, pale in color, sometimes embedded in hyaline material. Forms varying from the type may occur, like branching forms and those lacking the central filament. They are sometimes studded with epithelium cells, leucocytes, or Charcot-Leyden crystals. The spirals occur in certain forms of asthma and at times in other cases. Dittrich's plugs are opaque, whitish-yellow particles, foul in odor, in size from that of a millet to a mustard seed, occurring in sputum in putrid conditions (gangrene, foul bronchitis), and consisting of masses of bacteria mingled with fatty-acid crystals. They originate in small bronchi or tonsillar crypts. Fibrin, in the form of amorphous flakes or (rarely) of casts of the bronchi, may be expectorated in croupous pneumonia and the rare cases of croupous or fibrinous bronchitis. In the latter condition large casts of the ramifying bronchial passages are sometimes expelled. Crystals of various kinds are exceptionally present in sputum. The most significant are the Charcot-Leyden crystals, apparently crystals of spermin phosphate, which are small, greatly elongated and slender octahedral crystals, occurring chiefly in some cases of asthma. Scematoidin crystals or flakes sometimes appear in sputum after hemorrhagic extravasations. Patty-acid crystals are occasionally encountered. Crystals of cholesterin, leucin, tyrosin, soaps, calcium oxalate, calcium carbonate, and ammonio- magnesic phosphate have been observed, especially in putrefac- tive conditions. Calculi from the bronchi or lungs have been expectorated in rare instances. Foreign bodies, as pieces of bone, that have entered the air passages are occasionally coughed up. Particles of food from the throat or mouth frequently become mixed with sputum, and should not be allowed to mislead the observer ; particles of ani- Digitized by Microsoft® 128 A MANUAL OF CLINICAL LABORATORY METHODS. mal food, for instance, should not be erroneously regarded as Indicating pulmonary necrosis. EXAMINATION OF SPUTUM. Sputum is chiefly examined macroscopically and microscopic- ally ; exceptionally occasion may arise for its examination bac- teriologically (by cultures or animal inoculation) or chemically (as for traces of blood). The sputum expectorated early in the morning before breakfast is advantageous for examination, being usually abundant, representative, and unmixed with food parti- cles. In the case of thin iiuid sputum, the essential elements should be collected by allowing them to settle or by using the centrifuge, by which the sediment may be obtained. Macroscopic Examination of Sputum. — Simple inspection reveals much information as to the character of the sputum, its content of serum, mucus, pus, blood, and air, its consistency and form, color, odor, etc. , with all that these signify. In cases of profuse expectoration, the amount discharged in twenty-four hours, to be determined by measurement, is a useful clinical datum. To make a comprehensive search of a quantity of sputum for objects like fragments of tissue, etc., it may be pressed out into a thin layer between two sheets of glass, when suspicious portions may be readily picked out and further examined ; the use of a dark background aids in the examination. Microscopical Examination of Sputum. — The most important diagnostic data to be obtained from the sputum are afforded by microscopical examination. Sputum may be examined either fresh or after being appropriately stained. In examination in the fresh condition a small quantity is placed on a slide and a cover-glass applied, when it is ready to be placed under the microscope. In staining sputum it is necessary first to spread and fix it, which is mostly done by the same process for the different methods of staining. A small portion of suspicious parts of the sputum is taken on the point of thumb forceps or with a platinum loop, and placed on a clean cover-glass. Another cover-glass is then placed over the drop of sputum, and the two covers are pressed together between the thumb and forefinger or between the points of forceps, so that the sputum spreads out in a thin layer between the two cover-slips. The two slips are then slid Digitized by Microsoft® THE SPUTUM. 129 apart and separated, leaving a thin :film of sputum on one side of each. The films are then dried at the room temperature, or by gentle warming high above a flame ; if held in the fingers above the flame there is no danger of overheating. The films of sputum can also be spread by means of the wire loop on a glass slide or single cover-glass. The films being prepared and dried, they are next to be ' ' fixed, " preparatory to staining. For staining bacteria and most other purposes this is done by heating or "fiaming" the speci- men, holding it by forceps and passing it at medium speed through the fiame of a Bunsen burner or alcohol lamp three times, at brief intervals. Examination for Tubercle Bacilli.— This is the object for which the great majority of sputum examinations are made. The spe- cific method usually employed for demonstrating these bacilli depends on the fact that after being well stained by f uchsin they retain this stain even when treated by strong acids that decolor- ize all the other objects in the specimen. There are two or three steps in the process, staining with fuchsin, decolorizing with acid, and counterstaining. The fuchsin solution ("carbol-f uchsin") is prepared as fol- lows: Two stock solutions are kept on hand, a 5-per-cent aqueous solution of phenol, and a saturated solution of fuchsin in alcohol. The staining solution is prepared by mixing 9 parts of the phenol solution with 1 part of the fuchsin solution ; this solution deteri- orates after a few weeks, and hence should be freshly prepaied from the stock solutions at proper intervals. To stain, the cover-glass preparation, fixed by heat as above described, is held in self-retaining (Fig. 13) or thumb forceps and covered with as much of the carbol -fuchsin solution as it will hold. It is then heated carefully over a Bunsen burner, until the fluid just boils (bubbles) two or three times. After a minute or two the staining fluid is washed off with water. The specimen is next decolorized by immersing it in a 25 -per- cent solution of nitric or sulphuric acid in water, for ten to sixty seconds, until the red color is just discharged and the film just decolorized. It is then immediately washed with water. It is then counterstained with Loffler's methylene-blue solution, washed, and mounted temporarily on a slide with water, the upper surface of the cover-glass dried with filter paper, and ex- 9 Digitized by Microsoft® 130 A MANUAL OP CLINICAL LABORATORY METHODS. amined with an oil-immersion objective. Permanent mounts can be made with Canada balsam. When thus prepared, the tubercle bacilli appear conspicu- ously, stained red, while other bacteria and ceU-nuclei are stained blue. Gobbet's method : For routine work Gabbet's method is very largely used, being substantially the same as that just described, but with the decolorization and counterstaining combined in one process. The specimen is spread and fixed in the usual manner, and stained by carbol-fuchsin in the manner described. After being washed with water the cover-glass is covered with Gabbet's solution, consisting of 75 parts of water, 25 of sulphuric acid, and 2 of methylene blue. This is allowed to remain for fifteen to sixty seconds, until by washing and examining against a white background the red color is discharged and a pale blue appears. The specimen is then washed and mounted as before. Tubercle bacilli are stained red, nuclei and other bacteria pale blue. If a more decided counterstaining is desired, Lofler's methylene blue can be used after Gabbet's solution. When the tubercle bacilli are very scanty and difficult to demonstrate, it may in doubtful cases be advisable to adopt means to facilitate their detection. One method is by sedimentation. To make the sputum sufficiently fluid and remove the cellular elements it is boiled with three or four times its vol- ume of water together with a few drops of liquor sodae, and the sediment ob- tained for examination either by centrifugation or after allowing it to settle for twenty -four hours. Or the sputum may be mixed with several times its volume of water, made alkaline, a small amount of commercial pancreatin added to digest away the cellular elements, and the whole set away at a tem- perature of 37° C. in a conical glass for twelve to twenty -four hours ; the sedi- ment is then examined for the bacilli (and elastic fibres). By placing the sputum in an oven at 37° C. for a day or two the bacilli may increase in number so as to be more easily found. Or by injection of the suspected sputum into" a guinea-pig the presence of tubercle bacilli may be shown by the lesions developed after three or four weeks. In the great majority of cases of pulmonary tuberculosis the bacilli may be easily found at every examination. Failure to find the bacilli must not, however, be taken to negative the diag- nosis of tuberculosis, especially if only one or a few examinations are made. A probable negative diagnosis can be arrived at if the bacilli are not found after many trials repeated at various stages of the disease and with specimens of sputum and during Digitized by Microsoft® THE SPUTUM. 131 periods in which ordinarily the . bacilli would be expected to appear. Examination for Other Bacteria. — Bacteria other than the tu- bercle bacillus are ordinarily shown with sufficient distinctness after Gabbet's or other tubercle-staining process has been applied, and even (especially fungi) in unstained specimens. They are not ordinarily of sufficient importance to require special stain- ing, but if it is desired to demonstrate them carefully, the cover- glass preparations, duly spread and fixed, may be stained by the usual bacterial stains, as Loffler's methylene blue, weak carbol- fuchsin, gentian-violet, or Gram's method. The micrococcus lanceolatus, or pneumococcus, is next to the tu- bercle bacillus in clinical importance so far as the sputum is con- cerned, and may at times require careful examination by methods which bring out the capsule. Gram's method of staining is very satisfactory ; this demonstrates the capsule nicely and does not decolorize the diplococcus. The bacterium is often well shown even after Gabbet's method. The bacillus influenzce may be stained by the usual stains, espe- cially Loffler's methylene blue or a solution of carbol-fuchsin diluted to a pale-red color; it decolorizes by Gram's method. Examination for this bacillus is not in general use for clinical purposes. The cells of various kinds in the sputum are ordinarily suffi- ciently evident in the specimen stained for tubercle bacilli. If it is desired to study them especially, the sputum may be exam- ined microscopically in the fresh, moist, unstained condition, using dim illumination; the addition of acetic acid may clear the cells and make the nuclei more evident. Or the cover-glass preparations may be stained by appropriate methods, as with hsematoxylin-eosin or by the triple stain (to demonstrate the va- rieties of leucocytes). Mucin in stained specimens appears as streaks of amorphous matter, staining with basic stains; fibrin might be demonstrated by its oxyphile staining reactions. Tissue fragments may be teased or embedded and sectioned, and thus stained. Elastic fibres may be examined unstained by pressing suspicious particles of sputum out under the cover-glass into a very thin layer, so as to render them visible in the mucus. If a careful search for elastic fibres is needful, the sputum may be boiled with an equal amount of 10-per-cent solution of potas- Digitized by Microsoft® 132 A MAIJUAL OF CLINICAL LABORATORY METHODS. slum or sodium hydrate, then . diluted with four times as much water. After allowing it to settle twenty-four hours or using the centrifuge, the sediment is examined for the fibres. If found, especially in an alveolar arrangement, the existence of a destruc tive process is demonstrated. Crystals, myelin drops, protozoa, fragments of echinococci, ova, etc., may be examined for in the fresh unstained state of the sputum. Fat may be demonstrated by Sudan III. Digitized by Microsoft® VII. THE URINE. Examination of the urine in many cases affords information of great clinical and diagnostic value, if not positively at least negatively. Containing as it does a large part of the waste prod- ucts of metabolism, especially nitrogenous metabolism, the urine is a valuable index of the state of the metabolic processes in gen- eral ; both on the anabolic or assimilative side (as when it con- tains substances that normally are completely elaborated into protoplasm, but owing to abnormal conditions are arrested in an intermediate stage and so excreted), and on the katabolic side (as when unusual products are formed by perverted katabolism and so excreted) ; it reveals the existence of abnormal condi- tions of the urinary organs ; and in special cases affords infor- mation as to the local processes in other parts of the body besides the urinary tract, or as to food, poison, or adventitious sub- stances introduced into the system. A. COMPOSITION OF THE URINE. The constituents of the urine consist of liquids, solids, and gases in solution, and particulate or undissolved solid elements (either crystalline, organized, or amorphous). They originate chiefly from the waste products of metabolic activity, as a result of local or distant pathological conditions, or from the surplus or unused portions of ingested alimentary or drug materials. The chemical elements represented in the urinary materials are : among the acid-forming and non-metallic elements, oxygen, sul- phur, nitrogen, chlorine, phosphorus, carbon, silicon, and hy- drogen; traces of iron, and the alkaline and earthy bases po- tassium, sodium, ammonium, calcium, and magnesium. The individual substances composing the urine are very numerous, of which some are abundant and conspicuous, while many are minute in amount, obscure in nature, difficult to demonstrate, of inconstant or rare occurrence, and, usually, of no clinical importance. Digitized by Microsoft® 134 A MANUAL OF CLINICAL LABORATORY METHODS. The substances entering into the formation of the urine in normal and abnormal conditions are as follows : Water. IJfitrogenous bodies : Urea. Alloxur bodies : xanthin bases, uric acid, urates, allantoin. Hippuric acid ; benzoic acid. Creatin and creatinin. Leucin and tyrosin. Sulphur compounds : sulphates, mineral and conjugate ; unoxid- ized- sulphur compounds. Phosphoric acid and phosphates. Carbon dioxide and carbonates. Chlorides. Calcium oxalate. Pigments and chromogens. Fat, fatty acids, soaps, lactic acid, other organic acids, ferments, hydrogen dioxide, iron, ammonia, oxygen, nitrogen. Proteids: nucleo-albumin, serum-albumin, globulin, albumoses, fibrin. Carbohydrates: glucose, levulose, inosit, sucrose, lactose, mal- tose, pentoses, glycogen, dextrin, animal gum. Glycuronates. Alkapton. Acetone, diacetic acid, ;?-oxybutyric acid, alcohol. Blood and derived substances: proteids, red blood corpuscles, leucocytes, haemoglobin, methsemoglobin, hsematin, hsematoi- din, hsematoporphyrin, and other derivations of haemoglobin. Lymph. Pus: leucocytes, proteids. Bile : bilirubin, bile salts, cholesterin, nucleo-albumin. Substances producing the diazo reaction. Toxins. Adventitious iugesta. Epithelium. Fragments of tissue. Leucocytes. Spermatozoa. Casts. Cylindroids. Digitized by Microsoft® THE URINE. 135 Mucous threads. Granular and amorphous debris. Calculi. Parasites, animal and vegetable. Foreign bodies. Water is the most abundant and one of the most important constituents of the urine, and one to which too little considera- tion is frequently given in clinical work. The amount of water determines the quantity of the urine and its degree of concentra- tion or dilution, and its specific gravity and proportion of solids are intimately dependent on the amount of water. The water is very variable in amount according to circumstances, ranging or- dinarily from about 1,000 to 1,500 cubic centimetres daily. The water is increased, causing a diluted condition of the urine, or hydruria, after copious ingestion of fluids, decrease of perspiration, from the action of hydragogue diuretics, in diabetes insipidus and mellitus, in certain neurotic conditions, in chronic interstitial nephritis. Water is decreased when little fluids are ingested, when much water is lost by free perspiration, catharsis, or vomiting, in chronic parenchymatous and acute" nephritis, in febrile conditions, in yellow fever, in cirrhosis, and acute yellow atrophy of the liver, etc. Urinary Solids. — The total daily amount of solids excreted in the urine is more constant than the water and of much clinical importance. In some cases the amount of solids varies to a cer- tain extent with that of the water, but ordinarily the wide fluctua- tions of the water are entirely independent of the solids, and the amount of the latter must be determined and considered entirely apart from the amount of water. The specific gravity of urine and the percentage proportions of any of its constituents do not in themselves alone convey sufficient information for clinical pur- poses, since the amount of water is so variable ; but when taken in connection with the daily amount of urine passed they afford accurate and adequate information as to the daily amount of solids excreted, and hence as to the condition of the vital and metabolic processes that produce the excreted solids and as to the efficiency of the organs concerned in their excretion. The normal amount of solids daily excreted in the urine is about 55 to 75 grams. Urea (CON^HJ. — Of the normal constituents of the urine Digitized by Microsoft® 136 A MANUAL OF CLINICAL LABORATORY METHODS. nrea is much the most abundant and of the greatest physiological importance and clinical significance, being the chief representa- tive of the katabolism of the nitrogenous tissues. The three chief end products of body katabolism are H^O, CO^, and H^N ; H^O and COj represent the breaking down of carbohydrates and fats ; all three, and most characteristically H3N", represent the breaking down of the vital nitrogenous bodies. Urea may be regarded as a combination of CO, and H,N (CO, + 2Ti,lS = COJST^H, + H,0). CO, is excreted mainly by the lungs, H,0 by the lungs, skin, and kidneys, HjN" mainly in the form of urea by the kidneys. Urea is freely soluble and always appears in the urine in solu- tion. In concentrated solution it is precipitated by nitric acid in crystalline form as urea nitrate ; in the nitric-acid contact test with concentrated urines (with a urea content of about 5 per cent or over) a crystalline layer of urea nitrate frequently devel- ops at the junction of the two fluids. Under the influence of certain bacteria, which frequently find access to urine both before and after its passage, urea is converted to ammonium carbonate (CON,H, + (H,0), = (NHJ ,COJ. This is the ammoniacal fermentation of the urine, which may take place within the bladder in cystitic and paralytic conditions, or after passage and standing of the urine. Urea is decomposed into carbon dioxide and nitrogen by so- dium hypobromite or hypochlorite, which affords means for its estimation. It is similarly decomposed by nitrous acid, so that frequently iu the contact test with nitric acid bubbles collect in the fluids from decomposed urea, or on adding impure nitric acid to urine a slight amount of eifervescence occurs. The urea excreted originates partly from ingested proteid food, and to a greater extent from the katabolic metamorphosis of the nitrogenous body tissues. That derived from food being taken into account, its amount affords an index of the condition of the nitrogenous metabolism of the body. Since the liver is actively concerned in the elaboration of urea, and the kidneys in its excretion, the amount of urea excreted is also significant as to the efficiency of the liver and kidneys in these respects. The daily amount of urea ordinarily excreted in the urine ranges from about 25 to 35 grams, or from 1.5 to 2.5 per cent of the urine. It is increased during the augmented tissue change caused by bodily activity, febrile diseases, and other conditions, Digitized by Microsoft® THE URINE. 137 in diabetes mellitus, and witli a meat diet. It is decreased in cacliexias and conditions of lowered vitality, in uraemia, in kid- ney diseases with impaired renal excretory power, and in affec- tions of the liver (notably cirrhosis, yellow fever, and acute yel- low atrophy) in which the urea-forming functions of this organ are impaired. It is greater in males than female's and relatively greater in children. XantMn Bases. — The xanthin or alloxur bases are a group of closely related basic substances that enter into the formation of the nucleins or essential chemical constituents of the nuclei of the body cells, and appear during the katabolic breaking down of the latter. They occur in animal and vegetable tissues, and in the urine are derived both from ingested food and nuclear katabolism. Those that have been found in the urine are xan- thin (CjH^N^OJ, hypoxanthiu, heteroxanthin, paraxanthin, guanin, adenin ; they occur in the urine as such only in mijiute amount, the largest part of them being apparently converted by oxidation into uric acid and so excreted. "Ordinarily the quantity of the xanthin bases is only about a tenth that of the uric acid. To a certain extent (but not always) these bases therefore have the same origin, significance, and fluctuations as uric acid. These substances occur in the urine iu solution; xanthin very rarely appears in the form of undissolved, colorless, fusi- form crystals, or enters into the formation of vesical calculi. The quantity of the xanthin bases is so minute that satisfactory methods for their practical determination are not available; hence their clinical significance apart from that of uric acid is as yet unsettled. Uric Acid (C^H.N^Oj). — This substance together with the xan- thin bases make up the class called the alloxur bodies. Consid- erable clinical importance is attached to uric acid, but the ques- tion of its relation to disease processes is at present in a very unsettled state. As to its origin uric acid is now generally con- sidered to be an oxidation product of the xanthin bases, and the chief form in which these bases are excreted ; it is therefore the chief representative of the products produced by the katabolic breaking down of the cell nuclei of the body (of leucocytes espe- cially). In man and mammals, then, uric acid is chiefly derived from certain foods and from nuclear destruction. To a certain extent uric acid can also be generated synthetically in the organ- Digitized by Microsoft® 138 A MANUAL OF CLINICAL LABORATORY METHODS. ism, and it is also partially capable of further oxidization into urea ; these obscure and uncertain factors complicate the ques- tion of uric-acid formation and significance. In birds uric acid plays a larger part, taking the place of urea as the principal end product of nitrogenous katabolism. The amount of uric acid excreted daily in the urine ranges from .4 to .8 gram, bearing a normal ratio to the amount of urea of between 1 to 40 and 1 to 60. It is increased after the inges- tion of food rich in cell nuclei, as liver, kidneys, etc. (rather than muscle) ; in conditions attended with leucocytosis and hence with increased disintegration of nuclei, as in leukaemia (in which there may be a great increase of uric acid, up to 5 grams or more in tv.renty-four hours), in pneumonia (especially at the crisis), at the subsidence of digestion leucocytosis, etc. ; in febrile con- ditions ; in the uric-acid diathesis ; and in other conditions. It is diminished in anaemic and leucopenic conditions, chronic nephritis, and other cases. The relation of uric acid to gout and lithsemia is very obscure at present ; uric acid itself does not seem to be responsible for the toxsemic manifestations in these diseases ; yet it appears to fluctuate in amount parallel with the symptoms so as to serve as a clinical index of the toxic agen- cies, though itself innocuous. Uric acid is very feebly soluble in water, requiring 17,000 parts of water at 20° C. or 1, 900 parts of hot water for solution. It occurs in the urine both in uncombined form and in combina- tion with the alkaline bases as sodium, potassium, and ammonium urates ; each of these forms again may be in solution or in un- dissolved crystalline or amorphous state. As the urates are more soluble than uncombined uric acid, a larger amount of the acid can be held in solution in combined than in uncombined form. In strongly acid urine, or after the addition of strong acids (hydrochloric, acetic), uric acid is freed from the urates, and if in excess of the soluble amount, especially in the cold, is pre- cipitated in crystalline form. In alkaline or feebly acid urine, or after the addition of alkalies, the uric acid forms urates that are more soluble. The crystalline forms of uric acid that appear in the urine (Fig. 21), while exhibiting considerable diversity of form, are usually very characteristic and easily recognizable. The urine in which the crystals appear is commonly very distinctive, being Digitized by Microsoft® THE URINE. 139 concentrated, clear, of deep amber color, high specific gravity, strongly acid, and with strong urinous odor. The larger crystals are visible to the naked eye as minute reddish particles, resem- bling grains of red pepper or brick-dust on the bottom, or adher- ing to the sides of the receptacle containing the fluid. Under the microscope the crystals present varying forms; they are mostly of a more or less bright amber color, but are sometimes only faintly tinged or quite colorless ; they vary in size from 10 to 20 micromillimetres in length up to particles distinguishable macroscopically. In form the crystals are frequently flat, with two parallel plane surfaces bounded by a double convex or loz- enge - shaped contour; many deviations from this type appear, some slender and fusiform, some with curved out- lines, some rhombic, some crossed, some short and thick, appearing rectangular, barrel- shaped, or fusiform, ac- cording to the point of view. Often the crystals are united in radiating clusters. An- other type of uric-acid crystals is slender and prismatic. The crystals are very polymorphous, but usually characteristic enough to be easily recognized. The presence of undissolved uric-acid crystals in urinary sed- iment does not necessarily indicate an excess of uric acid in the urine ; their precipitation is caused essentially by concentration and strong acidity of the urine, with cold as an auxiliary, and may occur when uric acid is normal as well as when it is increased. Urates. — Uric acid occurs in the urine chiefly in the form of sodium, potassium, ammonium, calcium, and magnesium urates, except when from high acidity it is forced from these combina- tions. The urates are considerably more soluble than uric acid, and more soluble in warm than in cold fluids. Many urines which at the body temperature or higher are perfectly clear and bright amber in color, when cold become turbid and dirty yellow Fig. 21.— Uric-acid Crystals of Various Shapes. Digitized by Microsoft® 140 A MANUAL OP CLINICAL LABORATORY METHODS. and precipitate their urates in the form of au abundant fine white or pinkish sediment (brick-dust or lateritious sediment), wliich is quite distincti\-e in appearance. The urates (except of ammonium) are freely soluble in alkaline fluids, and urine tur- bid with urates becomes clear on the addition of alkali. The mixed sodium and potassium urates are the commonest forms, calcium and magnesium urates being i'S''i TD • exceptional and minute in amount; °'°i?'- Cj\ T/~ ammonium urate is formed in connec- J|o-»i| ^-^ ^ tion with ammoniacal fermentation of (~) O ^'^® urine. a Microscopically the mixed urates O (Fig- 22) usually appear as minute FIG. 23.-urates. a. Amorphous granules. Sometimes irregular and ImmoliTumS.*'""'^™'^'°' amorphous, sometimes spherical, very minute, and faintly amber. Very rarely sodium urate appears in crystalline form, as spherules, radiating prismatic crystals, or dumb-bell forms. Ammonium urate is occasionally formed and precipitated during ammoniacal fermentation. It is the only urate that re- mains undissolved in alkaline urine, the other urates being pre- cipitated only in acid media. Ammonium urate appears in the form of rather large amber-colored spherules, some with project- ing spicules (Fig. 22). AUantoin is a substance allied to uric acid that occurs in minute traces in normal urine, especially after meat diet ; it is more abundant in the urine of newborn infants, and after the administration of tannic acid. It has no clinical importance. Hippuric acid (CgHgNOs) is a constant ingredient of normal human urine, in small quantity, about .5 to 1 gram daily. It originates to a small extent from metabolic products, to a larger e.xtent from ingested food or drugs. It is much increased in amount after the ingestion of benzoic acid and some other substances, which are excreted as hippuric acid. It is also greater in amount on a vegetable diet, especially after the ingestion of fruits containing benzoic acid ; it is very abundant in the urine of herbivora. It is still excreted, how- ever, on a meat diet. It is increased in diabetes mellitus, chorea, acute fevers, and other conditions; decreased in chronic nephritis. Hippuric acid is somewhat more soluble than uric acid; but when in ex- cess, as after taking benzoic acid, its crystals may be separated in the form of long four-sided prisms with bevelled ends, or slender acicular crystals. The crystals are either separate or grouped together in radiating clusters. It is more soluble in alkaline than acid media, and occurs combined with the alka- line and earthy bases except when set free by strongly acid reaction. Digitized by Microsoft® THE URINE. 141 Benzoic acid is occasionally present in the urine along with or in place of hippuric acid, as in diabetes and decomposing urine. Creatin and Creatinin, — Creatin is a nitrogenous body. (O4H9N3OS) oc- curring in muscle tissue; creatinin is closely related to it, containing one molecule of H2O less. Both substances, especially creatinin, occur dissolved in the urine in small amount (.5 to .9 gram daily), derived partly from the body muscle tissue, partly from muscle ingested as food. The amount ex- creted increases on lean meat diet and in acute and certain other diseases ; and is diminished in certain cases. It is not customary to consider these substances for clinical purposes. lieucin and tyrosin occur in urine in appreciable amount only in ab- normal conditions. They are closely related nitrogenous bodies, and usually occur together in the urine. They originate from abnormal proteid decom- position, or when the process of urea-formation is interfered with, as in hepatic disease. They thus appear in the urine in acute yellow atrophy of the liver, in large amount; in smaller amount in extensive suppuration or gangrene, hepatic carcinoma, cirrhosis, and other affections, leuksemia, typhoid fever, etc. When in large amount they may replace or supplement urea. They remain in solution, except that when they pass the point of saturation they appear in undissolved form ; leucin as brownish spherules with fine con- centric and radiating markings ; tyrosin as fine acicular crystals arranged in sheaves, bundles, or radiating rosettes. Ammonia, H,N, may be regarded as the simplest ultimate end product of nitrogenous katabolism, and as such is excreted in large amount, almost entirely in a dehydrated combination with carbon dioxide as urea. In ammoniacal fermentation ammo- nium carbonates are generated in large amount. Aside from urea, ammonia compounds, similar to the corresponding sodium and potassium salts, are normally excreted in minute amount ; the nitrogen of ammonia is normally about 2 or 3 per cent of the total nitrogen. In certain acid toxaemias in which there is an excessive formation of acids in the system from perverted me- tabolism (as in diabetic coma), or where there is excessive inges- tion of acids, ammonia is excreted in the urine in excessive amount, in combination with these acids ; in such cases the am- monia nitrogen is about 18 to 25 per cent of the total nitrogen, and an approach to this ratio is a danger signal. The rationale of this is that the sodium or potassium bases are not sufficient in amount to neutralize all the acid, and the ammonia which would otherwise go to form urea combines with the excess of acid and is so excreted. Quantitative estimation of ammonium com- pounds in the urine therefore furnishes an index of katabolic acid formation. Digitized by Microsoft® 142 A MANUAL OF CLINICAL LABORATORY METHODS. The ammonium salts of the urine form "volatile" alkali, in contradistinction to the "fixed" alkalies, sodium and potassium. Total Nitrogen. — The substances thus far considered compre- hend the chief katabolic nitrogenous products, and sometimes their total nitrogen content is measured as an index of the body nitrogen katabolism more complete and representative than the amount of urea alone affords. Ordinarily the total nitrogen excreted in the urine daily ranges from about 15 to 21 grams, of which about four-fifths is furnished by urea. Sulphur Compounds. — The sulphur excreted in the urine, like urea, chiefiy results from the decomposition of albuminous mate- rial, either that ingested with the food, or that broken down in katabolic processes. The sulphur is excreted chiefly in the form of sulphates ; also in unoxidized combinations, in traces in nor- mal urine, in larger amount (cystin, hydrogen sulphide) in ab- normal conditions. Sulphates. — These are normally excreted in quantities of 1.5 to 3 grams daily. Their quantity in general fluctuates parallel with that of urea. They are increased by meat diet, the inges- tion of sulphuric acid or sulphates, exercise, in acute fevers, especially meningitis and rheumatism, in leukaemia, and other affections. The sulphates of the urine are of two kinds, mineral sulphates and the conjugate or ethereal sulphates. Mineral Sulphates. — These ordinarily constitute about nine- tenths of the total urinary sulphates. They consist chiefly of sodium sulphate, with a small proportion of potassium and am- monium and perhaps calcium and magnesium sulphates. These salts, excepting calcium sulphate, occur in the urine only in solution. Calcium sulphate rarely appears in urinary sediments, in the form of slender prismatic or acicular crystals, some in radiating clusters, or in the form of amorphous granules or small dumb-bell shapes. Conjugate or Ethereal Sulphates. — These are formed by the combination of potassium or sodium sulphate with indol, phenol, and related substances generated in the course of putrefactive decomposition of proteids. They are normally formed in the intestine from disintegration of proteid food material ; and at times originate abnormally in putrid, suppurative, or necrotic processes, or abscesses. Prom the place of origin they are ab- sorbed into the circulation and thence excreted by the kidney, Digitized by Microsoft® THE URINE. 14:3 constituting normally about one-tenth of the excreted sulphates. These substances therefore represent albuminous putrefaction, and are of diagnostic significance in that respect. They are di- minished or absent with a decrease or cessation of intestinal de- composition. They are increased, or their ratio to the mineral sulphates is increased, in conditions of increased gastric or intes- tinal decomposition, as in gastric carcinoma, gastritis, hypochlor- hydria, gastric and intestinal stagnation, intestinal obstruction, peritonitis; also in putrid, suppurative, or necrotic conditions outside the alimentary tract, as in cystitis, empyema, etc. ; and from the use of certain drugs. The conjugate sulphates consist chiefly of indoxyl-potassium sulphate, with ordinarily a less amount of phenol-potassium sul- phate, and traces of the potassium sulphates of cresol, catechol, and skatoxyl. These sulphates as such occur in the urine in solution. Indoxyl-potassium sulphate, or indican, is the most abundant and most conspicuous of the conjugate sulphates, and ordinarily representative of them all, so that increase of indican has the same significance as that of the total ethereal sulphates. By de- composition with hydrochloric acid or otherwise, along with oxidation, as by the simultaneous or subsequent action of nitric acid or other oxidizing agents, indican is converted into a red or a blue pigment (indigo-red and indigo-blue), according to the amount present and stage of oxidation. Earely this takes place spontaneously, or after urine has stood for a time, giving rise to blue or red urines. In decomposing urine indigo-blue is some- times deposited in the form of undissolved amorphous blue gran- ules, rarely in the form of delicate blue crystals ; and a very few instances are reported of indigo calculi being formed in the kidney. Unoxidized Sulphur Compounds. — Normally only traces of sul- phur compounds other than sulphates occur in the urine, such as sulphocyanides derived from absorbed saliva, other substances perhaps of katabolic origin and related to cystin, and possibly derivatives of taurocholic acid from absorbed bile. The latter is present in choluria. Two bodies rarely present in abnormal conditions, cystin and hydrogen sulphide, are of some impor- tance. Cystin (CgH^NSOJ rarely occurs in the urine. Its formation Digitized by Microsoft® 144 A MANUAL OF CLINICAL LABORATORY METHODS. is not well understood, but seems to depend on metabolic anom- alies or hepatic disorder, arising either from proteid katabolism or from taurin in the liver. Cystinuria has little or no clinical significance ; it may continue for years without material impair- ment of health, and is at times a hereditary anomaly. Cystin is present in the urine both in solution and as an un- dissolved crystalline sediment, and also sometimes forms calculi. The crystals are hexagonal plates, occurring separately or in superimposed forms ; they are soluble in the caustic alkalies and in oxalic and strong mineral acids; insoluble in ammonium- carbonate solution, and hence precipitated during ammoniacal fermentation. When occurring in solution only, without crys- tals to direct attention to it, its presence may not be suspected unless hydrogen sulphide arising from its decomposition is de- tected. To a certain degree cystinuria is apparently associated with the formation and excretion of certain toxic diamines. Hydrogen sulphide, H^S, is rarely encountered in the urine (hydrothionuria). Sometimes it is generated subsequent to its passage by decomposition processes or H^S-forming bacteria act- ing on the sulphur-containing substances of the urine. It is es- pecially apt to appear in urine containing cystin. At other times it is formed within the body economy, and then absorbed or ex- creted into the urine, as in putrefactive processes of the intestine (frequently associated with excess of indican), and in foul ab- scesses in which H.^S is generated. H,S injected into the rectum may appear in the urine. Phosphates. — The compounds of phosphoric acid excreted in the urine are derived from the surplus phosphates of the ingested food and from the waste products of the katabolism of the body tissues and substances containing phosphorus. As urea and its congeners represent nitrogenous katabolism, so the urinary phos- phates, aside from those of the food, represent and indicate phos- phoric katabolism. The principal body substances containing and yielding phosphorus are the nucleins, lecithin, and phos- phates. A portion of the excreted phosphorus is derived from nuclear disintegration (nucleins), and this portion should bear a definite ratio to the uric acid and xanthin bases derived from the same source. Another portion is yielded by the breaking down of tissues containing lecithin and phosphates, especially nervous tissue, bone, blood corpuscles, and muscle ; this portion Digitized by Microsoft® THE URINE. 145 is independent of the excretion of the alloxur bodies. The actual excretion of the phosphates is dependent on the functional power of the kidneys in the same manner as that of urea. The normal amount of phosphoric acid excreted daily is about 2.5 to 3.5 grams, measured as Ffi^. It varies with the amount of phosphates introduced with the food, being greater on animal than vegetable diet. Its fluctuations in disease are not com- pletely made out. The excreted phosphates should be increased by increased katabolism of nuclei, nervous tissue, bone, blood corpuscles, etc. ; or when (as in rickets) the ingested phosphates normally used up in the nutrition of the tissues (as the bones) are not utilized for this purpose, but instead are excreted unused. They should be diminished in decreased phosphoric katabol- ism, or when there is an increased consumption of the ingested phosphates for tissue formation (as in pregnancy or rapidly de- veloped leucocytosis), or when from impaired functional power of the kidneys or other causes the phosphates present in the blood are retained and not excreted in usual amount. Study of the ratio of the total amount of P^O^ to the total nitrogen ex- creted in the urine (normally 1 to 5 or 7) or of its ratio to the alloxur bodies, may reveal information significant as to the seat of the disturbance, whether due to abnormal metabolism of cell nuclei or of other structures. Clinically, the total excretion of phosphates is decreased in most acute diseases (from excretory failure), anaemias, preg- nancy, some nervous diseases, nephritis, some cases of hepatic disease (cirrhosis, acute yellow atrophy), and other conditions. The total excretion is increased in some bone diseases, in starva- tion, some nervous diseases, during sudden hsemocytolysis (as after rapid destruction of many red corpuscles or the subsidence of leucocytosis in convalescence from acute conditions), and in "phosphatic diabetes." The combinations in which phosphoric acid appears in the urine are glycero- phosphoric acid (in minute traces), alkaline phosphates, earthy phosphates, and ammonio-magnesium phos- phate. Alkaline Phosphates. — These consist chiefly of sodium phos- phates, with a small amount of potassium phosphates. Phos- phoric acid is tribasic, and forms three series of salts. In the case of sodium, for instance, these are neutral sodium phosphate 10 Digitized by Microsoft® 146 A MANUAL OF CLINICAL LABORATORY METHODS. (NajPOJ, which is alkaline in reaction, and monacid (l^a^HPOJ and diacid (NaH^POJ sodium phosphate, which are of acid re- action. All three of these may be present in the urine, but nor- mally the acid phosphates preponderate, and to these the urine chiefly owes its acidity. When from any cause the neutral so- dium phosphate increases in relative amount, the urine becomes proportionately less acid, neutral, or alkaline. The sodium and potassium phosphates of the urine are proxi- mately derived from those of the blood. Why these phosphates, which in the blood are alkaline, should pass into the urine in acid form is not altogether clear. These phosphates are freely soluble in aqueous fluids of any reaction, and occur in the urine only in solution. They are normally excreted to the amount of 2 to 4 grams daily. Earthy Phosphates. — These are the phosphates of calcium and magnesium: Ca3(POJ.^, normal or tricalcic phosphate, CaHPO,, the monacid phosphate, CaH,(POJ„ the diacid phosphate, Mg,- (POJ„ MgHPO,, and MgH,(POJ,. The amount of these daily excreted is normally 1 to 1.5 gram, the amount of magnesium phosphates being about double that of the calcium phosphates. The earthy phosphates are only sparingly soluble in pure water, quite soluble in acid fluids, even if only slightly acid, insoluble in alkaline media. In urine they are held in solution by NaH,PO„ COj, and NaCl ; in urine that is alkaline or made alkaline they are thrown down as a white precipitate. The acid salts are more soluble than the normal earthy phosphates ; and the three series of phosphates are readily transformed from one to another by changes in the surrounding conditions. Under certain conditions the earthy phosphates are precipi- tated from urine by heat. In many specimens of urine a white cloudiness or precipitate appears on being heated in a test tube, which clears up when a drop of acid is added — a reaction which distinguishes phosphates from an albuminous precipitate. If the urine be cooled after the cloudiness has been developed by boil- ing, the precipitate totally or partially redissolves, and the fluid clears either entirely or in part. The precipitate formed by the heat consists of phosphates of calcium and magnesium. The precipitation is mainly due either (a) to the chemical nature of the phosphates being altered by heat, less soluble salts being formed, while the process is reversed on cooling and the salts Digitized by Microsoft® THE URINE. 147 redissolve; or (6) to their being less soluble in hot than in cold media and being therefore thrown down by heat, redissolving on cooling. Another cause, which is perhaps especially manifest in those cases in which the turbidity persists after cooling, is the expulsion of CO^ and consequent loss of acidity caused by boiling, which diminishes the solvent power of the urine over the earthy phosphates. The precipitation by heat will occur whenever the earthy phosphates are in solution in such degree of concentration that when heated the point of saturation is passed. The degree of concentration depends partly on the amount of earthy phos- phates, partly on the acidity of the urine, and the concentration increases as the acidity diminishes. The precipitation by heat does not in itself necessarily indicate that the earthy phosphates are absolutely increased, and, in fact, is ordinarily due to a low degree of urinary acid- ity without any increase of phosphates. The earthy phosphates may occur in the urine either in solution (in acid urine) or (in ^,^ gS.-Calclum-phosphate crystals. weakly acid or alkaline urines) undissolved. In the latter case they usually appear micro- scopically in the form of masses of fine, amorphous, colorless granules, which dissolve on the addition of acid. Calcium phosphate (Fig. 23) appears rarely in the form of slender prismatic or broader wedge-shaped crystals, usually in radi- ating masses. Magnesium phosphate also rarely occurs as short crystals. In disease conditions the alkaline and earthy phosphates may fluctuate pari passu, or either may be increased or decreased in- dependently of the other, according to the source of the phos- phates at fault. Ammonio-magnesium phosphate (triple phosphate, MgNH^POJ is formed and precipitated whenever any dissolved phosphates, ammonium compounds, and magnesium salts are commingled in an alkaline medium. It is not excreted into the urine as such, but is formed in abundance in the process of ammoniacal fer- mentation of the urea, and is one of the characteristic features of that process. It is not soluble in alkaline fluids, and in am- Digitized by Microsoft® 148 A MAISTTJAL OF CLINICAL LABORATORY METHODS. moniacal urine appears (mixed with calcium phosphate) as an abundant undissolved, white, powdery sediment, which micro- scopically is in the form of both amorphous granules and crys- tals. The crystals of triple phosphate are very distinctive, being clear, colorless, triangular prisms with bevelled ends, mostly of relatively large size (Fig. 24) ; these are the forms found in the urine, and are produced when the triple phosphate or the crys- tals are formed ^'ery slowly. If the phosphate is suddenly precipitated, as by mixture of the necessary solutions in Fig. 24.-Ammonio-magaesie Phosphate Crystals. ^ test tube, delicate leathery and snowflake-like crystals are produced ; these after a time gradually change into the pris- matic crystals. Carbon dioxide and carbonates are ordinarily present in urine, originating from katabolism, from the food, and from ammoni- acal transformation of urea. Cajbon dioxide is one of the chief katabolic end-products, being excreted chiefly by the lungs, but to a small extent by the kidneys also. The food and other in- gesta are an important source, salts of organic acids in particular being excreted as carbonates. In ammoniacal fermentation, large quantities of ammonium carbonate are formed from urea. Both free carbon dioxide and carbonates are present in solution in the blood plasma, and each is capable of excretion as such into the urine ; but the free gas is best adapted to elimination by the lungs, the carbonates by the kidneys. Carbon dioxide is practically constantly present in urine in free form, and sometimes also in alkaline urines in combination as carbonates. The free CO.^ of the urine contributes materially to its acidity, and aids considerably in keeping the earthy phos- phates in solution. As it is a volatile acid principle, urine is diminished in acidity after its expulsion by boiling, agitation, or otherwise ; and if the acidity of the urine be entirely due to CO,, litmus paper reddened by it turns blue again on drying. The free CO^ originates partly from direct passage of the gas from Digitized by Microsoft® THE UEINE. 149 the blood into the urine, partly from decompositipu of carbonates excreted into acid urine. Carbonates and bicarbonates are frequently present in alkaline, neutral, or feebly acid urines, but cannot exist as such in acid urines, which immediately decompose them and set the CO^ free. The carbonates thus present are ordinarily those of sodium, po- tassium, and ammonium, the latter especially in ammoniacal fer- mentation ; calcium and magnesium carbonates may also be pres- ent. Calcium carbonate sometimes appears undissolved in the sediment of alkaline urine, in the form of small granules, spher- ules, or dumb-bell forms which effervesce on adding acid ; rarely it forms vesical calculi. The carbonates increase the alkalinity of urine. Ammonium carbonate is "volatile alkali," and is dis- sipated by heat ; litmus paper turned blue by dissolved ammo- nium carbonate reddens again on drying. Sodium and potas- sium carbonates are "fixed alkalies," and not affected by heat. The amount of free CO, present in acid urine under ordinary circumstances ranges from about 2 to 12 per cent by volume (.004 to .025 per cent by weight). Free or combined, the amount is increased in neutral and alkaline urines, after vegetable diet or the ingestion of alkaline carbonates or salts of organic acids, after drinking carbonated waters, and during conditions of heightened metabolism, as exercise or fever. After ingestion of large amounts of calcium hydrate, calcium carbamate appears in the urine, and decomposes, yielding CO,, CaCO,, and HjN. Chlorides, next to urea, form the most abundant single con- stituent of normal urine, 10 to 16 grams being excreted ordi- narily each twenty-four hours. The chlorides excreted consist chiefly of sodium chloride, with a small proportion of potassium, ammonium, calcium, and magnesium chlorides. They are de- rived from the surplus, over body needs, of chlorides introduced with the food, and are not products of body katabolism. The chlorides are excreted in diminished amount, even to practical absence, in most acute febrile diseases, the diminution being proportionate to the severity of the disease; and they in- crease again with the subsidence of the disease ; their decrease and increase in such cases are therefore of unfavorable and fa- vorable import respectively and correspondingly. The chlorides in the urine are also diminished in connection with excessive secretion of gastric juice or with the development of copious se- Digitized by Microsoft® 150 A MANUAL OF CLINICAL LABORATORY METHODS. rous exudations or transudations (the chlorides in such conditions being otherwise disposed of) and in various other affections. They are increased after the use of certain drugs, after a period of retention of the chlorides (as in convalescence from acute fevers), during the absorption of exudates and transudates, and in other conditions. They fluctuate also to a certain extent with the amount of urine excreted, being increased in daily quantity in polyuric conditions, and vice versa. Calcium oxalate, in small amount, frequently occurs in both acid and alkaline urines in the form of minute undissolved crys- tals. It originates both from food (especially acid fruits, etc.) <'\ t ) ''*-* 8 I F:g. 25.— Calcium-oxalate Crystals. I, Octahedral lonns. II, " Dumb-bell " rorms. a, a'. One crystal from two points of view ; b, 6', b", one crystal from tbree points ol view. and from metabolic processes in a manner not well understood. When constantly and abundantly present the crystals may indi- cate some nutritive or nervous disturbance, though their signifi- cance is not well defined ; often they appear in the urine of per- sons in good health without having any pathologic import at all. The crystals appear in two forms (Fig. 25). By far the commonest form is that of minute, highly refractile, colorless octahedra, varying in size from about 3 to 15 micromillimetres. Viewed from an apex, they present a very characteristic "envel- ope shape," the form of a square crossed by diagonals. Some of the octahedra are regular ; some, usually smaller in size, elon- Digitized by Microsoft® THE URINE. 151 gated in lozenge form ; some flattened. The crystals occur sepa- rate, attached together in masses or strings, or adhering to mu- cous threads, casts, etc. The other forms of calcium-oxalate crystals are the so-called "dumb-bell forms," which are uncommon. They vary somewhat in form, sometimes being oblong, with an annular constriction in the middle (dumb-bell shape). Often they are flattened spher- ical or oval form, deeply biconcave from a depression at opposite sides ; these forms have the dumb-bell appearance when viewed from one direction, and appear oval when viewed from another. They are sometimes minute, but are usually much larger than the octahedral forms, of duller lustre, and less refractile, and exhibit longitudinal lines arranged radially or concentrically, according to the point of view. Pigments and Chromogens. — The substances to which urine owes its color, and those (the chromogens) which while them- selves colorless under special circumstances or treatment develop coloring matter, comprise a group of diverse substances having little in common as to nature and significance except their color. Present knowledge and nomenclature of the normal and some abnormal urinary pigments are very confused and imperfect. The pigments and chromogens of urine, normal and abnormal, are about as follows: urochrome, uroerythrin, indican and indigo, an unnamed substance in febrile urines, bilirubin, a slightly al- tered form of bilirubin, haemoglobin and its derivatives, me- lanin, and adventitious coloring matters derived from ingesta. Some of the pigments and chromogens exhibit noticeable re- actions with nitric acid in the course of the tests for albumin, which direct attention to them. In the contact test, zones of color form at the junction of the fluids; or after heating the urine the addition of a drop or two of nitric acid causes color change. Urochrome. — This is the chief or only pigment of normal urine, to which its yellow color is due. By some it is called urobilin, while others apply the term urobilin to an abnormal pigment. The term urochrome, here adopted, seems admirably descriptive and suitable as a designation for the normal urinary coloring matter. Its composition and origin are not definitely determined, and it probably has not yet been isolated in purity. In normal amount it probably gives little or no color change Digitized by Microsoft® 153 A MANUAL OF CLINICAL LABORATORY METHODS. with nitric acid. It appears to be proximately a derivative of bilirubin, and through that of hsemoglobin. It is hence decreased (the urine being paler) in anaemic conditions, where the amount of hsemoglobin set free by erythrocytolysis is diminished ; and is increased in febrile conditions or during the absorption of hem- orrhagic extravasations, where there is an increased breaking down of red blood corpuscles. Uroerythrin is the rosy pigment which colors certain urinary sediments, especially undissolved urates. Urine containing it in excess may stain paper pink. Opinions differ as to whether it is present normally or abnormally, and as to the extent to which the high color of febrile and other concentrated urines is due to it. Indican, already considered, is a normal chromogen from which indigo-red and indigo-blue may be derived. Earely this takes place spontaneously, after standing, giving rise to red or blue urines. The pink color that appears in the nitric-acid tests with normal urine is probably due to indigo-red. The high color of febrile and other concentrated urines may be partly due to an increased amount of urochrome or uroery- thrin ; but in addition to these an unnamed and unknown sub- stance is perhaps present. Such urines in the nitric-acid contact test yield bright to dark red, purple, or dark color zones at the junction of the fluids, or the entire urine turns a similar color after being heated and treated with a few drops of nitric acid. Bilirubin and an altered form of bilirubin not capable of being oxidized to biliverdin appear in the urine in icteric conditions and choluria, imparting a brown or deep amber color (see below). Hsemoglobin and certain of its derivatives when present give the urine a color ranging from blood red to dark shades (see below). Melanin. — In some (not all) cases of melanosis or melanotic tumors the black pigment melanin may appear in the urine, either in solution or in the form of undissolved black granules. Usually the urine is normal in color on being passed, but turns dark or black on standing, the substance actually excreted being a chromogen (melanogen). In wasting diseases the urine some- times contains melanin. A black pigment (probably different from melanin) is sometimes voided with the urine in repeated or Digitized by Microsoft® THE URINE. 153 chronic malaria. Urines containing alkapton also turn dark on standing. Various ingested foods and drugs yield substances that affect the color of the urine (page 164). Fat is sometimes present in the urine in minute amount in the form of small globules discernible under the microscope ; rarely the fat is very abundant (lipuria), so that it runs together in oily masses or rises to the surface of the urine as a layer of fluid oil, a cream -like layer, or (on cooling) a solidified or coagulum-like layer. Urine containing much fat, suspended in fine globules, is whitish, opaque, and milk-like. Very rarely fatty calculi form in the bladder. In small amount fat may be present as droplets in or set free from epithelium cells that have undergone fatty degeneration in parenchymatous inflammations or fatty conditions of the kid- neys ; the fatty casts are of like origin. Pat has been observed in the urine, sometimes in large amounts, during the repair of fractures, fat embolism, heart disease, pancreatic disease, dia- betes mellitus, phosphorus poisoning, abscesses and necrosis of adipose tissue (liberating fat in abundance), especially when discharging into the urinary passages, during pregnancy and the puerperium, following the ingestion of oils (as oleum morrhuse), from the rupture and leakage of lymph vessels into the ureter or bladder, and in other conditions from causes imperfectly under- stood. Fat is most often present and abundant in the urine in chy- luria, a tropical affection mostly associated with filariasis and due to leakage of chyle into the urine. Chylous urine is white, opaque, and milk-like ; it contains large amounts of fat in veiy fine granular subdivision, along with leucocytes, albumin, coag- ulable fibrin, red blood corpuscles, and sometimes larval filarise. In cases of lipuria not chylous the fat occurs in larger glob- ules than in chylous urine. Fatty acids (formic, acetic, butyric, propionic) occur in tlie urine nor- mally in minute amount, increased at times in various conditions, as diabetic coma. They are derived from intestinal fermentations, and perhaps in some cases from perverted katabolism. In the course of cystitis in cases of glyco- suria they may originate from fermentation of the glucose. Earthy soaps are perhaps at times present in the urine in the form of radiating acicular crystals. Lactic acid has been found in the uiine after muscular exercise, in hepatic afiections, diabetic coma, and other conditions. Other organic Digitized by Microsoft® 154 A MANUAL OF CLINICAL LABORATORY METHODS. acids, ferments (pepsin, diastase, rennet), and hydrogen dioxide have been demonstrated in urine in traces, but are witliout practical importance. Iron is normally present in minute amount, probably entering into the formation of some of the pigments. Nitrates, derived from ingesta, sometimes appear in the urine in small amount. Traces of silicic acid may be present. Small amounts of free oxygen and nitrogen are normally present, derived from the inspired gases taken into the blood. The other gases of the urine, COs and HjS, have been already considered. A few cases have been reported in which a large amount of gas was pres- ent in the urine (pneumaturia), distended the bladder, or escaped from the urethra. Some cases are caused by the entrance of gases from the intestine through a vesico-enteric fistula. Others arise from the action of gas-forming bacteria on the urine in the bladder, being usually associated with cystitis. Many of these cases occur in glycosuric conditions, the sugar being fermented by microorganisms like yeasts and the colon bacillus, and yielding CO2, alco- hol, and fatty acids. Some cases occur in connection with sugar-free urines, the substance yielding the gas not being known ; the colon group, bacillus aerogenes capsulatus, and other bacteria are the active agents, and the gases produced are carbon dioxide, hydrogen, oxygen, nitrogen, and methane. Ex- ceptionally the site of gas generation is the kidney or ureter. Proteids. — The proteids that appear in the urine, chiefly in abnormal conditions, are nucleo-albmnin, serum -albumin, globu- lin, albumose, fibrin, haemoglobin. Nucleo-albumin, or urinary mucin, is often present in normal urine in minute amount, at other times absent or so scanty as to be undemonstrable by delicate tests. It may be in sohition, or it may in strongly acid urine be precipitated in the form of fine, colorless mucous threads, which play an important part in the formation of the light flocculeut cloud or nubecula that often forms in urine on standing. If in excess mucin sometimes ap- pears in the form of viscid, gelatinous, mucous masses, which set- tle to the bottom unmixed with the urine, or rarely may give the entire urine a gelatinous consistency. It is normally derived from the mucous lining of the urinary passages ; but as this mu- cous membrane lacks mucous glands and goblet cells, its muci- nogenous action ordinarily is very insignificant. In women it may be adventitiously introduced from the vagina. Mucin is increased in the urine by irritation or catarrhal involvement of the uriniferous tubules and urinary tract; also sometimes in acute nephritis, febrile and other albuminurias, choluria, leukae- mia, etc. Its increase is chiefly due to pathologically increased mucinogenous action of the renal or urinary cells, or to disinte- gration of these cells ; in some cases (as in choluria, leukaemia, Digitized by Microsoft® THE URINE. 155 and perhaps other conditions of lencocytolysis) it is probably introduced from the blood. Albumin (serum -albumin) is the most important urinary pro- teid. It is never normally present in urine, except possibly in traces so minute as to be demonstrable only by the most delicate tests, and even then with doubt. Its presence (at least in amount sufficient to respond to the ordinary tests) is always of serious pathological significance, and the test for albumin is one of the most important items in the examination of urine. Albu- min may and most frequently does appear in the urine as a result of impaired functional action of the parenchymatous renal cells, and is thus indicative of acute or chronic nephritis, renal conges- tion, toxic injuries to the renal cells (as in febrile toxaemias, etc. ), parenchymatous renal degeneration ; albuminuria may also result from circulatory conditions (as increased renal blood press- ure), alterations in the quality of the blood, and in other ways not well understood. Albumin may also enter the urine along with blood, lymph, or pus from lesions along the urinary pas- sages subsequent to the excretion of the urine from the kidneys. The greatest amount of albumin usually occurs in the urine in acute and chronic parenchymatous nephritis ; a less amount in other conditions. The amount of albumin excreted may range up to 10 or 15 grams per day. Globulin (serum-globulin) usually appears in the urine when- ever serum-albumin does, in large relative amount, sometimes indeed in greater quantity than the latter. Its origin and signifi- cance are about the same as those of serum-albumin. Egg-albumin sometimes appears in the urine after free inges- tion of eggs as food. Albumoses may appear in the urine in a variety of condi- tions, as in association with other albumins in albuminurias, in suppurations, bone diseases, hepatic affections, and many others. The definite clinical significance of albumosuria is not determined. Whether peptone ever appears in the urine is now in doubt, the reactions formerly taken as showing the presence of peptones now being regarded as indicative of albumoses and not of pep- tone. Fibrin appears in the urine in hsematuria, chyluria, lymphu- ria, or fibrinous inflammation of the urinary tract. The passage Digitized by Microsoft® 156 A MANUAL OP CLINICAL LABORATORY METHODS. of blood, chyle, or lymph into the urine necessarily introduces fibrin, with other substances. The fibrin may be in solution in the urine, in coagula of various size, or in microscopic flakes; sometimes the entire urine coagulates into a gelatinous mass. Other proteid substances, varying from the ordinary proteids in some of their reactions, are rarely found in the urine, as histon. Carbohydrates. — This urinary group includes one substance of prime importance, glucose, along with a number of rare or less important related substances, namely, levulose, inosit, su- crose, lactose, maltose, pentoses, glycogen, dextrin, animal gum. Glucose (dextrose, grape sugar), C^Hj^O^, is usually practi- cally absent from normal urine, though traces of it or some other reducing agent may at times be demonstrable in concentrated urines. It may appear temporarily in many diverse conditions, as febrile affections, meningitis, alimentary disturbances, hepatic disease, excessive ingestion of starchy or saccharine foods, or after the use of some drugs, as phloridzin ; such temporary oc- currence has no serious significance. Its persistent presence in large quantities in the urine is the cardinal and pathognomonic symptom of diabetes mellitus, of pancreatic, hepatic, nervous, or any other form, and indicates severe metabolic disturbance. According to its abundance glucose causes a marked increase in the specific gravity of the urine, frequently up to 1.040 or more ; it is sometimes, however, plentiful even with low specific gravities, down to 1.012. The amount eliminated in twenty-four hours ranges from zero up to 500 grams. With the glucose other allied carbohydrates may at times appear. Glucose when boiled with caustic alkali with access to the air is oxidized and turns dark-colored. It reduces the oxides of copper and bismuth. With phenylhydrazin it forms crystals of phenylglucosazon. It rotates polarized light to the right. Under the action of certain micro-organisms, as yeast and the colon ba- cillus, glucose undergoes fermentation, breaking up into carbon dioxide and alcohol (C,H,p,= 2C„H,0 + 2C0J ; fatty acids and other substances may also be formed in the process. At times in cases of glycosuria when the bladder becomes infected with the fermenting organisms, the glucose undergoes fermentation in the bladder, with the formation of carbon diox- ide, alcohol, and sometimes fatty acids, which are voided with Digitized by Microsoft® THE URINE. 167 the urine. Thus, in an intercurrent cystitis in the course of diabetes mellitus, the amount of glucose in the urine may dimin- ish, abundant gas, with alcohol, may be present, and the urine may be strongly acid. Such a decrease must not be mistaken for a real decrease in the renal excretion of glucose. Levulose, CeHuOs, sometimes appears in the urine in diabetes mellitus, usually together with glucose. Its chemical reactions are similar to those of glucose, except that it rotates polarized light to the left. luosit, CoHijOa.SHaO, is occasionally present in the urine in diabetes mellitus, diabetes insipidus, and chronic nephritis, and at times in other con- ditions, and even in traces in normal urine. It is quite different from glucose in its reactions, having no action on polarized light, not undergoing alcoholic fermentation vrith yeast, not giving the phenylhydrazin reaction, and not typically reducing copper. Sucrose (saccharose or cane sugar), C12H22O11, may appear in the urine in traces after the free ingestion of cane sugar. It is dextro-rotatory, and does not reduce copper, or ferment with yeast. By prolonged boiling with water (especially if acidulated) sucrose is inverted, or converted into a mixture of glucose and levulose, with a predominating Isevo-rotatory power. Xiactose (or milk sugar), CuHajOn-HaO, is frequently present in small amount (exceptionally in largo amount, constituting a sort of diabetes) at the end of pregnancy and during lactation, especially with amply' developed breasts or when the milk secreted is not freely evacuated, as just after labor, during mastitis, and at the weaning period. Lactose reduces copper, is dextro- rotatory, gives (in dilute solution) a negative phenylhydrazin reaction ; it does not undergo alcoholic and OO2 fermentation with yeast, but is more subject to lactic-acid fermentation. It may be converted into glucose and galactose. Maltose, CuHjaOu, has been observed in urine along with glucose. Its reactions are similar to those of glucose. Maltose is the first sugar formed in the diastatic digestion of starch; it is closely related to glucose, into which it is converted by further action of diastase or other treatment. Pentoses. — This is a group of sugars based on the formula CbHjoOb, some of which (arabinose, rhamnose, xylose) may appear in the urine after the ingestion of certain food (pears and other fruit, beer, etc.), in diabetes, and in other conditions. Glycogen, nCsHioOs, has been observed in the urine, in diabetes and other conditions. Bextrin, nCeHioOj, has alsp been observed in the urine in diabetes mellitus. Animal gum is said to be present even in normal urine in minute amount. Grlycuronic acid, CeHi oOt, is related to the carbohydrates, and may occur in the urine in combination with alkaline or with ethereal bases (indoxyl, phenol, etc.). Glycuronates sometimes appear in normal urine in traces and in increased amount'after the ingestion of turpentine, camphor, chloral, chloro- form, morphia, phenol, and other drugs. Of their quantitative variations in disease conditions practically nothing is known. Glycuronic acid in the Digitized by Microsoft® 158 A MANUAL OF CLINICAL LABORATORY METHODS. urine probably has little clinical significance, but it is of practical importance in that it reduces copper salts and is hence apt to be erroneously taken for glucose. Alkapton.— Cases are rarely met in which the urine contains a reducing substance of obscure origin and composition, but different from the sugars, glycuronic acid, or certain reducing ingesta. Various names have been given this substance, as pyrocatechin, urrhodinic acid, oxyphenic acid, glycosuric acid, homogentisinic acid, etc. ; but until its definite nature is ascertained the term "alkapton," employed In the first case studied, may be conveniently re- tained for the substance or substances responsible for the reactions presented. Urines containing alkapton may be of normal color on being passed ; but, especially when alkaline or made alkaline by fermentation or the addition of caustic alkali, they turn dark from the surface of the urine downward, by a process of oxidation ; they reduce salts of copper, but not of bismuth, do not ferment with yeast, do not afEect polarized light, and give no phenylhydrazin reaction. Alkaptonuria has been observed both in healthy and diseased per- sons, but is probably in itself an anomaly devoid of pathologic significance. It may persist for years, has been discovered in both children and adults, and may appear in several individuals of the same family. Practically it is im- portant not to mistake it for glycosuria. COMPABATrVK TABLE OP REACTIONS OP CARBOHTDKATBS AISTD ALLIED Bodies. Substances. II 8 Undergoes alcoholic fermentation with yeast? Rotatory action on polari2ed light. Gives phenylhydrazin reaction ? Melting-point of osazon crystals in phenylhydrazin test. Reaction with KOH In contact with air. Yes Yes Not typi- cally... No Yes Yes Yes Not typi- cally . . . No Yes. . . Right, 56° Left, 106° at 14° C. Yes 204° C... 150° Darkens when Yes.. No.. No .. No . Yes . No.. boiled. No. No hoiled. Eight, 74° Right, 59° Right, 150° .... Some to right, others inactive Right, 311° .... Right, 138° ... . Right Lactose Not In dilute solution. Yes None. 190° Slightly discol- ored by boiling. Pentoses Glycogen Dextrin Some, yes. No. No Animal gum . . . Glycuronic acid. Glycuronates... Alkapton No. Yes Yes Yes No.. No.. No .. Yes. Wo. No Left drnary tem- perature. Acetone, Diacetic Acid, /3-Oxybutyric Acid. — These three sub- stances are closely related chemically and pathologically. They occur separately or associated together, especially in diabetic coma and other manifestations of the "acid intoxication"; they Digitized by Microsoft® THE URINE. 159 also at times appear in other conditions. Their precise relations to one another and to the disease process are not as yet deter- mined. Their presence in abundance is usually accompanied by an increase of ammonia, which forms ammonium salts with these acids and is abstracted from that which would otherwise form urea. Acetone seems to be a product of disintegration of fat or pro- teid, either from food or body tissue, and in minute amount may appear in normar urine. It may be increased on a fat or meat diet, with insufficient carbohydrate food, during starvation, high fevers, nervous and mental affections, carcinoma, alimentary dis- turbances; and pre-eminently in diabetes. Acetone imparts a fruity odor to the urine, is volatile, and expelled by boiling. Diacetic acid and ;3-oxybutyric acid are never present in nor- mal urine ; their presence and that of acetone in excess are of serious import. Diacetic acid yields similar reactions to those of acetone, but it is not volatile ; under certain conditions it breaks up and yields acetone, alcohol, and carbon dioxide. Beta-oxy- butyric acid is prominently characterized by its Itevo-rotatory power. Alcohol may rarely be found In the urine, in traces after free ingestion of alcohol, in connection with diacetic acid as one of its decomposition pro- ducts, and in cases of glycosuria when the glucose has undergone alcoholic fermentation in the bladder. Blood (hsematuria). — Blood, as such, enters the urine by hemorrhagic extravasation in some part of the urinary tract. All the elements of the blood are thus introduced into the urine, plasma, corpuscles, and pigment. Hsematuria is manifested by the concurrent presence of proteids (albumin, globulin, some- times clots of fibrin), red corpuscles, leucocytes, and hsemoglobin or its derivatives. The characteristics and appearance of urine containing blood depend upon the amount of blood present, its source, the length of time it has been mixed with the urine, and the changes which it may have suffered. If in large amount and recently shed, the urine is manifestly bloody, bright red in color, and with a sedi- ment of corpuscles and clots. With a smaller amount of blood the color ranges from red through brownish or reddish amber to the normal color of the urine. If the blood has remained a long time in the urine, the haemoglobin may undergo transformation Digitized by Microsoft® 160 A MANUAL OF CLINICAL LABORATORY METHODS. to derivative substances that give tlie urine more of a smoky, or brown, or dark color. In cases of hEematuria it is important to determine if possible the seat of the hemorrhage. Hsematuria originating from the kidney occurs in acute nephri- tis, congestion, chronic nephritis, especially with sclerosis and degeneration of the renal vessels, traumatisms, malignant dis- ease, tuberculosis, conditions like purpura, scurvy, and haemo- philia, malaria, and from the action of cantharides and other poisons. The urine in cases of renal haematuria has the blood intimately mixed with it, and is of a darker and more smoky color than in vesical haematuria. Eenal tube-casts may be pres- ent and the occurrence of blood casts is quite pathognomonic. Blood clots are absent or small, or may be cylindrical casts of the ureter. The red corpuscles are apt to be considerably al- tered, decolorized, or even disintegrated altogether. Haematuria originating from the ureter or renal pelvis is caused by the presence or passage of calculi and other causes. From the ureter the hemorrhage is usually slight ; cylindrical blood clots moulded in the ureter may be passed. Haematuria originating from the bladder results from ulcers, cystitis, vesical calculi, malignant disease, tuberculosis, vascular tumors, rupture of varicose vesical veins, etc. In vesical haema^ turia the blood is usually less intimately mixed with the urine than in the renal forms, the color is brighter red, clots may be more abundant, larger, and irregular. Features pointing to the bladder as the seat of trouble may be present. Hemorrhage from the urethra is caused by traumatisms, ure- thritis, neoplasms. The blood may be passed independently of the urine, or in the first portion of the urine voided. During menstruation blood is apt to find its way into the urine of women from the vagina. Haematuria from various parts of the urinary tract may also originate from parasitic conditions, as from filaria, schistosoma haematobium, or protozoa. Red blood corpuscles in the urine may exhibit their ordinary form, or they may be more or less altered by the action of the urine. They may be creuated, irregularly contracted or de- formed, or they may become decolorized ("shadow corpuscles") ; or in alkaline urine they may be destroyed and disappear. Digitized by Microsoft® THE URINE. 161 Haemoglobin may occur iu the urine in two conditions, either alone and unaccompanied by other ingredients of the blood (hsemoglobinuria), or along with other blood elements in hsema- turia. HsBmaturia is common, hsemoglobinuria uncommon. The distinction between the two rests definitely on the presence of red corpuscles ; if these are present the condition is hsematuria, if haemoglobin is present but the corpuscles are absent it is hsemo- globinuria ; and before settling on the latter diagnosis it must be considered that red corpuscles if in small number may become disintegrated and escape detection. Hsemoglobinuria is a result of hsemoglobinsemia, the excess of haemoglobin iu the blood plasma, derived from excessive disinte- gration of red blood corpuscles, being excreted by the kidneys. It'is thus indicative of excessive erythrocytolysis (also perhaps of inability of the liver to transform hsemogloftin into bilirubin), and as such is rather a serious symptom. Hsemoglobinuria oc- curs at times after the ingestion of various poisons, transfusion of animal blood, burns, in paroxysmal hsemoglobinuria, acute fevers, etc. In hsematuria, haemoglobin occurs not only in the red corpus- cles, but passes into solution in the urine. Haemoglobin in the urine imparts its red color to the fluid, and also, being a proteid, gives the reactions of albumin. Instead of haemoglobin, deriva- tives from it may appear in the urine, as methaemoglobin, hsema- tin, hsematoidin, hsematoporphyrin, and perhaps others. Methaemoglobin is often present in the urine in the same condi- tions (haematurias and haemoglobinurias) in which haemoglobin appears. It may be excreted from the blood as methaemoglobin, or it may be formed from haemoglobin in the urine. It is closely related to oxyhsemoglobin, but is of a brown color, and gives the dark color to urine containing altered blood. Haematin is rarely present in the urine, arising from the trans- formation or decomposition of haemoglobin (which consists of a combination of a globulin with haematin). Hsematoidin is rarely seen in urine, in connection with condi- tions similar to those causing hsematuria and haemoglobinuria. It is a derivative of haemoglobin, is ordinarily regarded as iden- tical with bilirubin, and occurs in undissolved crystalline or amorphous form. Hsematoporphyrin is formed by the action of acids on blood 11 Digitized by Microsoft® 162 A MANUAL OF CLINICAL LABORATORY METHODS. pigment. It may be demonstrated in many normal urines in minute amount. Earely it appears in large quantity (haemato- porphyrinuria) giving the urine a brownish-red color (sherry or port- wine color) ; and such urines may darken on standing. Hsematoporphyrinuria occurs especially after excessive use of sulfonal and similar drugs, in some cases of gastric and intes- tinal hemorrhages, at times in rheumatism and other conditions, and even after the use of food containing much haemoglobin. Lymph would appear in the urine (lymphuria, chyluria) from rupture of lymph vessels into the urinary tract, as in filariasis. Lymphuria would be manifested chiefly by the presence of fibrin and albumin; chyluria by the presence, also, of fat (page 153). Pus in the urine (pyuria) enters in connection with catarrhal or suppurative processes or abscesses along or communicating with the urinary tract. It is manifested by the presence of large numbers of leucocjd^es, albumin, and nucleo -albumin. The pus may be intimately mixed with the urine, especially if the latter is acid, causing a general turbidity or subsiding as a white granular sediment. If the urine is ammoniacal the leucocytes disintegrate and a mucoid, viscid, tenacious, opaque white sedi- ment is formed. It is important to determine the source of the pus. Pus derived from the kidney or renal pelvis may be either scanty or abundant ; unless the affection is bilateral the urine is usually acid and may have a characteristic odor. The presence of casts indicates renal involvement, and pus casts are quite pathognomonic of renal suppuration. The pyuria may be inter- mittent owing to temporary ureteral obstruction. Urine containing pus of vesical origin is usually ammoniacal and foul. Pus is passed with the urine in urethritis, especially in the portion of the urine first voided, the latter portion of the urine being clear or at least clearer than the first. In this condition microscopical mucous filaments studded with leucocytes ("gonor- rhoeal threads ") appear and are very characteristic. In women pus from leucorrhceal discharges may become mingled with the urine. Bile, or its characteristic constituents, occurs in the urine (choluria) in icteric conditions. The constituents of bile that appear in the urine are bilirubin (and its derivatives), bile salts, Digitized by Microsoft® THE URINE. 163 cholesterin, and nucleo-albumin. Urine containing bile elements is very characteristic in its appearance and properties ; it is of a clear, deep amber or brown color, very frothy upon being shaken, the foam also being golden or brown in color, and such urine leaves a yellow stain on paper or cloth. Bilirubin, the primary bile pigment, is diagnostically the most important and conspicuous element of the bile in urine, on which the diagnosis of choluria is made. It imparts to such urine a characteristic deep amber or brown color. Under the action of oxidizing agents, as nitrous acid, hydrogen peroxide, and others, bilirubin is converted successively into a series of pigments ex- hibiting a marked contrast of colors. The rich amber bilirubin is first transformed into biliverdin, a bright-green pigment ; this in turn changes into a blue substance, followed by a purplish- red, and then, it is said, a yellow substance ; on these reactions are based the tests for bilirubin. The pigment excreted into the urine is bilirubin ; such urine on standing may turn green from the formation of biliverdin. Bilirubin very rarely appears in urine in undissolved crystalline or amorphous form. Altered Bilirubin. — It occasionally happens that the urine from icteric patients, although presenting precisely the same color and other characteristics that are shown by urine containing normal bile pigment, does not give the reactions of bilirubin. Instead of green, a red, brownish, or even blackish color appears on treatment with nitric or other mineral acids. In these cases the bilirubin has undergone some slight alteration, of obscure nature, to a substance incapable of oxidation to biliverdin. This altered bilirubin is perhaps related to the pigment of febrile and similar urines. Bile salts and acids appear in urine associated with bilirubin in icterus and possibly under other circumstances. Their demon- stration is difficult. Cholesterin, in the form of undissolved crystals, has been ob- served in the urine in a very few cases in chyluria and nephritis (not in jaundice). Diazo Reaction. — This is a color reaction of the urine obtainable in certain conditions by means of diazo -benzene-sulphonic acid. The substance which yields the reaction is entirely unknown. The diazo reaction usually occurs in typhoid fever, measles, acute tubercidosis, severe stages and terminal stages of chronic Digitized by Microsoft® 164 A MANUAL OP CLINICAL LABORATORY METHODS. tuberculosis, and perhaps in smallpox; only exceptionally in other diseases, acute or chronic. It is of some diagnostic and prognostic value, and worthy of clinical consideration, epecially in typhoid fever. The reaction usually appears at some time in the course of typhoid fever, and often early enough (during the first week) to be of great significance. Its absence does not, however, negative the diagnosis. Toxins. — Toxic substances varying in amount and kind are excreted in the urine in both normal and abnormal conditions. They originate from normal or abnormal katabolism (leuco- mains), in the intestine, as products of bacterial growth in infectious conditions, or products of decomposition. They are mostly alkaloidal bodies of the nature of ptomains or diamines, some of which have been isolated, as putrescin, cadaverin, etc. A constitutional tendency to the formation and excretion of certain diamines sometimes appears to be associated with cysti- nuria. Toxicity of the Urine. — The poisonous ingredients of the urine impart to it various toxic properties, as myosis, dyspnoea, narco- sis, diuresis, salivation, hypopyrexia, convulsions, etc. The toxic powers of the urine vary in kind and degree in different conditions, and are determined by intravenous injection of the urine into rabbits or other animals ; the amount required to pro- duce special symptoms or to cause death furnishes a quantitative index of the toxicity of the urine. Animals are killed by nor- mal urine injected intravenously in the average proportion of 45 cubic centimetres of urine to 1 kilogram of body weight. The toxicity of the urine is diminished (larger amounts being re- quired to kill) when the excretory power of the kidney is im- paired (as in uraemia), the toxins not being excreted. Its tox- icity is increased or new toxic symptoms are produced, in most acute infectious diseases, and leuksemia, unless the functional power of the kidneys is impaired. The urine in such cases (as in tetanus and cholera) may reproduce the prominent phenomena of the disease itself. As the urine in typhoid fever may yield the Widal reaction, it is evident that agglutinins as well as other bacterial products are excreted by the urine. Adventitious Ingesta.— Many food and drug substances intro- duced into the system by the alimentary tract or otherwise are Digitized by Microsoft® THE URINE. 165 excreted in the urine, either in their original or a modified form. Some of these affect the appearance of the urine, some become evident during the application of the usual urinary tests, others may be demonstrated by special methods. At times diagnostic information of value is afforded as to poisons ingested. The ingestion of rhubarb, senna, or santonin turns the urine bright yellow if acid, or red if alkaline. Various dyes, as methylene blue, indigo, madder, etc., impart to the urine their respective colors. Salicylic acid is excreted as salicyluric acid, which if in large amount colors the urine green or smoky. Urines containing the derivatives excreted after free ingestion of phenol are green or greenish- black, and turn darker on standing. Excreted iodides form a sort of chromogen, yielding the brown color of free iodine on adding nitric acid. • Most of the alkaline salts of organic acids are excreted as carbonates. After the in- gestion of drugs like copaiba and turpentine the resinous sub- stance is excreted in the urine and in the nitric-acid tests appears as a white precipitate, which, however, differs from albumin in being soluble on the addition of alcohol. A case is recorded in which coal dust temporarily appeared in the urine in the case of a cleaner of stoves. Epithelium cells desquamated from the urinary passages are usually present in the urine, varying from very scanty numbers to a quantity sufficient to cause general cloudiness. Large num- bers of cells also enter from the vagina, so that the cells are usually much more numerous in the urine of women than of men. The cells may be separate and single, or attached together in as yet unbroken aggregations. As to the form, the epithelial cells in the urine can be classed as squamous, spherical, and elongated or irregular. The sc[uamous cells are large, expanded, iiat pavement cells. The body protoplasm is more or less granular, and in the centre is a round granular nucleus ; sometimes two nuclei are present. The cells are often curled or rolled up. Cells of this kind are desquamated from the most superficial layers of the renal pelvis, ureters, bladder, and vagina, and have little or no pathological significance. The spherical cells frequently seen in urine, smaller than the flat cells, are rounded or spheroidal in shape, finely granular, each with a large central spherical nucleus. They differ from Digitized by Microsoft® 166 A MANUAL OP CLINICAL LABORATORY METHODS. leucocytes in their larger size and single round nucleus. Some of them may originate from the renal tubules, such as the cells forming epithelial casts ; they are mostly, however, derived from the deeper or germinal layers of the epithelial lining of the renal sinus, ureters, and bladder. Those of recognizably renal origin in connection with other renal symptoms indicate disease of the kidney. Those derived from the urinary passages, being cast off rather prematurely in immature form, to that extent indicate slight irritation or catarrh ; in small number, their significance in this respect is trivial, but in large numbers they may accom- pany catarrhal conditions. The term mucous corpuscle is used in urinological literature in a rather vague sense ; if used at all, it is best applied to designate the spherical epithelium cells cast off immaturely from the germinal layers of the urinary mucosa. Elongated, columnar, fusiform, pyriform, or irregular cells some- times appear in the urine ; their origin and significance are simi- FiG. 26.— Epithelium Cells in Drlne; lar to those of the spherical cells, and they are derived from the kidney or from the deeper layers of the epithelial lining of the urinary passages. It is not often possible to determine with certainty, from the form of the epithelial cells, the precise region from which they are derived. Precisely the same kind of epithelium lines the renal pelvis, ureters, and bladder ; the squamous cells of the vagi- na cannot practically be distinguished from those of the urinary passages; and round or elongated cells from the kidney, when not in casts and after being altered by the urine, are practically identical in appearance with those from the urinary passages. Digitized by Microsoft® THE URINE. 167 Fragments of tissue, as neoplasms, are rarely passed with the urine and may afford valuable diagnostic information. Leucocytes are frequently present in small numbers in normal urine, with no clinical significance, being wandering cells that have worked their way through the tissues. They are present iii increased number in local inflammations, haematuria, lymphuria, and in very large number in the severe inflammatory, catarrhal, and suppurative conditions of the urinary organs attended with pyuria. They may also be derived from vaginal discharges. They are mostly of the polynuclear variety ; sometimes exhibit amoeboid movements ; may become altered or disintegrated by the action of the urine, especially if ammoniacal ; and by strong al- kalies are converted into a viscid mucoid mass. Spermatozoa appear in the urine after coitus, masturbation, or emission from other causes, in spermatorrhoea, and sometimes after epileptic convulsions or in other local or general affections. They may appear in the urine of women after coitus. Casts. — Tube casts or cylinders are moulds or casts formed in the renal uriniferous tubules, and are of great diagnostic signifi- cance, being almost always distinctly pathological and always at least suspicious. They are long, cylindrical bodies, 20 to 50 mi- cromillimetres in diameter, unbranched, usually straight, but sometimes irregularly curved as if following the course of a con- voluted tubule. They are composed of various materials, ac- cording to which they are classified as follows: hyaline casts, epithelial casts, granular casts, fatty casts, waxy casts, leucocyte casts, blood casts, bacterial casts, urate casts. Cylindroids are objects somewhat similar to the casts. Hyaline casts are the forms most frequently seen. They are pale, colorless, clear, hyaline, and faintly outlined in the micro- scopic fields. Sometimes they contain scattered granules or a few epithelium cells, or a portion of the casts may be granular or epithelial, indicating a transition to granular and epithelial casts. Granules, crystals, or cells may also become attached to their surface subsequent to their discharge from the kidney. They seem to be formed by proteid material from the blood be- coming coagulated in the tubules and thus forming a mould of them. They are abundant in acute and chronic diseases of the kidney, and are strongly indicative of renal congestion, acute or chronic nephritis, or degeneration, especially when, as is usually Digitized by Microsoft® 168 A MANUAL OP CLINICAL LABORATORY METHODS. b the case, they are accompanied by albiuniuuria and other renal symptoms.' At times, however, they appear in the urine in very small numbers without concomitant albuminuria and in individ- uals without demonstrable renal or other lesions. Epithelial casts are moulds of the tubules formed of the cells of the tubes cemented together by hyaline material. The cells may exhibit their original form, little changed, with nuclei dis- tinct, or they may be degenerated, becoming granular or fatty. They indicate renal disease of the same nature as do hyaline casts. Granular casts are composed of granules, coarse or fine, dark or light colored, cemented together in the form of tubules. The granular material is derived from the debris of broken- down epithelium, blood, or pus cells. Their significance is the same as that of hyaline and epithelial casts. Fatty casts originate from epithelium cells that have un- dergone extensive fatty de- generation, and contain abun- dant fatty globules. Their surface may exhibit fatty-acid ciystals. They indicate fatty degeneration of the kidney. Waxy casts resemble the hyaline casts, but are larger, rather yellowish, more retractile, more conspicuous, and more substantial in appearance than the hyaline casts. Sometimes they yield the amyloid reaction with iodine, sometimes not. They are uncom- mon. Their mode of origin and significance are not definitely set- tled. They have no exclusive association with amyloid disease of the kidney ; that is, they occur in other conditions as well as amyloid kidney, and do not occur in all cases of amyloid kidney. Leucocyte casts, or pus casts, are composed of leucocytes ce- mented together in the form of casts. They are rare, occurring in suppurative conditions of the renal parenchyma. Blood casts consist of, or contain, red blood corpuscles, and are indicative of hemorrhagic conditions of the renal substance. They are rare. Casts composed of granules of hsematoidin or haemoglobin have been observed in eases of haemoglobinuria. Fig. 27. — Casts from Urine, a. Hyaline cast; /*, hyalo-granular cast ; c, granular cast ; (J,liyalo- epithelial cast ; e, epithelial cast. Digitized by Microsoft® THE URINE. 169 Fi&. 28.— Cyllndrolds (a) and Mucous Threads li;;. Bacterial casts, consisting of an aggregation of bacteria, may occur in infectious, septic, or embolic conditions of the kidney, and are of serious import. Urate casts, composed of amorphous urates cemented together, may appear in the urine of new-born infants, and in cases of gouty kidney. Cylindroids are long, pale, hyaline, faint band-like formations somewhat similar to, and indeed at times difficult to distinguish from, hyaline casts. They are of greater length than the casts; at one end they taper to a point or filament, and at the place where they taper usually exhibit a character- istic indented or scalloped outline. They are generally regarded as being composed of mucoid material, and as being formed within the uriniferous tubules. Granules and cells may be adherent to them. They are frequently associated- with mucous threads, and at times with hyaline casts. They are frequently observed and are not of much pathological significance, only a slight degree of renal irri- tation being necessary to produce them. Mucous threads are fine, colorless filaments, long and slender, often matted and tangled together ; they consist of the trace of nucleo- albumin or mucin in the urine precipitated in threads by the action of the acid principles of the urine, and appear most markedly in highly acid and concentrated urines. Spirals analogous to Curschmann's spirals of the sputum have been observed in the urine in one or two instances. Granular and amorphous debris may be present in the urinary sediment, derived from broken-down cellular material or insol- uble chemical constituents. Calculi.— The commonest calculi derived from the urinary or- gans, kidney or bladder, are composed of earthy phosphates, cal- cium oxalate, uric acid, or urates. Very rarely urinary calculi are. composed of xanthin, indigo, cystin, calcium carbonate, fat or soap (urostealith), cholesterin. Prostatic and urethral cal- culi are sometimes encountered, as well as renal and vesical concretions. Digitized by Microsoft® 170 A MANUAL OF CLINICAL LABORATORY METHODS. Parasites. — The organisms appearing in the urine are both ani- mal and vegetable. Animal Parasites. — Three or four species of protozoa, tricho- monas, amcebse, and others, have in a few instances been observed in the urine. Trichomonas is probably the commonest, usually wandering into the bladder or urethra from the vagina. Some of these parasites are harmless, others more or less patho- genic, being especially associated with hsematuria. Vermes: Schistosoma hwmatoMum is common in Egypt and certain other localities ; the parasite inhabits the blood-vessels in the vicinity of the urinaiy organs, and the ova and sometimes the embryos appear in the urine, along with blood. Larval fllarice may appear in the urine, with chyluria and hsematuria, in filariasis. In hydatids of the urinary organs, the cysts, hook- lets, or portions of the larval echinococcus maybe voided with the urine. Dioctophyme renale, a large nematode, 30 to 100 centi- metres in length, usually occupying the pelvis of the kidney, has occurred in man in a very few cases, it or its ova appearing in the urine. Intestinal parasites, as ascaris, very rarely find their way into the bladder through intestinal fistulse. The larval arachnoid pentastoma denticulatum has been observed in the urine. Animal parasites are exceedingly rare in the urine in this country. Vegetable parasites of many species and in large numbers are frequently present in urine, but in the great majority of cases they are introduced and develop rapidly after the urine is voided. Certain species are capable of causing marked changes in urine, as those producing ammoniacal transformation of urea, alcoholic or acid fermentation of diabetic urine, pneumaturia or hydrothionuria. The vegetable micro-organisms that may be present in the urine when voided originate from the urethra, the bladder, ureter, or renal sinus, or from the renal parenchyma ; and they may be either pathogenic or innocuous. Bacteria are apt to be normally present in the urethra. In the bladder they are ordinarily absent, though at times a few may be there pres- ent without causing trouble. They are present in large numbers in the bladder, ureters, or renal sinuses in infected or inflamma- tory conditions of these passages or in vesical atony and stagna- tion; in such cases they usually cause decomposition of the Digitized by Microsoft® THE URINE. 171 urine while in the bladder. From the kidney substance bacteria may be derived from infected areas of the organ or by a process of excretion in systemic infections. The vegetable parasites of the urine belong to the fungi and bacteria. The higher fungi found in the urine are mostly contaminations subsequent to its being voided. Earely pathogenic fungi, as actinomyces, aspergilli, etc. , are derived from the urinary organs themselves. The fungi appear on microscopical examination in the form of mycelia or stellate forms. Saccharomycetes or yeast fungi do not often appear in the urine ; they are most apt to be present in glycosuria. Bacteria are the most abundant and most important organ- isms in the urine. Many species, some pathogenic, some causing fermentation, some harmless, are frequently present, though not normally to any material degree in freshly passed urine. The pathogenic bacteria are chiefly derived from local infections of the genito- urinary organs, such as the gonococcus, tubercle bacil- lus, the ordinary pyogenic bacteria (staphylococci, streptococci, colon bacilli), and these or other micro-organisms concerned in the causation of cystitis, pyelitis, etc. In some general infec- tious diseases the causative bacteria may be excreted into the urine through the kidneys, without there being any local infec- tion of the urinary organs by the germs ; thus in typhoid fever, croupous pneumonia, erysipelas, etc, the typhoid bacilli, pneu- mococci, streptococci, etc., may appear in the urine without there being any nephritis, pyelitis, or cystitis present. Earely bacteria are voided in the urine in large numbers without any local or general infection of consequence being demonstrable ("idiopathic bacteriuria"). The determination of pathogenic bacteria in the urine may afford information of diagnostic value. Foreign Bodies. — Extraneous objects, as fibres, particles of various kinds, etc., are apt to appear in the urinary sediment, varying in amount according to the cleanliness of the parts or the receptacles. Starch granules from toilet powders used on the genitals (female) are sometimes abundant. Such extraneous bodies should not be allowed to cause error. In cases of vesicointestinal fistula faecal matter may be voided with the urine ; such an occurrence is diagnostic. Digitized by Microsoft® 172 A MANUAL OF CLINICAL LABORATORY METHODS. B. PHYSICAL CHARACTERS OF URINE. The physical characters of the urine depend on its chemical composition, and a consideration of these characters yields im- portant information. The quantity of urine passed daily is normally about 1,000 to 1,500 cubic centimetres, but fluctuates greatly according to the amount of water excreted. It is increased (polyuria) after free ingestion of liquids, when perspiration is not active, under the use of diuretics, in diabetes mellitus and insipidus, chronic in- terstitial nephritis, certain nervous and other conditions. It is decreased (oliguria) with lessened drinking of fluid, with increased perspiration, in hepatic diseases (cirrhosis, acute yel- low atrophy, yellow fever), in most renal affections, in acute fevers. The daily amount of urine is an important item for clinical consideration, since without it no adequate idea can be formed as to the amount of excreted products. Complete sup- pression (anuria) may occur. Specific Gravity. — This depends upon the relative proportion of dissolved solids, and for the most part varies inversely to the fluctuations of the water. It is normally from about 1.013 to 1.025 (pure water being 1.000). It is lowered in most condi- tions of polyuria and hydruria, ranging down to 1.000; it is in- creased in concentrated urines, as in fever, and in diabetes mellitus. The greatest increase of specific gravity occurs in diabetes mellitus, not infrequently exceeding 1.040, though ex- ceptionally glucose is present in urines of specific graidty as low as 1.012. Concentrated urines other than diabetic do not often exceed a specific gravity of 1.035. Total Solids. — The total solids excreted daily in the urine amount normally to about 60 or 70 grams, of which the most abundant ingredients are the following : Urea about 30 grams. Other nitrogenous bodies 3 Pigments 6 Chlorides 15 Phosphates 5 Sulphates 3 The amount fluctuates according to the ingesta, metabolic activity, renal sufficiency, and the presence of abnormal sub- stances (glucose, albumin). Digitized by Microsoft® THE URINE. 173 The undissolved solids of the urine merit special consideration since as a distinct group they affect the appearance of the urine (its transparency) and require special methods of examination (microscopical ) . When present to the extent of about . 2 per cent by bulk, or more, the undissolved particulate solids in the urine are sufi&cient to cause general cloudiness. In fresh normal acid urine the undissolved solid bodies are extremely scant, consisting of little more than a few scattered epithelium cells. After standing a light flocculent cloud or nubecula may appear in acid urine, and gradually settle to the bottom ; this is formed by delicate mucous threads precipitated by the acid principles, with which may be entangled cells, granules, or crystals. Acid urines in the cold (as in winter or in an ice-box) deposit an abundant white or pink granular sediment of urates, or if strongly acid a more scanty sediment of uric-acid crystals ap- pearing like red-pepper grains. Alkaline urines usually exhibit a turbidity or sediment of pale, granular earthy or triple phos- phates. The development of large numbers of bacteria causes a diffuse cloudiness; the bacteria do not settle to the bottom, even with the centrifuge, and cannot be removed with filter- paper. The occurrence of various abnormal substances, as pus, leuco- cytes, blood corpuscles, clots, an excess of epithelium, sperma- tozoa, or calcium oxalate, and others less common, may produce cloudiness or sediments. In lipuria, the oil present rises to the top. Transparency. — Fresh normal acid urine is perfectly clear and transparent. The urine becomes cloudy when undissolved solids are diffused through it, the turbidity varying from slight cloudi- ness to dense opacity. Turbidity of alkaline or weakly acid urine, increasing with heat, and becoming clear on the addition of a drop or two of acid, is due to earthy phosphates. Turbidity of acid urine, clearing with heat or addition of alkali, is due to urates. Other causes of turbidity will be revealed by microscop- ical examination. Most turbid urines become perfectly clear on filtration, and clear or nearly so after settling ; the cloudiness caused by bacteria is practically undiminished by ordinary fil- tration, and does not clear by sedimentation. The sediment in urine also depends entirely on undissolved solids, and is associated with cloudiness of the urine, the sub- Digitized by Microsoft® 174 A MANUAL OF CLINICAL LABORATORY METHODS. stances causing turbidity usually settling to the bottom as a sediment. The reaction of the urine may be acid or alkaline, in varying degree, neutral, or amphoteric, according as acid or alkaline principles preponderate or balance each other. ISTot only the kind of reaction, but also in many cases the degree of acidity or alkalinity is clinically important, as whether the urine is strongly, moderately, or slightly acid ; by titration the degree of acidity can be determined with quantitative precision. The chief acid principles of the urine are the acid phosphates of sodium and potassium and carbon dioxide ; rarely free fatty acids also. The principal substances giving urine an alkaline reaction are the carbonates and neutral phosphates of sodium, potassium, and ammonium. Of these, carbon dioxide and ammonia compounds are volatile ; they are dissipated by heat- ing, correspondingly affecting the quantitative reaction of the urine ; and litmus paper changed by them resumes its original color again on drying. The sodium and potassium compounds are "fixed " and non-volatile acid and alkaline principles. The mixed twenty-four-hour urine is normally moderately acid ; individual passages may be neutral or alkaline (from fixed alkali) without being materially abnormal. The acidity is in- creased after the ingestion of acids, meat diet, and muscular exer- cise, in concentrated urines, gouty and other conditions, certain neurotic cases, and after acid fermentations. Its acidity is di- minished or it may become alkaline from the ingestion of veget- able foods and certain drugs, as a result of mental work or nervous influences, or ammoniacal fermentation. The degree of acidity fluctuates at different times of the day, diminishing after meals. The quantitative acidity of the normal twenty-four-hour urine ranges from about 10 to 40, averaging about 25. The color of the urine normally is yellow, ranging from light yellow to bright amber. In concentrated and febrile urines the color is a deep amber to reddish. In dilute urines of hydruria and polyuria the color is paler, ranging down to nearly colorless. In auEemic conditions the color is paler, owing to the deficiency of haemoglobin from which the urinary pigment is ultimately derived. Various alterations of color are produced by abnormal ingre- dients. Bile pigments produce a rich deep or brownish amber ; on standing such urines occasionally turn green. Haemoglobin Digitized by Microsoft® THE URINE. 1Y5 and its derivatives impart a red, brown, or dark color. Hsema- toporphyrin produces a brownish-red color, darkening on stand- ing. Melanin imparts a dark color, or darkens on standing. Indican is ordinarily colorless, but on decomposition rarely turns urine red or blue. Urines containing alkapton or derivatives of phenol may darken on standing. Various drugs and food-stuffs impart their color to the urine. In turbid urines the color is modified by the undissolved solids present ; thus, urates cause a dirty yellow color, which on warming or filtering becomes a normal amber ; chylous urine is white and milky. Urine, especially ammoniacal, is fluorescent. Consistency. — Normal urine is thin and fluid like water. A large proportion of solids, especially albumin or glucose, makes it slightly less fluid. An abundance of mucin, pus, or fibrin makes urine much thicker, sometimes fairly gelatinous. Chyl- ous urine is also thick. Frothiness. — After agitating normal urine a certain amount of froth appears on the surface which subsides more or less slowly. The frothiness is greater in concentrated urines, less in dUute urines. It is much increased in urine containing albumin, mu- cin, or bile. Its causes and variations are the same as those of the emulsifying action of urine. Emulsifying Properties. — On agitating urine with a fluid not miscible with pure water, as chloroform, ether, or oil, it will be found after the liquids separate that the oleaginous fluid remains coarsely emulsified to a variable extent, being either broken up into globules through its entire mass or with an emulsified layer at the contact zone. Normal urine possesses a distinct and some- times marked emulsifying power over such liquids ; this action is increased in concentrated urines, diminished or abolished in di- lute urines, and disappears after the urine is treated with lead acetate or decolorized with carbo animalis. Albuminous urine possesses strong emulsifying power. The emulsifying action of normal urine has been by some ob- servers regarded, erroneously, as an evidence of the presence of minute amounts of albumin in normal urine. Albumin in such dilution as to give no response to the ordinary tests possesses no material emulsifying power ; this power in normal urine is there- fore not due to, or a proof of, the presence of albumin, but is caused by other substances, perhaps urochrome. Digitized by Microsoft® 176 A MANUAL OF CLINICAL LABOEATOBY METHODS. The odor of normal urine is characteristic ; it increases or di- minishes according to the density of the urine. Ammoniacal and decomposing urine has a foul odor, acetone imparts a fruity odor, hydrogen sulphide a disagreeable odor. In pyelitis the urine may have a peculiar offensive odor. Some foods and drugs impart special odor to the urine, as asafoetida, turpentine, copaiba, asparagus, garlic. The freezing point of urine varies according to the presence of certain organic material, so that determination of the freezing point (cryoscopy) has recently been utilized for the purpose of obtaining information as to these substances. Variation of Urine.— The composition of the urine varies from time to time during the twenty-four hours, according to body activity and ingestion of food. ISTo one passage is exactly rep- resentative of the entire day's urine. Albumin, sugar, urea, •etc., are increased after exercise or meals, while they are often diminished or even absent in the morning urine. After meals the acidity of the urine is decreased. During some con- ditions, as the passage of renal calculi, or at the crisis of acute diseases, the urine may change markedly in the course of a few hours. Permeability of the Kidneys. — By this is meant the time elapsing after the ingestion of various substances until they appear in the urine, and again until they are entirely excreted and disappear from the urine. Methylene blue, potassium iodide, phloridzin, and similar easily recognizable substances are employed for test purposes. The time to the beginning and end of excretion rep- resents the functional activity and quickness of the kidney. Normally, methylene blue appears in the urine within one -half to two hours, and is entirely eliminated by the third day. In interstitial nephritis the methylene blue often does not appear for twenty-four hours, and is not entirely eliminated until about the fifth day. Changes on Standing. — With the lapse of time wcme is prone to undergo a variety of changes. Such changes may occur while the urine is still in the bladder, in infected, paralytic, atonic, or obstructive conditions causing stagnation of the urine in this vis- cus ; or they may develop after the urine is voided. It is there- fore necessary to examine urine as fresh as possible, both to determine what changes it may have undergone in the bladder Digitized by Microsoft® THE URINE. 177 and to avoid being misled by subsequent alterations, which are often needlessly alarming to patients. Nubecula-formation. — The appearance of a light cloud of tan- gled mucoid threads shortly aft«r passage of urine, especially if strongly acid, is a normal' occurrence. Bacterial Growth. — Bacteria are very prone to develop in urine. This may occur in the bladder in infected conditions of the organ ; or urine passed clear may very quickly become contaminated and clouded by enormous bacterial growth, especially in warm weather. Certain bacteria further cause marked transformation, fermentation, or decomposition of the urinary constituents. Ammoniacal fermentation is one of the commonest changes to which urine is subject under the action of contaminating bac- teria. It very frequently occurs in the bladder, and is one of the chief features of cystitis or urinary stagnation in this organ ; it may also develop in urine subsequent to its passage. It is caused by certain bacteria which have the power of changing urea to ammonium carbonate, thus, CON,H, + (H^O), = (NHJ, CO3. Urine undergoing ammoniacal fermentation is very dis- tinctive in its characteristics. It has a foul ammoniacal odor ; it is strongly alkaline, turbid from the precipitation of earthy phosphates, effervesces with acid from the presence of carbon- ates, and under the microscope shows large amounts of granular amorphous phosphates, numerous ammonio-magnesic phosphate crystals, many bacteria, and sometimes calcium carbonate and spherules of ammonium urate. Carbohydrate Fermentation. — Exceptionally urine containing glucose or other carbohydrates may undergo fermentation, of either the alcoholic or lactic-acid form, under the influence of micro-organisms of the yeast or lactic-acid bacillus type respec- tively. This may occur in the bladder or subsequent to mic- turition. Effect of Cold. — After being voided, urine exposed to cold (as in winter) deposits its urates as an abundant sediment. Some urines after standing for a sufficiently long time deposit crystals of uric acid. The nature and cause of this deposition, and the associated changes in the other urinary constituents, are not well understood. Urines containing hsematoporphyrin, alkapton, or deiivatives of phenol darken on standing. Urine containing bilirubin some- 12 Digitized by Microsoft® 178 A MANUAL OF CLINICAL LABORATORY METHODS. times turns green, while decomposition of indican may cause urine rarely to turn blue or red. Decomposition of cystin gen- erates hydrogen sulphide. Action on Polarized Light. — l^ormal urine rotates polarized light slightly to the left (10°). Polarized light is rotated tow- ard the left by indican if in sufficient amount, serum albumin (56°), other proteids, levulose (106°), glycuronates, and /J-oxy- butyric acid. It is rotated toward the right by glucose (56°), lactose (59°), sucrose (74°), dextrin (138°), maltose (150°), glycogen (211°), free glycuronic acid, and some pentoses. Cer- tain drugs or their derivatives excreted into the urine also affect polarized light. C. EXAMINATION OF URINE. The examination of the urine comprises a variety of proced- ures — simple inspection, physical, chemical, microscopical, etc. The large number of ingredients of the urine requires a corre- spondingly large number of processes for the detection and estimation of them all ; only a few of these substances are, how- ever, considered in ordinary clinical work, and the limits of this work permit only the most important and most frequently used procedures of urine analysis to be detailed. Some such blank as that on the following page will be found convenient for recording and reporting th'e results of urine ex- amination. Collection of Specimens of Urine. — In examining urine it is usu- ally by far the most satisfactory to use a sample from the entire passage of the twenty-four hours mixed together. The urine may vary considerably during the day, and no one passage can be taken as representative of the entire twenty-four-hour urine. That passed after meals or exercise contains an amount of kata- bolic and ingested products above the average, while that of the morning is below the average. Of all the passages of urine dur- ing the day, that of the morning on arising is least representa- tive and satisfactory; for instance, in slight albuminuria or glycosuria, the repose of the night may cause a disappearance of albumin or sugar from the urine, when it would appear after exercise. The collection of the urine for the entire day also affords a means of measuring the amount for the twenty-four Digitized by Microsoft® THE URINE. 1Y9 Date, ,190 . Examination of Uhine. Name of patient Amount in 34 hours c. c. Color Transparency Reaction Specific gravity (at 15° C.) AllDumin Sugar per cent; grams in 34 hours. Urea per cent; grams in 34 hours. Uric acid per cent; grams in 34 hours. Haemoglobin Indican Bile pigment Diazo reaction Undissolved solids. Microscopical examination : Casts Cylindroids Mucous threads Epithelium Leucocytes Red hlood corpuscles , Bacteria Spermatozoa Uric acid Urates Phosphates Calcium oxalate (Signature) Digitized by Microsoft® 180 A MANUAL OF CLINICAL LABORATORY METHODS. hours, which in. itself is a \al liable clinical datum, and enables the daily amount of the individual excreta to be determined. To collect the twenty-four-hour urine, the bladder should be emptied at a given hour, say 7 a.m., and the urine thus passed thrown away. All the urine voided in the ensuing twenty-four hours, including that passed at stool, should then be scrupulously saved in a clean, large, stoppered bottle or jar. At the stated hour on the following day the bladder should be again emptied, and this portion of the urine saved and added to the rest. By this means the renal secretion for exactly tweuty-four hours is collected. By the addition of phenol or otlier antiseptic, bac- terial changes will be prevented. In some cases in which rapid changes are in progress, as during the passage of a renal calculus or other obstruction of the ureter, or at the crisis of acute disease, it may sometimes be useful to examine the urine at short intervals (every few hours) in order to follow the changes going on. In order to distinguish between vesical and urethral affections, the first part of the urine voided at a micturition may be passed in one receptacle, and the last part in another. If the first portion contains pus or other abnormal elements while the second part contains less or none, disease of the urethra is indicated. If the second portion is more abnormal than the first, disease of the bladder is indicated. It is sometimes useful to obtain urine secreted by each kidney separately, in order to ascertain in case of unilateral disease which kidney is affected, or whether either kidney is absent or its outlet obstructed, or whether the kidneys are affected at all. In the case of women an expert gynaecologist can catheterize the ureters and thus obtain a suflcient amount of the secretion of each kidney separately for test purposes. With men catheter- ization of the ureters is scarcely practicable. Instruments have, however, been devised for introduction into the bladder to col- lect separately the secretion from the two sides. Preservation of Urine, — When urine cannot be examined fresh, or in collecting the twenty-four-hour urine in warm weather, bacterial growth and chemical changes can be prevented, by the addition of preservatives or antiseptics, such as phenol, of which three to five drops may be used for every hundred cubic centi- metres of urine. Some of these to a slight degree reduce copper Digitized by Microsoft® THE URINE. 181 salts, but do not afford other glucose reactions. The urine may also be kept in the cold; this precipitates urates, but these may be redissolved by warming at the time of examination. Anti- septics should not be added to urine that is to be subjected to the fermentation test for sugar. Clarification of Urine.— For many purposes in urine analysis it is not necessary that turbid urine be clarified ; but in some in- stances, as in testing for albumin, it is important that the urine be clear. Turbid urine can usually be clarified by filtering ; but sometimes, especially if the cloudiness is due to bacteria, ordi- nary filter-paper will not clear the urine. In such cases, filtra- tion after adding insoluble substances like talc, chalk, magnesia, etc., or after adding alkali, lime water, etc., to precipitate phosphates, may clarify the fluid ; this procedure, however, also removes a portion of any albumin that may be present, or if in small amount may remove it all. Urine can be completely clari- fied by the use of Chamberland's filter. Urine may sometimes be clarified by allowing the sediment to settle or by throwing it down with the centrifuge. Phosphatic turbidity is cleared by adding a little acid; turbidity due to urates may be cleared by heating. At times the color of the urine is so dark as to interfere with some of the color tests. This may be obviated by diluting it, or by decolorizing it with lead acetate or charcoal. A strong solu- tion of neutral lead acetate added freely to urine precipitates the chlorides, phosphates, sulphates, carbonates, the normal urinary pigments (not indican), bilirubin, and all or a part of albumin present, leaving the fluid colorless. Urine shaken with a large proportion of powdered animal charcoal, allowed to stand a few minutes, and then filtered, is decolorized, the pigments and a part or all of the albumin present being removed. Quantity. — This is determined by measurement. The method of obtaining the exact twenty-four-hour product of the kidneys has been given above. Color. — This is noted by simple inspection, no exact colorime- tric methods or uniform scale of color notation having yet come into general use. In practice, each observer adopts a scheme of color notation of his own, and different observers frequently, do not employ the same terms to denote the same shades of color, and vice versa ; so that the terms of different observers (as straw- Digitized by Microsoft® 182 A MANUAL OF CLINICAL LABORATOPY METHODS. color, amber, etc.) do not always mean precisely the same shade. The intensity of the color varies according to the depth of the urine looked through, and here again each observer adopts his own standard. If the urine is turbid, the color of both the unfiltered fluid and the clear filtrate may be noted. The transparency of the urine, —whether it is clear, or if cloudy the degree and cause of turbidity, — should be noted. Undissolved Solids.— These, if present, may be noted both with regard to the character and degree of the UrUdity which they produce, and with regard to the appearance, nature, and amount of the sediment which collects after settling or the use of the cen- trifuge. The nature of the undissolved solids is determined chiefly by microscopical methods. If in the routine centrif uga- tion a graduated tube be used the proportionate bulk of the sedi- ment thrown down (as -^, ^) may be noted; this affords a measure of the undissolved solids, useful for purposes of record or comparison. Centrifugation. — The centrifugal machine has been alluded to in a general way (page 9). In urine analysis two (or four) glass tubes of 10 or 15 cubic centimetres capacity, with conical bottoms, and (for some purposes) graduated to tenths of cubic centimetres, are employed as the urine containers (Pig. 29). The tubes should be filled alike, to preserve an even balance. Undissolved solids or various precipitates can be thrown down as a compact sediment, and their relative bulk can be determined from the graduations. The reaction of urine is determined by means of blue or red litmus paper. According to the intensity of the change of color of the test paper, the degree of acidity or alkalinity can be roughly judged and noted, as weakly, moderately, or strongly acid or alkaline. Sometimes it is useful to determine the acidity of FIG. 29.— urine quantitatively. This can be done most easily by centriiu- titrating a measured amount of urine with decinormal gal Tube. =■ iBausoii sodium-hydrate solution, using phenolphthalein as an indicator, in the same way as in determining the acid- ity of gastric fluid (page 89). The color of urine interferes somewhat with the delicacy of the end reaction with pheuol- Digitized by Microsoft® THE URINE. 183 phthalein, but ordinarily the results are sufficiently reliable for practical purposes. The specific gravity of urine is ordinarily taken by means of the so-called " urinometer " (Fig. 30). This floats at different depths, varying according to the specific gravity of the fluid, which is shown by means of a scale graduated from 1.000 to 1.040 or more (1.000 being the specific gravity of pure water). The specific gravity of the urine is indicated by that marking of the scale which is even with the upper level of the urine. The reading should be made from just above the surface of the urine, and not from below, froth being removed. The portion of the instrument that is immersed in the urine should have no air bubbles adherent to it, and the stem above the surface of the fluid should be dry, in order not to falsify the reading. The urinometer jar, or cylindrical container for the urine, should be large enough to permit the urinometer free play up and down. The urinometer should be one carefully graduated and standardized, otherwise the results will be unreliable. If the quantity of urine available is too small to float the urin- ometer, it should be diluted to a sufficient and known degree, and the specific gravity of the diluted fluid multiplied by the degree of dilution to give the density of the original urine ; this procedure should be employed only when absolutely necessary, as the error of reading is multiplied. The specific gravity varies materially with the temperature of the fluid, so that urine cooled in an ice-box will give a speciflc gravity reading about six points higher than the same urine at body temperature. For accurate work the specific gravity ob- served at any temperature should be corrected or reduced to some standard temperature. The urinometers sold are graduated for definite temperatures, usually either 25° or 15° C. The corrections to be made vary with the temperature and also with the density of the urine, but are approximately as follows : Fig. 30.— Urinometer and Jar. (Bausch & Lomb.) Digitized by Microsoft® 184' A MANUAL OP CLINICAL LABOEATOEY METHODS. Tempeifature at which observation Correction to be applied to Observed Speoipic GRAviTy TO Eeduce it to— Temperature at which observation is made. CORRECTION TO BE APPLIED TO OBSERVED Specific Gravity TO Keddce it to— Is made. 15° C. 25° 0. 15° C. 25° C. 0° C 10 13 15 18 -0.8 - .7 - .4 .0 + -5 -3.8 -3.7 -3.4 -3.0 -1.5 31° C 35 39 33 35 + 1.0 + 3. --3. --4. --5. - -1.0 0. r3. For very accurate determination of tlie specific gravity, the pyknometric method may be used, the weight of any accurately measured volume of urine being obtained with delicate scales. From these data the specific gravity is calculated, and reduced to standard temperature. Total Solids. — The amount of the total solids of the urine is most exactly determined by accurately weighing the residue ob- tained after complete evaporation of the water from a definite volume of urine. For practical clinical purposes, however, the total solids may be calculated with approximate correctness from the specific gravity, which necessarily bears a definite ratio to the proportion of solids in solution. To make this calculation, mul- tiply the last two figures of the specific gravity as ordinarily ex- pressed by .233 (.2 according to some authorities) ; the product gives the percentage weight of solids present. The total solids in twenty-four hours can then be calculated from the daily amount of urine. Urea. — The quantitative estimation of urea is one of the more important procedures of urine analysis. Urea is determined from the volume of nitrogen given off in the decomposition of urea by the action of sodium hypobromite (or sodium hypochlor- ite) with an excess of sodium hydrate. The reaction which takes place is about as follows : CON",H, + (NaBrO), + (NaOH), (HP)3 + N, (NaBr)3 + m,C03 + The purpose of the NaOH is to take up the CO, derived from breaking up of the urea, leaving nitrogen as the only free gas evolved, from the volume of which the amount of urea can be calculated. Digitized by Microsoft® THE URINE. 185 Since the molecular weight of urea is 59.95, of which 38.03 is nitrogen, the weight of the urea' is 3.1395 times that of the nitrogen evolved. The weight of 1 cubic centimetre of nitrogen at a temperature of 0° C. and a barometric pressure of 760 millimetres of mercury is .001255 gram; therefore each cubic centimetre of nitrogen evolved in the test at 0° C. and a net press- ure (after deducting tension of watery vapor) of 760 mm. mercury theoretic- ally represents .003685 gram of urea. According to numerous observers, however, the total amount of nitrogen theoretically required is not actually given off in the test ; but the different observers are not agreed as to the pre- cise deficiency, the deficit found ranging from zero to 8 per cent. Hiifner's results, tliat urea yields only about 95 per cent of its nitrogen in the test, are adopted by some urine analysts as a basis; according to this, 1 cubic centi- metre of nitrogen (0° C, 760 mm.) would represent .00383 gram of urea. In urine analysis, uric acid, proteids, and other nitrogenous substances yield a small amount of nitrogen with the hypobromite method, and as this may be assumed to offset the deficit from urea, the coefficient required by theory may be accepted in practice. In practice, for the most accurate results the temperature, barometric pressure, and tension of aqueous vapor must be taken into account in calculat- ing the amount of nitrogen and of urea. While in localities near the sea-level slight variations of the barometric pressure may perhaps be ordinarily disre- garded, in high altitudes it must be taken into account or a very large error (up to 100 per cent of the true value in the highest inhabited regions) wiU be added. Assuming that all the nitrogen is given off by the urea, and that the aqueous vapor within the gasometric tube is saturated, the amount of urea in the portion of urine tested may be determined by the following general formula, in which U = the weight of urea, in grams, in the urine tested ; N = the volume, in cubic centimetres, of nitrogen actually observed ; T = the temperature. Centigrade, at which the observation of the vol- ume of nitrogen is made ; B = the barometric pressure at the time of observation, in millimetres of mercury ; A = the tension of aqueous vapor at the temperature T : ■ 008685 y(B- A) " 760 (1 + .003666 T)' This formula may be simplified as follows : .000003533 y (B - A) ^- 1 + . 003666 T ' "' U = .000003533 N (B - A) G^, 003666 t ) The maximum tension of aqueous vapor in millimetres of mercury, and the value of nmiWd T ^* "^^n°"s temperatures, are as follows: Digitized by Microsoft® 186 A MANUAL OF CLINICAL LABORATORY METHODS. Temperature. Maximum Temperature. Maximum Value of • tension of aqueous 1 tension ol aqueous 1 C. F. vapor. 1 + . 003666 T. C. r. vapor. 1 + . 003666 T. 0° 32.0° 4.6 1.0000 25° 77.0° 28.5 0.9160 10 50.0 9.2 .9646 26 78.8 25.0 .9130 11 51.8 9.8 .9612 27 80.6 26.5 .9099 12 53.6 10.5 .9579 28 82.4 28.1 .9069 13 55.4 11.2 .9545 29 84.2 29.8 .9039 14 57.2 11.9 .9512 30 86.0 31.5 .9009 15 59.0 12.7 .9479 31 87.8 33.4 .8979 16 60.8 13.5 .9446 32 89.6 35.3 .8950 17 62.6 14.4 .9413 33 91.4 37.4 .8921 18 64.4 15.4 .9381 34 93.2 39.5 .8892 19 66.2 16.3 .9349 35 95.0 41.8 .8863 20 68.0 17.4 .9317 36 96.8 44.2 .8834 21 69.8 18.5 .9285 37 98.6 46.7 .8805 22 71.6 19.7 .9254 38 100.4 49.8 .8777 23 73.4 20.9 .9223 39 102.2 52.0 .8749 24 75.2 22,8 .9191 40 104.0 54.9 .8721 At various temperatures, under a barometric pressure of 760 mm. of mercury and allowing for water- vapor tension, the weight of urea correspond- ing to each cubic centimetre of nitrogen evolved in the hypobromite test is as follows : Temperature. Weight, in grams, Temperature. Weight, in grams. of urea corresponding to corresponding to C. F. • 1 CO. of nitrogen. C. F. 1 c.c. of nitrogen. 16° 60.8° .00349 28° 83.4° .00334 IS" 64.4 .00247 30 86.0 .00333 20 68.0 .00244 33 89.6 .00229 22 71.6 .00242 34 93.3 .00226 34 75.2 .00240 36 96.8 .00233 26 78.8 .00237 At ordinary temperatures and pressure, the coefficient is about .0035 for each cubic centimetre of nitrogen. Ordinarily, therefore, the percentage of urea can be quickly and approximately calculated by dividing the number of cubic centimetres of gas by 4 (1 c.c. of urine having been used for the test). In brief, the volume of the nitrogen evolved being deter- mined by the methods given below, the corresponding weight and percentage of urea may be calculated by one of the follow- ing methods: (a) For a simple and approximate mode of calculation, di- vide the volume of nitrogen evolved from 1 cubic centimetre of urine, expressed in cubic centimetres, by 4 ; the quotient is the percentage of urea, by weight. Digitized by Microsoft® THE URINE. 187 (6) Where the barometric pressure is near 760 millimetres of mercury, so near that slight variations can be disregarded, mul- tiply the volume of nitrogen obtained, in cubic centimetres, by the coefficient for the temperature at which the observation was made, as given in the table on page 186 ; the result expresses the weight, in grams, of the urea in the volume of urine used in the test ; from which the percentage can be readily calculated. (e) If the most accurate results are desired, calculate by the formula U = .000003533 N (B-A) ( Y ^ ^ VI + . 003666 T; That is, from the barometric pressure at the time of the test subtract the tension of aqueous vapor at the given room tem- perature (see table on page 186) ; multiply together this result, the coefficient .000003533, the volume of nitrogen evolved in the test, and the value of at the temperature of the test 1+. 003666 T ^ (see table on page 186). The result gives the weight of urea, in grams, in the portion of urine tested; from which the weight percentage can be calculated. For example, if 10 cubic centimetres of gas is evolved from 1 cubic centimetre of urine, at a room temperature of 20° C. and barometric pressure of 750 millimetres of mercury, the corre- sponding amount of urea calculated by the three methods would be 2.5, 2.44, and 2.41 per cent respectively. Another method that may at times be useful is to make a control test with a 2-per-cent solution of urea, and calculate the urea of the urine from the volume of gas evolved from the urea solution of known strength. The hypobromite test solution employed in the urea test must be prepared fresh each time before use, as follows : To 10 or 15 parts by measure of a 40-per-cent stock solution of sodium hydrate add 1 part of bromine ; mix thoroughly and then add an equal volume of water. To minimize the irritating fumes, the stock of bromine in its bottle should be kept covered with a layer of water, and the proper amount of bromine removed with a pipette. To carry out the test special arrangements of apparatus are necessary for decomposing the urea and measuring the nitrogen generated. Nimierolis forms of apparatus have been devised for Digitized by Microsoft® 188 A MANUAL OF CLINICAL LABORATORY METHODS. this purpose, all more or less efficient and convenient, of which the following may be commended : 1. Doremus's ureometer (Pig. 31) consists of a graduated closed tube with an overflow bulb ; it is accompanied by a pipette with a mark representing 1 cubic centimetre. The hypobromite fluid is poured into the bulb, and the instru- ment tilted so that it runs into and fills the long arm. Sufficient fluid should be used so that when held upright the tube is entirely full, with enough fluid in the bulb to prevent the access of air to the tube. The urine to be tested is then drawn into the pipette to the 1 c.c. mark. The curved point of the pipette is passed to the bot- tom of the long arm of the ureometer and the urine slowly expelled into this arm by compressing the rubber bulb. Brisk effervescence occurs, and the evolved nitrogen rises to the top of the tube, where, after allowing a sufficient time for the completion \3a\TX'i<^a ^^ *^® reaction, the equalization of Fig. 31. — Doremus's Ureometer, with the temperature, and the subsi- Support. (Lentz & Sons.) , ^ j_i j. j.i j_i - i dence of the froth, the gas is read from the graduation. The instrument is graduated to show directly either fractions of a gram of urea in each cubic centi- metre of urine, or grains of urea to the ounce of urine ; from this, if desired, the percentage can be easily calculated. The apparatus has a range up to 3 per cent. Urine containing over 3 per cent of urea should be diluted with an equal amount of water, the mixture tested, and the results obtained multiplied by 2. With albuminous urine the froth subsides very slowly. This apparatus gives results sufficiently reliable for ordinary clinical purposes', although not precise enough for very exact determinations. A certain amount of gas usually escapes while the urine is being discharged from the pipette, which interferes with great accuracy. It should be noted that the 1 c. c. mark on the pipette is relative only to the instrument which it accom- panies, and does not necessarily indicate exactly 1 cubic centi- metre; hence with each ureometer only the particular pipette pertaining to it (or one of like volume) should be used. Digitized by Microsoft® THE URINE. 189 2. Author's Ureometer (Pig. 32). — The author has had an ap- paratus constructed for the estimation of urea which is very easy to manipulate and accurate in itr results. It consists of a graduated closed tube c, an overflow bulb b, and an open arm a. Projecting obliquely downward from the tube c is a side arm d, which is closed off from the lumen of the main tube by a glass stopcock e containing a perforation n. The capacity of d and n together is exactly 1 cubic centimetre. To make the test, the glass stopper is turned so that its perforation n opens communication between the side tube d and the main tube c. A small amount of the urine is poured into the open arm a, and the instrument, tilted so that the urine fills d and n; this urine, which is in- troduced for the purpose of rinsing out the tube d, is then drained away. When suffi- ciently rinsed, a portion of the urine is introduced into the side arm, completely fill- ing it and the perforation in the stopper; the stopper is then turned so as to close off the side arm from the main tube. In this manner, exactly 1 cubic centimetre of urine is segregated in the spaces d and n, which should be entirely free of air bubbles. The main tube is then rinsed out with water, and sufficient hypobromite so- lution introduced to fill the graduated arm c completely when in an upright position. The stopper e is then turned so as to open communication between arms d and c, and allow the urine to be acted on by the test fluid ; the rapidity of the reaction can be controlled with the stopcock. Effervescence, occurs, and the nitrogen evolved rises and fills the upper end of the graduated Fig. 33.— Author's Dreometer. Digitized by Microsoft® 190 A MANUAL OF CLINICAL LABORATORY METHODS. arm. Wlien the nitrogen is completely evolved and the tem- perature of the gas and licLuid has subsided to that of the room, water is poured into the open tube a until the level of the fluid in the two arms is the same, thus equalizing the hydrostatic ten- sion on the gas. The volume of gas is then read off, in cubic centimetres and tenths; from which and the data as to temper- ature and barometric pressure the percentage of urea can be cal- culated in the manner given above. The grad- uation covers a range up to 4 per cent of urea. If desired, the graduation could be made to show the approximate percent- age of urea at ordinary room temperature and sea-level atmospheric pressure. 3. A very efficient ar- rangement for estimating urea can be improvised from ordinary laboratory apparatus (Fig. 33). The test solution is placed in a wide-mouthed bottle. A measured amount of urine (1 or 2 cubic centi- metres) is put into a slen- der cylindrical glass re- ceptacle of suitable size, which is then set upright inside the glass bottle ; this keeps the two fluids separate until the time arrives to mix them. The bottle is closed by a stop- per penetrated by a glass tube, which is connected by a long rubber tube with a burette clamped in an inverted position with its major 'portion in a tall, cylindrical glass vessel of water. After arranging the test fluid and urine in the bottle, inserting the stopper tightly, and waiting until the temperature becomes equalized, the burette is moved up or down until the water within it is at the same level as that without ; the graduation on Fig. 33.— Apparatus for Estimation ol Urea. Digitized by Microsoft® THE URINE. 191 the burette corresponding to this level is noted. The bottle is then tilted so that the urine in the container inside mixes with the hypobromite solution. The water inside the burette is de- pressed to an extent equal to the volume of nitrogen evolved. After waiting ten or fifteen minutes or until the temperature is equalized, the burette is raised until the water inside it is on a level with the water outside, when the corresponding graduation is again read. The difference between the two readings gives the volume of nitrogen generated, from which the urea can be calculated. 4. Squibb' s apparatus can also be readily improvised. The arrangement for mixing the urine and test fluid is the same as in that just described. The volume of nitrogen is determined from the volume of water displaced by the gas generated. Uric Acid. — Deposits of uric acid or urates are ordinarily easily recognizable by their microscopical appearance and their solubility with alkalies or heat. Occasionally, chiefly in xjonnec- tion with calculi, chemical qualitative tests are required to deter- mine if suspected material contains uric acid. For this purpose the following tests may be used : Mureocide Test : In a porcelain dish dissolve a small portion of the dry material or residue from evaporation in two or three drops of nitric acid ; evaporate by gentle heat ; when cool add a drop or two of aqua ammonise, when if uric acid or urates are present a spreading purple-red color appears. Some of the xanthin bases give a similar reaction ; but on the addition of a drop of sodium-hydrate solution, if uric acid is present, the color changes to a reddish blue and disappears with heat. Silver Test : Place a drop or two of a solution of silver nitrate on filter paper, and add a like amount of sodium-carbonate solu- tion in which the suspected material has been dissolved; the formation of a black or grayish color indicates the presence of uric acid. This test is said to be very delicate. Quantitative Estimation : A number of methods of determining the amount of uric acid have been presented, all quite compli- cated and none closely accurate. One of the easiest and most satisfactory of these is the following modification of the Hop- kins method : In 50 or 100 cubic centimetres of the urine, accurately meas- ured, dissolve 5 or 10 grams, respectively, of powdered ammo- Digitized by Microsoft® 192 A MANUAL OF CLINICAL LABORATORY METHODS. nium sulphate, and add sufficient ammonia to make the fluid barely alkaline. Allow the fluid to stand two or more hours, with occasional shaking, when the uric acid will be all precipitated as ammonium urate. Filter through a small, thin filter-paper, col- lecting the precipitated urate on the filter. Then thoroughly wash the filter-paper and precipitate free from chlorides, etc., by passing a 10-per-cent solution of ammonium sulphate through it several times. Break a hole through the bottom of the filter with a glass rod, and with a pipette or wash-bottle blow 100 cubic centimetres of hot water in forcible jets upon the filter paper so as to wash the precipitated ui-ate entirely from it into a beaker or wide-mouthed bottle beneath. Add 15 cubic centi- metres of strong sulphuric acid to the fluid containing the pre- cipitate, and titrate the mixture immediately, while hot, with one-twentieth normal solution of potassium permanganate. Un- til the uric acid is completely saturated the permanganate is instantly decolorized on being added to the titrated fluid; the titration should be continued until the fluid being tested, thor- oughly mixed with the permanganate, remains of a pale-pink color throughout for a few seconds. This is not a very sharp end reaction, but it is sufficient to obtain practical results. Then multiply the number of cubic centimetres of the perman- ganate solution employed in the titration by .00375 ; the product expresses the weight, in grams, of the uric acid present in the amount of urine taken for the test. The twentieth-normal test solution is prepared by dissolving exactly 1.577 grams of pure dry potassium-permanganate crys- tals in 1 litre of water. It should not at any time be allowed to come in contact with organic material; it should therefore be kept in glass-stoppered bottles, and used in a burette controlled by a glass stopcock, without a rubber tube attache!^. Indican.— The amount of this substance can be estimated roughly, but with sufficient accuracy for clinical purposes, by the following simple test of Heller : With 4 cubic centimetres of strong hydrochloric acid in a porcelain capsule or a test tube thoroughly mix 5 to 20 drops of urine. With the amount of in- dican normally present a characteristic pink color appears. If indican is in excess a violet or blue color develops immediately or within three or four minutes; the greater the intensity or rapidity with which the blue color develops, and the smaller the Digitized by Microsoft® THE URINE. 193 amount of urine that produces it, the greater is the amount of indican. If, on the contrary, the pink color is produced tardily and faintly, indican is diminished ; and if no pink tinge appears, especially after adding a drop or two of nitric acid or other oxi- dizing substance, indican is practically absent. If bilirubin is present or the urine is highly colored, the addition of lead acetate will decolorize the urine and make the indican test more practicable. Phosphates. — All the phosphates of the urine, earthy and alkaline, are precipitated together by adding about one-third of its volume of "magnesian fluid," which consists of magnesium sulphate 1 part, ammonium chloride 1 part, aqua ammonise 1 part, water 8 parts. The precipitate consists of a mixture of earthy and ammonio-magnesic phosphates. The total amount of this precipitate can be determined gravimetrically by adding filtered magnesian fluid to a measured amount of clear urine (50 or 100 cubic centimetres), and then collecting the precipitate on a filter-paper previously exactly weighed after being well dried in a hot oven. The precipitate and filter-paper are then re- peatedly and thoroughly washed with an abundance of pure water made markedly alkaline with ammonia, to remove all other substances. The filter-paper and precipitate are then thoroughly dried in a hot oven as before, and carefully weighed. The increase in weight indicates the weight of the precipitated phosphates in the volume of urine employed for the test. The earthy phosphates when present undissolved in urine are recognizable from the microscopical appearance and their prompt solubility on adding a few drops of any acid. They can be completely precipitated by themselves from urine by making it strongly alkaline with sodium, potassium, or ammonium hy- drate, and then heating. Thus precipitated from a measured amount of clear urine (as 100 cubic centimetres), their quantity can be determined gravimetrically by collecting them on a weighed filter, washing thoroughly with ammoniacal water, dry- ing and weighing again, in the manner just described for the total phosphates. The alkaline phosphates can be precipitated separately, in the form of ammonio-magnesic phosphate, by adding maignesian fluid to the filtrate obtained after precipitating and removing the earthy phosphates ^s above. The precipitate thus obtained from 13 Digitized by Microsoft® 194 A MANUAL OF CLINICAL LABORATORY METHODS. a definite amount of urine can be collected on a filter and weighed in the same manner as just described ; the result expresses the weight of triple phosphate formed from the sodium and potassium phosphates. These gravimetric methods give results sufficiently useful for approximate comparative purposes, but as the precipitate con- sists of a mixture of different phosphates, the amount of con- tained phosphoric acid cannot be exactly determined. Volumetric Estimation of Phosphoric Acid. — The amount of phosphoric acid, calculated as P,0„ may be estimated volu- metrically. The solutions required are as follows : a. A standardizing solution of 10.085 grams of Na^HPO^ in 1 litre of water. The phosphate must be weighed in the form of dry, well-formed crystals, which have not lost any of their water of crystallization. This solution contains 0.2 per cent of P^O,. 6. A solution containing 35.511 grams of pure and well- formed crystals of uranic nitrate, TJO^ (^Os)^ + 6 H^, in a litre of water. This may be prepared as nearly as possible by weight, or slightly stronger than desired, and then brought to the stand- ard after titrating it with 50 cubic centimetres of the foregoing solution of sodium phosphate in the sam.e manner, described be- low, as urine is titrated. Twenty cubic centimetres of the uranic solution should be equivalent to 50 cubic centimetres of the phos- phate solution. Each cubic centimetre of the uranic solution therefore represents .005 gram of P,Oj,. e. Sodium-acetate solution, consisting of sodium acetate 10 parts, glacial acetic acid 5 parts, water sufficient to make 100. d. Saturated potassium-ferrocyanide solution, freshly pre- pared. To make the estimation, add 5 cubic centimetres of the so- dium-acetate solution to 50 cubic centimetres of urine, and heat in a water bath to 90° or 100° C. Titrate, while thus heated, with the uranic-nitrate solution. A precipitate of uranic phos- phate falls until the urinary phosphates are exhausted, after which the excess of uranic nitrate will give a reddish-brown pre- cipitate with potassium ferrocyanide. From time to time during the titration, transfer a drop of the urine, well stirred, to a porcelain dish and touch it with a drop of the potassium-ferro- cyanide solution ; as soon as this shows a reddish-brown color, discontinue the titration. Multiply the number of cubic centi- Digitized by Microsoft® THE UHINE. 195 metres of the uranic-nitrate solution required to saturate the urinary phosphates by .005; the product expresses the weight, in grams, of P^O^ in the 50 cubic centimetres of urine tested. It may be found convenient to make two titrations each time, the first to determine the approximate result, the second to arrive at an exact figure. To estimate the P^O^ of the earthy phosphates alone by this method, precipitate them by alkali and heat, collect them on a filter and wash with ammoniacal water, as above indicated ; then break a hole in the bottom of the filter, wash the precipitate from the paper with 50 cubic centimetres of water, in jets, and dissolve it by adding as little acetic acid as is necessary. Add 5 cubic centimetres of the sodium -acetate solution and titrate while hot with uranic-nitrate solution in the manner described. The P3O5 of the alkaline phosphates may be determined by subtracting the amount found combined with earthy bases from the total amount found ; or by titrating the urinary filtrate after removing the earthy phosphates. Carbonates. — ^The presence of carbonates is shown by the oc- currence of brisk effervescence upon adding acetic or other acids. In the nitric-acid contact test for albumin, bubbles of nitrogen slowly collect on the sides of the test-tube from decom- position of urea ; this is much slower than the effervescence of carbonates and does not occur with other acids than nitroso- nitric. Albumin. — Many tests for albumin in the urine have been in- troduced, some too delicate, some not sensitive enough, most of them not needed. Only two tests need ordinarily be used, which have been in use for many years, answer all requirements as to sensitiveness and facility, and permit quantitative estimations sufficiently approximate for practical comparative purposes; these are the nitric-acid contact test and the heat test. For applying these tests satisfactorily it is important that the urine be perfectly clear. Large amounts of albumin are easily shown even if the urine be turbid ; but small amounts can be demonstrated with certainty only with clear urine. Fresh urine not contaminated with bacteria can be clarified by filtration. If bacteria or very fine particles are abundant the turbidity due to them cannot be removed by filter-paper. In such cases filtration after shaking the urine with insoluble powders like talc, etc., Digitized by Microsoft® 196 A MANUAL OF CLINICAL LABOEATOBY METHODS. may clear it ; but this procedure also removes a portion or all of the albumin present, and hence is likely to lead to error. Vigor- ous centrifugation may clear urine sufficiently for the test. Fil- tration through Chamberland's filter will completely clarify urine. By passage through filter paper urine or pure water may take up vegetable albumin, but under ordinary circumstances only in amount so minute as not to react with the tests here described or with any but the most extremely sensitive tests for albumin. Nitric-acid Contact Test (Heller's test). — This test is performed by placing a small amount of strong nitric acid in a test tube or conical glass, and then carefully overlaying it (see page 10) with the urine to be tested, which should be clear. If albumin is present, a sharply defined white opaque layer or disc several millimetres in thickness appears at the junction of the two fluids. If only a minute trace of albumin is present there may be only a faint cloudiness at the contact zone, instead of a sharp white disc. In this test, not only serum-albumin, but also acid or alkali albumin, globulin, and albumose are precipitated. The reaction is best observed against a dark, unlighted back- ground, the tube itself being well lighted. The reaction is some- times slow in developing; hence the fluids should be observed for fifteen to thirty minutes before a negative conclusion is ar- rived at. The sensitiveness of the test is probably increased by using hot urine, or by warming the test-tube containing the fluids in hot water ; heat thus applied clears away any trouble- some cloud of urates. Sometimes the albuminous disc may be more or less colored by urinary pigments. This test is very sensitive to albumin ; and while other tests are more delicate, they are also untrustworthy, and the contact test is sufficiently sensitive for all clinical purposes. It affords little reliable information, even approximate, as to the amount of albumin present. The method is slightly more sensitive with alkali-albumin than with acid-albumin, though under the condi- tions ordinarily presented by the urine the difference is scarcely appreciable. This contact test presents a number of other features with which the urine examiner should be familiar. Nucleo-albumin or mucin, if abundant, may be precipitated in a diffuse cloud in the urine just above the contact zone. It is Digitized by Microsoft® THE URINE. 197 important not to mistake this for albumin, which forms usually a sharply defined white disc at the contact plane. Still, great difficulty may be found at times in distinguishing a mucoid from a faint albuminous reaction. After the ingestion of certain resinous substances, as copaiba or turpentine, the urine when tested by the nitric-acid contact method yields a white disc closely resembling that of albumin. This resinous precipitate is redissolved on the addition of alco- hol, wherein it differs from the albuminous precipitate. If the urates are concentrated in the urine, the nitric-acid contact method may cause them to be gradually precipitated in the form of a faint cloud in the upper part of or throughout the urine. An excessive or rapidly produced cloudiness of this kind may roughly indicate an excess of uric acid. If a cloud of urates interferes with the albumin test, it may be obviated by introduc- ing the urine hot, or by warming the test-tube in hot water or, carefully, oxqv a flame ; or the urates may be kept in solution by adding alkali to the urine. With most urines in the performance of this test numerous bubbles of gas slowly form at the contact zone, and rise to the surface or adhere to the sides of the glass. These are caused by the action of traces of nitrous acid on the urea and uric acid, which are decomposed with the evolution of nitrogen and carbon dioxide. This, effervescent action is most marked when impure nitric acid of a yellow tinge and containing considerable nitrous acid is used ; but even pure colorless nitric acid causes a certain amount of bubble formation. If carbonates are present, brisk effervescence from that source occurs. If the urine is cloudy froni phosphates, it clears in the vicin- ity of the acid. When the urine contains a large proportion of urea (4 or 5 per cent or more), the contact-test is apt to cause a white crystalline disc of urea nitrate to form slowly at the junc- tion of the fluids. This can hardly be mistaken for albumin, since its crystalline structure is easily recognizable, and on shaking it up with the urine or diluting with water it redis- solves. Mtric acid applied by the contact method produces marked color reactions with the pigments and chromogens of normal and abnormal tirines. In normal urine a diffuse pink color forms at Digitized by Microsoft® 198 A MANUAL OF CLINICAL LABORATORY METHODS. the junction of the fluids, fading away above ; this seems partly at least to be due to the action of the acid on indican. In cer- tain abnormal urines, as when concentrated or in febrile condi- tions, the color reactions are strongly marked, a darker color layer appearing, sometimes reddish, often a deep brown, diffuse or sharply defined ; this may be partly due to indican, partly, probably, to excessive or abnormal pigments. If bilirubin is present, its characteristic series of colors, beginning with green, appears at the contact plane ; this test therefore serves to show the presence of both albumin and bilirubin. If iodides are pres- ent in the urine the liberation of iodine causes the formation of a brown layer. Heat Test. — This test consists in boiling the urine to coagulate any albumin present, followed by the addition of nitric acid to increase the proteid reaction and eliminate other reactions. A column of clear urine 5 to 10 centimetres in depth is boiled in a test-tube; first the upper part of the fluid may be boiled, in order to detect any cloudiness that may appear by comparison with the lower part. Later the entire urine may be boiled to precipitate all the albumin. The development of a cloudiness or precipitate on heating will be due to precipitation of either earthy phosphates or albumin. A drop or two of nitric acid is then added ; if the turbidity is due to phosphates it will immedi- ately clear up, while if due to albumin it will remain or increase. Whether a cloudiness appears or not, nitric acid is added, two or three drops at a time, until a precipitate forms or ten or fifteen drops have been added. Sometimes the albumin is not precipi- tated until several drops of the acid are added. A precipitate appearing after the urine cools, redissolving on again heating, is uric acid or albumose. By the combined action of heat and nitric acid, albumin is precipitated in fine granules or feathery flakes, sometimes white, sometimes colored by urinary pigments. As a qualitative test, this method is rather less delicate than the contact test, as it may fail to react when the latter gives de- cided response. It is useful as an approximate quantitative test, sufficient for comparative purposes. For this purpose the boiled and acidulated urine is set aside upright in a test-tube, or better a graduated tube, and the precipitate, which should be broken up into granular portions by shaking, allowed to settle for twelve to twenty-four hours, or thrown down immediately with Digitized by Microsoft® THE URINE. 199 the centrifuge. Wlien the precipitate is compactly sedimented in the bottom of the tube, its relative volume compared with the volume of the entire urine (as |, ^, etc.) is noted. It should be observed that this is the amount by bulk and not by weight ; the weight percentage of albumin in urine is always compar- atively small (rarely over two and usually under one per cent), while the corresponding amount by relative bulk appears large and conspicuous. The bulk ratio is but a rough quantitative method, but is suf&cient for comparison of the progress of the case. For more accurate quantitative measurement of the albumin precipitated by heat and acid, it may be collected on a weighed filter, thoroughly washed, dried, and weighed ; by deducting the weight of the filter paper, the amount of albumin is obtained. In practical work, it is convenient to apply the contact method first as a qualitative test, and then if this yields a posi- tive result to employ the heat test for quantitative purposes. The precipitate thrown down by this method contains serum- albumin, globulin, and when cool albumose. E"ucleo-albumin, or mucin, likewise forms a diffuse cloud with heat and acid, and must be carefully distinguished from albumin. If the cloudiness be due to mucin, a similar cloud can be produced in unboiled urine by the addition of acetic acid, which does not so react on albumin. In doubtful cases, sodium chloride may be added to the urine to hold the mucin in solu- tion, and the boiling and acid method tried, when a positive re- sult will indicate albumin. Eesinous drugs may cause a cloudiness of the urine in this test as in the contact test; the precipitate is re -dissolved with alcohol. Urates are not thrown down in the hot urine. An evolution of gas bubbles from decomposition of urea by nitroso- nitric acid occurs as in the contact test. Decided color reactions similar to those of the contact test take place on adding nitric acid to hot urine, pink or darker colors being produced through- out the fluid; or a bright' green develops, followed by other ■colors, if bilirubin is present. If it is desired for any purpose to remove albumin from the urine coagulate it well by boiling, and then filter. ITucleo-albumin, or mucin, is detected chiefly by its being pre- cipitated on the addition of an excess of acetic acid. It is some- Digitized by Microsoft® 200 A MANUAL OF CLINICAL LABORATORY METHODS. times advisable to dilute the uriue before testiug, to preveut precipitation of uric acid, and by diluting the chlorides of the urine to diminish the solubility of the mucin. The importance of distinguishing between mucin and a faint albuminous re- action has been considered above. Sugar. — Numerous tests, qualitative and quantitative, are available for the sugars, especially glucose, chiefly based on their reducing power over copper or bismuth salts, their prop- erty of undergoing alcoholic fermentation with yeast, their action on polarized light, and their reaction with phenylhydrazin. Oc- casionally a combination of tests is necessary to differentiate the different kinds of sugar from one another or from other reducing substances. Copper Test. — This test depends on the power possessed by glucose and other sugars of reducing cupric oxide, CuO, to ciiprous oxide, Cu^O. This reaction is yielded alike by glucose, levulose, lactose, maltose, pentoses, glycuronic acid, alkapton, and certain adventitious substances derived from ingesta. The cupric oxide is prepared by mixing copper sulphate with an ex- cess of caustic alkali, with some organic substance to make the cupric oxide thus formed soluble in the alkali. The test is cap- able of both qualitative and quantitative application. Several formulae for the test have been introduced, as Fehling's, Pavy's, Trommer's, etc. The glycero-cupric test may be recommended as probably the best of the copper tests for glucose. For qualitative purposes the solution may be prepared as follows: Dissolve 4 grams of pure copper sulphate in 30 cubic centimetres of watei-, and add 30 cubic centimetres of pure glycerin. When the test is to be made mix about 1 cubic centimetre of this solution with 5 cubic centimetres of liquor potasste or sodse (about 5-per-cent solution of potassium or sodium hydrate) . Boil the test fluid thus freshly prepared for a few seconds, to determine if the solution itself is good, in which case it will undergo no change. Then add two or three drops of the urine to be tested, and boil again for fifteen to thirty seconds (not too long) ; if no reaction appears, add two or three more drops of urine and boil again ; continue thus until a reaction develops, or until two or three cubic centimetres of urine are added. Sometimes the reaction does not develop until after several minutes. If glucose or other reducing substances Digitized by Microsoft® THE URINE. 201 are present in material amount, the blue color of the test fluid changes to a yellow or orange, and a copious orange or red pre- cipitate is thrown down ; this reaction should appear promptly after the addition of 2 to 10 drops of the urine. The more abun- dant the sugar the less is the amount of urine required to produce a vigorous reaction. If the glucose or other reducing substance is in minute amount, it will require a larger quantity of urine (over 10 drops) to give a reaction; which in this case will con- sist simply in a change of color of the test fluid to amber or green, without a precipitate. So slight a reaction as this indi- cates only a minute amount of glucose or some other reducing agent besides glucose, and can be fairly disregarded clinically as being within a normal limit. The test should always be begun with a very few drops of urine. During the procedure the earthy phosphates are always pre- cipitated by the alkali and heat, in a whitish or grayish cloud, which should not be (but sometimes is) mistaken for a sugar reaction. Albumin, if present, may give the biuret reaction in this test, turning the fluid a violet color ; albumin does not often interfere with the operation of the sugar reaction, but if it is suspected of so doing or is in excessive amount it may first be re- moved. As a qualitative test this method is adequately sensitive, but has the disadvantage of reacting with other substances (which are, however, rarely present or sufficient to give a vigorous re- action) besides glucose. The glycero-cupric and other copper methods ma}' be employed for quan- titative glucose tests, the sugar being computed from the amount of urine re- quired exactly to decolorize a solution containing a known amoimt of copper salt. The method has not proven very satisfactory, partly from the instabil • ity of the solutions, partly from the indefiniteness of the end reaction. For ordinary purposes the fermentation method is preferable. Pvrdy's Metlwd, which is typical of and probably as good as any of the quantitative copper tests, is as follows; Dissolve 4.742 grams of pure copper- sulphate crystals and 38 c.c. of pure glycerin in 200 c.c. of pure water, with gentle heat; dissolve 23.5 grams of pure potassium hydrate in another 200 c.c. of water; mix the two solutions; when cooled add 450 c.c. of pure aqua am- moniae fortior (U. S. P., specific gravity .9), and dilute the whole with water to exactlv 1 litre. Thirtj'-five cubic centimetres of this standard solution is exactly reduced and decolorized while boiling by .02 gram of glucose. Place exactly 35 c.c. of the solution in a sufficiently large flask, dilute with about twice as much pure water, and bring thoroughly to the boiling-point. From Digitized by Microsoft® 202 A MANUAL OF CLINICAL LABORATORY METHODS. a burette above, the urine to be tested is discharged drop by drop into the boiling test fluid until the latter is just completely decolorized. The amount of urine required for this result contains exactly .03 gram of glucose; from which the percentage can be calculated. If the urine contains much sugar it Is best to dilute it accurately to a known degree, and, after titrating with it thus diluted, to make the proper allowance. Fermentation Test. — Under the action of yeast fungi, glucose, levulose, and maltose undergo alcoholic fermentation, breaking up into alcohol and carbon dioxide. This affords a useful quali- tative test for differentiating glucose from all other reducing substances except levulose and maltose, and the most practicable quantitative method of estimating glucose in the urine. For qualitative purposes the test is best applied by means of a "fermentation tube" (Fig. 41), consisting of an upright glass tube closed above and opening into an overflow bulb below (similar to Doremus's ureometer, which can be used for the pur- pose). Sufficient urine to fill the tube is well mixed with one or two grams of fresh yeast, and then introduced into the tube so that in the upright position the tube is completely filled with urine, with no air at the top. The tube is then set aside for eighteen to twenty-four hours at a temperature of 30° or 35° C. to allow fermentation to take place. If glucose is present it is de- composed, and the carbon dioxide copiously evolved rises in the tube, progressively depressing the level of the fluid. A small bubble of gas ordinarily appears at the top of the tube even in normal urine; but the accumulation of more than such a bubble, as shown, if needed, by comparison with a control test with nor- mal urine, indicates the presence of a fermentable sugar, ordi- narily glucose. Antiseptics should not have been added to the urine for preservative purposes, or the action of the yeast will be inhibited. In default of a fermentation tube a test-tube can be completely filled with the yeast-urine mixture and inverted, still filled, in an open glass containing urine. As .004 gram of glucose yields 1 cubic centimetre of CO^ (at 0° C. and 760 milli- metres pressure) it does not require a very large proportion of glucose completely to fill the tube with gas, and the test is quite delicate. For quantitative purposes the differential specific-gravity method is the best, the fermentation tube not affording satisfac- tory results. The principle is this, that the loss of CO. causes a Digitized by Microsoft® THE URINE. 203 decrease of the specific gravity of the urine, and from this de- crease the amount of CO, and of glucose can be calculated. In practice, after taking the specific gravity 75 or 100 cubic cen- timetres of urine (not containing antiseptics) is mixed with two or four grams of yeast and set aside in a warm place, at about 30° or 35° C, in a bottle with a hole through or a notch cut alongside the cork to permit escape of air. Fermentation is usually complete after twelve to twenty-four hours ; its conclu- sion will be shown by a negative response to the copper test. When fermentation is complete the specific gravity of the fer- mented urine is again taken, at the same temperature at which it was taken before fermentation. The difference between the two, multiplied by .23, gives approximately the percentage of glucose by weight. Thus, if the specific gravity was 1.036 be- fore and 1.007 after fermentation, the difference, 29, multiplied by .23, indicates that there is 6| per cent by weight of glu- cose present. The method involves a delay of twenty-four hoars, and is only approximate in its results, but it is easy to carry out and is sufficiently accurate for ordinary clinical and comparative purposes. In routine urinary work, sugar can be conveniently tested for qualitatively by the glycero-cupric method, and if this shows the presence of sugar, its amount can then be determined by the specific-gravity fermentation method. Polariscopic Tests. — The differentiation between different kinds of sugars and other reducing substances is aided by tests of their action on polar- ized light, as to the existence, direction, and amount of their rotatory action (see page 158). This method also affords an approximate means of quantita- tive estimation of sugar. Phenylhydrazin Test. — Glucose, levulose, maltose, some pentoses, and glycuronic acid when treated in a certain way with phenylhydrazin yield characteristic microscopic osazone crystals, which differ from one another somewhat in appearance and melting-points. Some authorities commend the method as delicate and reliable; but as originally practised it is a rather troublesome process and does not appear to offer any advantages over the methods given above as to sensitiveness, or as to ability to differentiate be tween the various reducing substances. Acetone. — Several tests for acetone have been presented, aside from its odor, among which the following qualitative methods may be mentioned : Legal' s Test : Make a few cubic centimetres of urine distinctly Digitized by Microsoft® 204 A MANUAL OP CLINICAL LABORATORY METHODS. alkaline with a little liquor potasste or sodse ; add a few drops of a rather strong solution of sodium nitro-prusside made just at the time of using. The mixture becomes red, and then rapidly fades. On adding a few drops of strong acetic acid a purple or violet-red color (also transient) develops if acetone is present. Lieben's Test: Add about 1 per cent of phosphoric acid to the urine and distill 500 or 1,000 cubic centimetres. To a few cu- bic centimetres of the first 10 to 30 cubic centimetres of the dis- tillate add a few drops of liquor potassae or sodse and a few drops of dilute Lugol's solution (page 11). If acetone is present a yellow precipitate of iodoform appears, recognizable by its odor when heated. Alcohol, which might exceptionally occur in the distillate, and lactic acid also give this reaction. Diacetic acid may be tested for qualitatively as follows : To a few cubic centimetres of fresh urine add drop by drop -a rather strong watery solution of ferric chloride ; if phosphates are pre- cipitated filter them out and continue adding the ferric chloride. If a Bordeaux-red color appears, boil another portion of the origi- nal urine and repeat the test. If no reaction is obtained in the second test, treat a third portion of the urine with sulphuric acid and extract with ether; apply the ferric-chloride test to the ethereal extract. If this extract gives a positive reaction, the color disappearing in twenty-four to forty-eight hours, diacetic acid is present, especially if acetone is abundant. Beta-oxybutyric acid may be suspected to be present if after removal of sugar by fermentation the urine rotates polarized light to the left. The presence of diacetic and /5-oxybutyric acids in large amount is indicated if there is found an excess of ammonia in the urine or a high ratio of ammonia nitrogen (15 per cent or over being of serious significance). Blood. — The presence of blood (htematuria) is shown by the concurrent presence of red corpuscles, albumin, and haemoglo- bin ; the corpuscles alone are pathognomonic. Hsemoglobin may be conveniently tested for in the urine by the guaiacum test (page 70). Heller's test may also be em- ployed as follows: Add liquor potassse or sodse (or if the urine is alkaline and the earthy phosphates are already precipitated add a few drops of magnesian fluid), and heat, so as to precipi- tate the phosphates ; if blood pigment is present the precipitate is colored red. The hsemin test (page 70) may be employed. Digitized by Microsoft® THE URINE. 205 being applied to a portion of the dried phosphatic precipitate. The spectroscopic method may also be used, especially for recog- nizing and differentiating the precise nature of the various derivatives of haemoglobin (as hsematoporphyrin). Pus is recognized by the presence of a large number of leuco- cytes, with more or less albumin. The sediment may or may not be viscid and tenacious. Bilirubin may be satisfactorily recognized by the formation of a series of different-colored pigments, beginning with green, (biliverdin), on treatment with an oxidizing reagent. The test is best made with nitroso-nitric acid, that is, impure or so-called "fuming" nitric acid, having a yellow tinge from the presence of nitrous acid. If a sufficiently impure nitric acid is not at hand, "pure" or colorless nitric acid will answer; or a minute portion of copper or mercury may be added to nitric acid to generate nitrous acid ; or a fresh mixture of equal parts of nitric and hydrochloric acids may be employed. If the urine is very dark it may be advisable to dilute it. Three to fifteen drops of the acid may be mixed with 5 to 10 cubic centimetres of the urine to be tested ; or the urine may be underlaid with the acid. The presence of bilirubin is indicated in the former case by the urine turning a bright or dark green, perhaps subsequently changing to blue and then red. In the contact test, if bilirubin is present a green layer appears, gradually extending upward in the urine ; beneath the green are usually blue and red zones. The test frequently recommended of allowing a few drops each of acid and urine to flow together on a white dish is much less sensitive than the two methods just specified. The develop- ment of a green color is absolutely significant of bilirubin, but of green only ; no other substance in the urine produces a green color, while other colors than green are produced by various other urinary pigments and chromogens when treated with nitric acid. In practice the presence of bilirubin is ordinarily sufficiently shown incidentally in the application of the tests for albumin, especially if a yellowish nitric acid is used, so that the same pro- cedure serves for both. If bilirubin is present the characteristic color changes appear at the contact zone in the contact test for albumin, or the albuminous precipitate inay be green ; and in the heat test, the hot urine readily turns green on the addi- Digitized by Microsoft® 206 A MANUAL OP CLINICAL LABORATORY METHODS. tion of sufficient nitric acid, or tlie albumin may be precipi- tated green. If altered bilirubin (page 163) is present, the urine from an icteric patient exMbits all th§ macroscopic characteristics of the presence of bilirubin, but yields no green reaction with nitric acid. Diazo Reaction. — The application of this test requires three solutions: 1. A saturated solution of sulphanilic acid (para- amido-benzene-sulphonic acid), in a 5-per-cent solution of hydro- chloric acid ; sulphanilic acid is soluble to the extent of about 2 per cent. Sulphanilic-acid crystals deteriorate so that after being kept for twelve to eighteen months they cannot be relied on to make an effective test solution. 2. A one-half-per-cent solution of sodium nitrite in water ; the solution deteriorates, so that it should be freshly prepared every few weeks at least. 3. Aqua ammonise. To make the test, mix 1 part of the sodium-nitrite solution with 40 parts of the sulphanilic-acid solution ; add an equal vol- ume of urine and mix ; then add about a tenth or a fifth as much ammonia, and mix the fluids. With urine yielding a negative response to the test, the mixture turns a brown or vinegar color ; if the diazo reaction is present, the mixture turns a carmine or bright garnet-red color. Microscopical Examination. — The purpose of microscopical ex- amination is to determine the character of the undissolved solid elements of the urine, or occasionally to examine precipitates artificially produced. The material for the examination, the undissolved solids, may be concentrated and collected either by sedimentation or centrifugation. To sediment the urine, shake the bottle containing the urine to diffuse the solids thoroughly, and place a quantity of the fluid in a glass receptacle with a conical bottom, adding a few drops of phenol to prevent bacterial growth. Allow it to stand thus for twelve to twenty-four hours, when the solids will have settled to the bottom of the glass and be compacted within a small space. This method is quite efficient, but requires time. To concentrate the solids by centrifugation, place a portion of the urine, well shaken, in the centrifugal machine and revolve it rapidly. This compacts the sediment densely in the bottom of the tube. This method is very effective, and enables immediate microscopical examination to be made. Digitized by Microsoft® THE URINE. 207 For very searching investigations, the two methods may be combined. A large quantity of urine may be sedimented, prefer- ably in a large receptacle with contracted bottom, as a percolator or corked funnel. The undissolved solids of a large volume of urine are thus concentrated at the bottom of the glass. This lower portion of the fluid is then removed by a pipette, or by removing the cork beneath, and further concentrated by the cen- trifuge ; in this manner the sediment from several hundred cubic centimetres of urine can be concentrated within the space of a few drops. The sediment being concentrated at the bottom of the glass by one of the above-mentioned methods, it is removed for exami- nation by means of a pipette or glass tube, drawn to a slender point at one end. The large end of the tube being tightly closed with the finger-tip, the pointed end is passed to the bottom of the glass containing the sedimented or centrifuged urine, and by carefully and slightly raising the finger-tip the urine and sedi- ment at the bottom are allowed to flow into the pipette till a sufficient amount is collected ; the finger is again pressed tightly against the top of the pipette, the tube withdrawn, and a few drops of the urine and sediment are allowed to flow out on a glass slide. The material thus deposited on the slide may or may not be covered with a cover-glass. If the examination is made with a low-power objective, a cover-glass is not needed, unless the fluid oscillates so as to interfere with observation. If a strong objec- tive is used, a cover-glass is advantageous. The examination should always be begun with a weak objec- tive (about 2 centimetres), with a strong ocular and the tube lengthened to increase magnification ; with this power the sub- stage illumination should be very dim, the iris diaphragm almost completely closed, and the condenser lowered if necessary. An excess of light drowns out most of the faint objects in urine ; but they are brought out conspicuously by very dim light. With any power the illumination should be adjusted till the optimum effect is attained. "With the low power, epithelium, casts, and most of the urinary objects can be made out perfectly well, or even better than with the high power, while the larger field en- ables a more rapid and thorough search to be made. The mi- nutest objects, as leucocytes, red blood corpuscles, bacteria, and Digitized by Microsoft® iJ08 A MANUAL OP CLINICAL LABORATORY METHODS. calcium-oxalate crystals, are best distinguislied with a high power ; and if with a low power small objects are seen but not clearly recognized, the higher objective should be applied. Sometimes one kind of object will be so abundant and pre- dominating as to obscure and hide everything else in the field, as with precipitated phosphates or urates, or leucocytes ; it will then be found useful to clear away the superabundant and ob- scuring material. The phosphates can be easily dissolved and gotten rid of by slightly acidulating the urine before using the centrifuge. The urates can be easily cleared away by heating the urine just before centrifugalizing. Other substances, as leucocytes, it is impracticable to remove. Sometimes micro-chemical tests are applied, the effects of the addition of reagents being observed under the microscope. A drop or two of the test fluid may be mixed with the urinary sedi- ment before or without applying the cover-glass ; or after the cover is applied, a drop of the reagent may be placed at its edge and allowed to flow under. Various procedures have been suggested for staining the cells and casts, and so making them more conspicuous, as by adding iodine or alizarin solution. These methods have not, however, come into general use. Staining methods may be required for specific purposes, as in examining for bacteria, like the tubercle bacillus or gonococcus ; in such cases the centrif uged sediment or a portion of pus present is spread on a cover-glass, dried, fixed, and stained in the usual Avay for differentiating these or other bacteria. The objects most commonly seen in microscopical examina tion of urine are casts of various kinds, cylindroids, mucous threads, epithelium, leucocytes, red blood corpuscles, sperma- tozoa, bacteria and other vegetable micro-organisms, uric acid, urates, amorphous phosphates, ammonio-maguesic phosphate, calcium oxalate, debris, and foreign particles. These may be recognized by their characteristic appearances as already de- scribed. Sometimes consideration of other characteristics of the urine throws light on the nature of doubtful objects; thus, an amorphous granular deposit in alkaline urine is probably phos- phatic, in acid urine uratic. Micro-chemical tests may be ap- plied. Thus, leucocytes may be cleared and their nuclei made prominent with acetic acid ; urates dissolve with alkalies, or on Digitized by Microsoft® THE URINE. 209 adding nitric or acetic acid crystals of uric acid may slowly form ; all forms of phosphates dissolve without gas formation on adding acetic or nitric acid. The following objects are unusually or rarely seen, and may be distinguished by their form, or chemical or micro-chemical tests: xanthin, hippuric acid, leucin, tyrosin, calcium sulphate, •indigo granules or crystals, cystin, calcium or magnesium phos- phate, calcium carbonate (dissolves with formation of bubbles of gas on adding acid), melanin granules, fat (reacts with Sudan III.), fibrin flakes, hsematoidin, bilirubin (gives green reaction on adding nitroso-nitric acid), cholesterin, tissue fragments, animal parasites. Bacteriological examination of the urine is made by the usual cultural methods, the urine being collected with strict aseptic precautions. 14 Digitized by Microsoft® VIII. MISCELLANEOUS SECEETIONS AND BODY FLUIDS. The most important body fluids, from a clinical standpoint, have been considered in the previous chapters. The remaining glandular products or other natural fluids of the body may occa- sionally be required to be subjected to laboratory investigation for diagnostic purposes. Saliva consists of a small amount of solids (of which the most notable are ptyalin, mucin, potassium and sodium sulpho- cyanide, chlorides, phosphates) dissolved in water, along with adventitious epithelium, leucocytes, vegetable micro-organisms, and food particles. It is ordinarily odorless and colorless, and varies in consistency from a thin serous fluid to a more viscid mucinous fluid. It is normally slightly alkaline, but may range from strongly alkaline to acid under morbid conditions. Its specific gravity is ordinarily 1.003 to 1.009. The daily quantity is normally about 1,500 cubic centimetres, but fluctuates greatly under the action of drugs, local or general affections, mental and nervous influences, etc. Saliva may be obtained for examination by chewing a rubber band and collecting the secretion thus stimulated. The amount that can be secreted in a given time may be thus determined. To test for ptyalin or the amylolytic power of saliva, add a small amount of saliva to a solution of starch, and after keeping it for a short time at a temperature of about 37° C. test as to the presence of starch, erythrodextrin, and sugar by means of the iodide and copper tests. To test for sulphocyanides, acidulate saliva, concentrated by evaporation if necessary, with a little hydrochloric acid, and heat with weak ferric -chloride solution; the sulphocyanides cause the formation of a red color. Certain ingested drugs are freely secreted in the saliva, not- ably iodides, and their detection in the saliva is a feature of cer- tain clinical tests. Digitized by Microsoft® MISCELLANEOUS SECRETIONS AND BODY FLUIDS. 211 In abnormal conditions, local or general, variations may take place in the various constituents of the saliva ; thus, ptyalin may be decreased in fever, urea increased in nephritis. Acid fermen- tation in the mouth may generate acids and make the saliva acid, with injurious effects on the teeth. Putrefactive processes about the mouth cause a foul odor. Microscopically, saliva exhibits large, squamous epithelium cells, cast off from the oral mucous membrane ; leucocytes and lymphoid cells (salivary corpuscles) that have wandered out of the adjacent tissues and are more or less altered by the action of the saliva; foreign particles derived from food, etc., and large numbers of bacteria. The vegetable micro-organisms found in the mouth and vicin- ity are exceedingly abundant, most of them being innocuous, some (as the leptothrix) quite characteristic of this locality, some either actually or potentially pathogenic. The micrococcus lanceolatus, streptococci, and other pathogenic bacteria are fre- quently present in the mouth of persons in health as well as in those with the corresponding infections. In local infectious con- ditions the causative bacteria are present, notably diphtheria bacilli, streptococci, pyogenic bacteria, and among pathogenic fungi the oidium albicans, actinomyces, and others. The demon- stration of these micro-organisms in pseudo-membranous deposits about the mouth and pharynx,, by appropriate cultural and staining methods, is of high diagnostic value. Bile. — ^The presence of bile elements in gastric fluid, fseces, and urine has been considered. Bile itself obtained from the gall bladder at autopsy or operation has been scarcely studied pathologically, except as to the presence of bacteria. Nasal Discharge. — There is very little or none of this nor- mally. In nasal affections it appears, varying in amount and character; similar considerations and methods of examination apply to it as to sputum and catarrhal exudates in general. Expired Air.— Theoretically the composition of expired air should afford clinical data analogous in all respects to those fur- nished by the urine ; that is, it should yield information as to body metabolism and as to local lesions of the respiratory tract. Of the three chief end products of katabolism, NHj, CO2, and H2O, am- monia, the characteristic representative of nitroa^enous katabolism, is chiefly- excreted by the kidneys ; carbon dioxide, tlie representative of carbohydrate Digitized by Microsoft® 212 A MANUAL OF CLINICAL LABORATORY METHODS. katabolism, chiefly by the lungs ; while the excreted water is divided between the kidneys, lungs, and perspiration. The composition of inspired air varies within slight limits, that of expired air within wide limits. Their average composition Is about as given in the following table, the figures for expired air being representative of a healthy adult on full diet under conditions of active exercise: Gas. Nitrogen Argon Helium Oxygen Carbon dioxide. . . Ammonia and other gases. Watery vapor Oxygen Oxygen in carbon di oxide Total oxygeh .... By Weight. Inspired air, per cent. 75.97 .97 Trace. 23.01 .05 Traces. Variable 100.00 Expired air, per cent. 76.40 .97 17.67 Slightly increased. Usually to saturation. 101.72 Differ- ence. + .43 -5.34 + 6.63 + ■1.72 By Volume. Inspired air, per cent. 78.36 .80 20.81 .08 100.00 Expired air, per cent. 78.80 .80 16.00 4.40 100.00 Differ- ence. + .44 -4.81 + 4.37 23.01 17.67 -5.34 20 81 16.00 .04 4.86 + 4.82 .03 4.40 23.05 22.53 -.52 20.84 20.40 -4.81 + 4.37 -.44 Respiratory quotient = IH = ^'^^ X '^^^ = ^ ■. 4.81 5.34 5.34 .90. The principal change in expired air Is a decrease of free oxygen and an increase of carbon dioxide and water. The decrease in oxygen (4.81 per cent by volume in the illustration given) represents the amount absorbed into the blood. The increase in carbon dioxide is produced by oxidation processes in the body tissues and excreted by the lungs. It will be observed that more total oxygen (free, and combined in COj) Is inspired than is expired; fhat is, more free oxygen is absorbed into the blood (4.81 per cent) than is given out by the blood in the form of carbon dioxide (4.37 per cent); the difference Is utilized in the body, amounting in the example given to .44 per cent by vol- ume or .52 per cent by weight. The proportion of the absorbed oxygen that is given out in the carbon dioxide excreted is called the "respiratory quotient." This is found by divid- ing the volume of carbon dioxide given out by the blood by the volume of oxygen absorbed by the blood (thus, 4.37 -f- 4.81 = .9); or it may be found by dividing the weight of oxygen in the COj (= weight of CO2 multiplied by .727) by the weight of the absorbed oxygen (thus, 6.63 x -727 -i- 5.34 = .9). The respiratory quotient is increased or diminished as oxidation in the body Increases or decreases, and is therefore an expression of body oxidation. Digitized by Microsoft® MISCELLANEOUS SECRETIONS AND BODY FLUIDS. 213 The total amount of oxygen daily absorbed into the blood under ordinary conditions is about 700 to 900 grams in a resting condition, or 900 to 1,100 grams if actively exercising. The daily amount of carbon dioxide given off by the lungs is about 900 grams in the resting condition, 1,000 to 1,300 grams during exercise. The respiratory quotient is about .7 to .8 during rest, .8 to 1 during exercise. These figures all vary according to age, sex, diet, rest or exercise, body condition, and in disease. The amount of water excreted by the lungs is very variable, ranging from 500 to 3,000 grams daily. These considerations illustrate the class of data that analysis of the ex- pired air would yield with respect to metabolism in disease ; but while the subject has been studied for physiological purposes, it has not yet been elabo- rated in its clinical and pathological relations. Volatile or gaseous substances in the blood may be excreted by the lungs, as after the use of onions, turpentine, ether, etc. ; acetone in acetonaemia, alco- hol after its excessive ingestion. Small amounts of ammonia, hydrogen, methane, and nitrogen (in excess of that ingested) are expired. According to some authorities toxic organic substances are normally excreted from the blood into the expired air, which if allowed to accumulate poison and vitiate the atmosphere ; other observers have not found proof that such is the case, but attribute the harmful effects of air vitiated by over-breathing and insutflcient ventilation to excess of carbon dioxide or watery vapor, or to decrease of oxy- gen. In. renal insufficiency an increase of ammonia is said to be expired. Necrotic, suppurative, or putrefactive processes in the lungs, air pas- sages, or tributary cavities generate organic and other gases that contaminate the expired air and may impart to it a foul odor; as in pulmonary gangrene, putrid bronchitis, foul rhinitis, decaying teeth, or decomposition of food in an uncleaned mouth. The increase of ammonia and other organic gases in ex- pired air is largely derived from such local conditions. In ordinary quiet respiration epithelium cells, bacteria, or other particles are not expelled with the breath, though in coughing or sneezing they may be ejected. Perspiration. — Sweat is one of the three chief excretions of the body, and as such, were it easily investigated, should throw light on body metabolism. The daily amount of sweat is very vari- able, according to external circumstances and body conditions, but may be stated to average about a litre daily. The perspir- ation is composed chiefly of water, containing 1 to 2 per cent of dissolved solids, — urea and other nitrogenous bodies (total nitro- gen from .02 to .20 grams daily), sodium chloride (the most abundant solid), small amounts of other inorganic and organic salts, fat, fatty acids, etc. The fatty elements originate from the sebaceous glands. The reaction is sometimes alkaline, but ordinarily acid, from the fatty acids present. The odor is due to these volatile acids or fermentative jjroducts. Special ex- Digitized by Microsoft® 214 A MANUAL OF CLINICAL LABORATORY METHODS. creted substances cause the peculiar odor of tlie sweat from the axillEe, perineo-inguinal region, and between the toes. Fermen- tative and putrefactive processes from bacterial action may cause sour or foul odors. The significance of the changes of the perspiration in disease have not been systematically worked out. Urea may be much increased in renal insufficiency, sufficient to give the sweat a urinous odor. Earely colored sweat is excreted (chromidrosis), red, yellow, blue, black, green, caused by indican products, in- gesta, blood and bacterial products, or unknown substances. Phosphorescent sweat, due perhaps to bacterial growth, has been observed. Semen normally consists of a large proportion of actively mo- tile spermatozoa suspended in a fluid (liquor seminis), together with granular, crystalline, and adventitious matter. The liquor seminis is a muco-albuminous fluid containing sundry chemical ingredients. It exhibits abundant fine granular matter (cellular and protoplasmic d6bris), and, in small number, cast-off epithe- lium cells, stray leucocytes, clear "hyaline bodies" from the seminal vesicles, fat and lecithin particles, and small prostatic concretions or "amyloid bodies." After standing, crystals are deposited in semen, especially slender, octahedral crystals of spermin phosphate (Charcot-Leyden crystals). To determine as to the presence and activity of the sperma- tozoa, the semen, kept warm, is examined as freshly as possible by placing a drop or two on a warm slide, applying a cover- glass, and examining microscopically with a high power. Thus examined, the spermatozoa may be found absent, or if present they may be motionless, in sterility of the male. Blood may be discharged with the semen, from excessive coitus, or congestive or inflammatory conditions. For medico-legal purposes suspected stains on clothing, etc., are subjected to examination to determine as to the presence of seminal fluid. If scales can be removed from the stain, they may be macerated and teased in a drop of water or dilute stain- ing fluid on a glass slide, and examined directly with high mag- nification for the presence of spermatozoa. Or a portion of the cloth containing the stain may be cut out and soaked fifteen to sixty minutes in water or dilute stain (such as TJnger's stain, methyl green .2 gm., hydrochloric acid 3 drops, water 100 c.c.) Digitized by Microsoft® MISCELLANEOUS SECRETIONS AND BODY FLUIDS. 215 The fluid in tlie piece of cloth is squeezed out on a slide and examined with the microscope. Florence's test for seminal stains, which has been highly com- mended as a supplement to the above method, is as follows : On a slide mix a drop of a watery extract of the suspected stain with a drop of a solution of iodine 1.65, potassium iodide 2.54, in water 30. The iodide must be in excess. With seminal mate- rial this fluid promptly precipitates an abundance of microscopic dark brownish-red, long, slender, rhombic or acicular crystals, single or in rosette or other combined forms. The crystals much resemble hsemin crystals. They soon redissolve and disappear. This reaction is not afforded by other body fluids, so far as known. The ingredient of the semen yielding the crystals has Hot been definitely determined. Vaginal Discharges. — The secretion of the normal vagina is a scanty mucous fluid, often acid, containing abundant squamous epithelium cells, with a few leucocytes and bacteria. It is quite strongly germicidal, and not ordinarily favorable to the presence of pathogenic bacteria. The amount is increased during coitus, pregnancy, etc. Spermatozoa are present and demonstrable for a considerable period after coitus. Leucorrhceal discharges are of the nature of catarrhal fluids, and are of a muco-purulent or purulent character, containing leucocytes in proportion to the suppurative process going on. In local inflammations and infections the causative bacteria are demonstrable by staining or cultural methods, as gonococci, staphylococci, streptococci, etc. A protozoan, the trichomonas vaginalis, has been frequently found in the vaginal fluids, espe- cially in some localities ; it appears to have little or no pathogenic action in the vagina, and has been also found in the faeces and urine. Large shreds or membranes from the vaginal mucosa are sometimes discharged. Uterine DischBiTges.— Menstrual fluid consists chiefly of blood, with some vaginal secretion, vaginal and uterine epithelium cells, and granular and fatty d6bris from the uterine mucosa. The lochia for the first two or three days after labor are abun- dant, thin, and red, and chiefiy composed of blood, with vaginal epithelium and decidual cells. The amount steadily decreases, the red blood cells diminish, the leucocytes and epithelium in- crease, and the color changes to rusty-brown, gray, or white. Digitized by Microsoft® 216 A MANUAL OF CLINICAL LABORATORY METHODS. Bacteria are always abundant. The odor of uncontaminated lochia is bloody; in the presence of decomposing clots or re- tained portions of the afterbirth or septic processes the odor becomes fetid; sometimes an ammoniacal odor develops from ammoniacal fermentation, which is innocuous. In septic or infected conditions of the uterus the discharges become purulent, and the causative bacteria may be demon- strated. Masses, shreds, or fragments of tissue may be expelled from the uterus, consisting of clots, moles, embryos, decidua, dysmenorrhceal membranes, etc. Amniotic fluid rarely exhibits abnormalities, which although interesting are scarcely of practical importance. Milk consists of a large number of minute fat globules sus- pended in a watery solution of proteids, lactose, and salts. Cellu-- lar elements are ordinarily absent ; bacteria are usually present. The composition of normal human and cow's milk varies somewhat, but averages about as follows : . Human mili. Cow's mUk. Proteids (chiefly casein) 2.0 3.5 5.8 .3 3.3 Pat 40 Lactose 5.0 Salts (phosphates, etc.) .7 Total solids 11.5 88.5 13.0 Water 87.0 100.0 100.0 Human milk is alkaline in reaction, has a specific gravity of 1.026 to 1.035 (averaging about 1.032), and in twenty-four hours amounts to ^ to 1|- litres. Numerous substances ingested by the mother or elaborated by maternal metabolism are excreted in the milk. Abnormal changes in the milk may be caused either by local morbid conditions of the mammary apparatus or by general sys- temic disturbances. In inflammatory, suppurative, or hemor- rhagic conditions of the breast, leucocytes and red blood corpus- cles may appear in the milk. Bacteria in the milk may originate from the maternal tissues or be introduced after withdrawal. Some of these bacteria are pathogenic, as staphylococci, strepto- cocci, typhoid bacilli, micrococcus lanceolatus, etc., originating Digitized by Microsoft® MISCELLANEOUS SECRETIONS AND BODY FLUIDS. 217 in tlie corresponding infections; others induce fermentative changes in the milk, especially formation of lactic acid from lactose and coagulation of casein; many micro-organisms in milk are indifferent and harmless. The agglutinins of typhoid fever may appear in the milk. Variations in the composition of milk are caused by various general conditions of the mother, as fevers, lowered general nu- trition. The changes in special diseases have not been much studied, . and analyses of milk are made chiefly in connection with infant feeding. Milk for examination is collected with a breast pump. A drop of the milk under the microscope exhibits a large number of minute rounded fat globules 1 to 5 micromilli- metres in size. To search for cellular elements, leucocytes, etc., centrifuge the milk and examine the sediment under the micro- scope. Bacteria may be searched for by staining the centrifuged sediment, cultural methods, or animal inoculation. The two chief points to be determined in the analysis of milk are the specific gravity and the amount of fat. These two data ordinarily afford a suf&ciently good idea of the nutritive strength of the milk, and from them the total solids can be approximately calculated. The specific gravity of milk is determined by the pyknometric method, or by a sensitive "lactometer" on the hydrometer prin- ciple, especially constructed for the purpose. Temperature cor- rections should be made. The amount of total solids is determined by drying a definite quantity of the milk and weighing the residue after its weight has become constant. The total solids can also be calculated (for cow's milk) by adding 1.2 times the percentage of fat to one-fourth of the specific gravity at 15° C. ; the siun is approxi- mately the percentage by weight of the total solids. The estimation of the amount of fat in milk is one of the chief procedures of milk analysis, since the other solid constituents usually correspond closely with the fat in amount. For this purpose a number of simple methods of sufficient approximate correctness have been devised, of which Babcock's sulphuric- acid centrifugal method is in common use. In Babcock's test equal carefully measured amounts of milk and strong sulphuric acid are thoroughly mixed ; the acid dissolves all but the fat ; on centrif ugation in a special bottle with a graduated neck, the fat Digitized by Microsoft® 218 A MANUAL OF CLINICAL LABORATORY METHODS. is forced to the top and from the graduation its weight percen- tage is directly read off. The relation between the volume and the weight of the butter fat is such that at 50° C. each 1 per cent by weight indicated on the graduated scale occupies 1.1428 per cent by volume of the amount of milk originally taken for the test. With care this method yields quite accurate results. By using a mixture of equal parts of pure amyl alcohol and strong hydrochloric acid (of specific gravity of 1.16 or over), in amount equal to one-fifth of that of the milk used, in addition to the sulphuric acid, a sharper separation of the fat is obtained (Leflfmann-Beam method). Commercial establishments use a large centrifugal machine for the Babcock test, but for clinical purposes it is more conven- ient to use a smaller centrifugal tube adapted to the ordinary laboratory centrifuge (Fig. 34). To make the test, the requisite amount of milk, 5 cubic centimetres, is accurately measured in a pipette and introduced into the tube. One cubic centimetre of the amyl-alcohol and hydro- chloric-acid mixture is then added and thor- oughly mixed ; this procedure may be dispensed with, but it facilitates reading the final result. Strong sulphuric acid (specific gravity 1.83 to 1.84) is added till the tube is filled to the shoul- der, and the fluids are thoroughly mixed. When the mixture becomes dark and homo- geneous, add sulphuric acid to the top of the graduated scale, and again mix the fluids. Ee- volve the tube in the centrifuge for a couple of minutes. The liberated fat is concentrated at the top of the tube, and its amount is read off at once from the scale. If from cooling the fluid contracts below the gradu- ation, the tube should be placed in hot water until the fluid expands sufficiently to enable the result to be read. The column of fat should be free from bubbles and undissolved casein. Commercial sulphuric acid of specific gravity 1.82 to 1.83 may be used if the tube containing the milk-acid mixture is kept in hot water at about 95° C. for a few minutes before centrifuga- tion. Proteids in milk are determined gravimetrically after preclpi- FiG. 34.— Centrifu- gal Tube and Pi- pette lor Estima^ tion of Fat in Milk. (Bauscli & Lomb.) Digitized by Microsoft® MISCELLANEOUS SECRETIONS AND BODY FLUIDS. 219 tating them, or estimated by multiplying the amount of nitrogen found by Kjeldahl's method by 6.33. Lactose in milk is determined quantitatively by the polari- metric or copper-reduction methods. Articular Fluids. — Synovial fluid is normally a muco-serous fluid, glairy from the presence of mucin. In articular lesions, inflammatory, suppurative, etc., it undergoes corresponding alterations variously exhibiting fibrinous coagula, pus and leuco- cytes, blood and erythrocytes, and bacteria. These foreign ele- ments are demonstrable with the aid of the centrifuge, the microscope, and cultural methods. Cerebro-spinal Fluid. — Examination of this fluid is capable of affording valuable diagnostic information in cases of suspected cerebro-spinal meningitis and other affections. The fluid is obtained for examination by means of " lumbar puncture," as follows: With the patient sitting, lying on the side, or doubled face downward over the knees, the body and back being bent forward as far as possible, with strict asepsis, and under local or if necessary under general ansesthesia, a strong aspirating needle is introduced in the interval between the second and third, third and fourth, or fourth and fifth lum- bar vertebrae. A good guide for the insertion of the needle is the line connecting the crests of the ilia. The needle, introduced in this line and about a centimetre to one side of the spinous proc- esses, is pushed forward and slightly inward and upward until its point enters the subarachnoid space, which is shown by escape of the fluid. The depth to which the needle must be passed varies from two or three centimetres in infants to eight or ten in adults. The syringe may be attached to the needle as a handle. The fluid may be allowed to flow out spontaneously to the extent of a few cubic centimetres, or may be withdrawn by very gentle suction with the syringe, and is collected in a sterile receptacle. The needle and syringe should be sterile, but should not contain any antiseptic at the time of use. formal cerebro-spinal fluid is a clear, limpid, non-coagulat- ing, colorless or slightly yellowish (sometimes accidentally tinged with blood), alkaline serous fluid containing about 1.3 per cent of various solids in solution, and of a specific gravity of 1.005 to 1.007. The most abundant solids are albumin (normally about .1 per cent or less) and chlorides. A few endothelium ceUs, Digitized by Microsoft® 220 A MANUAL OF CLINICAL LABORATORY METHODS. leucocytes, and fibrin filaments may appear microscopically. As to the amount of fluid normally obtainable by lumbar puncture no definite statement can be made, but ordinarily several cubic centimetres can be easily and safely withdrawn. In abnormal conditions the cerebro-spinal fluid may become modifled in consequence of inflammatory, suppurative, hemor- rhagic, and dropsical processes, etc., in the cerebro-spinal struc- tures. The chief abnormalities of practical diagnostic signifi- cance consist in variations of amount, appearance, specific gravity, quantity of albumin, the presence of i)us and leuco- cytes, of bacteria, and of blood and erythrocytes. The amount of cerebro-spinal fluid obtainable is increased in hydrocephalus, meningitis, etc., in which there is an increased exudation of fluid and increased tension. It is decreased or un- obtainable altogether when fibrinous or purulent exudations, ad- hesions, or pressure of tumors obstruct the passages along which the fluid flows. In fibrinous or suppurative meningitis the cerebro-spinal fluid becomes turbid, in hemorrhagic conditions it is bloody, in serous meningitis, tubercular meningitis, hydrocephalus, etc., the fluid is clear and colorless. In local inflammatory (but not in other) conditions the fluid clots, either in the form of a delicate fibrin network (tubercular meningitis) or as solid coagula. Simple inspection of the cerebro-spinal fluid therefore affords valuable diagnostic information. As a rule the amount of albumin, and with it the specific gravity of the fluid, is greater in meningitis than in non-meuin- gitic conditions (as hydrocephalus), the infiammatory processes or exudations introducing a larger proportion of proteids than is present in the normal fluid or simple transudations. In suppurative meningitis pus appears in the fluid, varying from a slight admixture' to almost pure pus. In such cases the fluid is cloudy, and microscopically exhibits leucocytes in num- bers corresponding to the proportion of pus present. In infectious cerebro-spinal conditions the causative bacteria are present, and their demonstration, which is possible in most cases, establishes the specific diagnosis. The bacteria chiefly responsible for meningitis are the tubercle bacillus, diplococcus meningitidis intracellularis, and micrococcus lanceolatus, with staphylococci, streptococci, typhoid bacilli, and others in a small Digitized by Microsoft® MISCELLANEOUS SECRETIONS AND BODY FLUIDS. 221 proportion of cases. Of these the tubercle bacillus is associated with non-suppurative meningitis, the cerebro-spinal fluid being clear and serous, while the other bacteria cause suppurative inflammations and a cloudy, purulent fluid. Blood, manifested by the color, microscopical presence of red blood corpuscles, and chemical reactions, appears in the cerebro- spinal fluid in hemorrhagic conditions, aside from traces that may be introduced accidentally in the process of lumbar punc- ture. In cerebral and intraventricular hemorrhage, a large ad- mixture of blood with the cerebro-spinal fluid takes place ; while in extradural bleeding little or no blood enters the fluid. The examination of the fluid obtained by lumbar puncture is accomplished by inspection, microscopical methods, bacteri- ological methods, or chemical tests. Simple inspection reveals much information as to the color, clearness, or turbidity of the fluid, and as to fibrin formation, with all that these imply. Microscopical examination is for the purpose of investigating as to the bacteria and cells present. Unless the fluid is very thick and turbid, the sediment should be concentrated and col- lected with the centrifuge, and then be examined either fresh or after staining. For the demonstration of bacteria the various appropriate staining methods should be employed. Tubercle bacilli, if present, are often difiicult to demonstrate. If they cannot be found after the use of the centrifuge, the fluid should be allowed to stand in the cold for several hours until a fibrinous network or coagulum forms. These fibrin filaments, in which the bacilli are apt to be entangled, are removed with a platinum loop, spread on a cover-glass, fixed, and stained in the usual manner for tubercle bacilli. Bacteriological examination of the cerebro-spinal fluid, in cases in which the microscopical examination is not regarded as suffi- cient, is carried out by cultural methods or animal inoculation in the usual manner. For this purpose, the fluid must have been collected and preserved with absolute asepsis, and without con- tamination with antiseptics. Chemical tests are rarely called for, but if desired can be car- ried out, as for the amount of albumin, presence of blood, specific gravity, etc. Digitized by Microsoft® IX. PATHOLOGICAL FLUIDS. This class embraces fluids produced ouly in consequence of pathological processes. These fluids may be classified according to their composition, as serous, sero-mucous, mucous, muco- purulent, purulent, etc. , or according to their cause, as inflam- matory, catarrhal, suppurative, cystic, etc. The pathological body-fluids will here be classified and considered as follows : Transudates, dropsical. Cyst fluids. Exudates, inflamma- tory: serous, fibrinous, sero-fibrinous, cancerous. Hemorrhagic, chylous, and fatty effusions. Pus. Catarrhal exudates. Granu- lation exudates. K"ecrotic fluid. Transudates. — This term is applied to dropsical effusions into the serous cavities or areolar tissues of the body, in contradis- tinction to exudates of similar appearance and location produced by inflammatory processes. The possibility of making a distinc- tion between transudates, cyst fluids, and exudates may aid in making a diagnosis between dropsical, cystic, and inflammatory conditions. Transudates most often occur in the subcutaneous tissues in general anasarca, in the peritoneum in ascites, in the pleura or pericardium in hydrothorax and hydropericardium, or the cerebro -spinal cavities in hydrocephalus. Transudate fluid is readily collected by means of the trocar and canula, or from the subcutaneous tissues by incising the skin and collecting the fluid as it drains away. Dropsical transudates usually consist of a sterUe, clear, light- yellow, serous fluid, of alkaline or neutral reaction. Chemically they are composed of a solution of about 1 to 5 per cent of solids in water. The solids consist of various mineral and organic sub- stances, among which sodium chloride and proteids are much the most abundant. Variations in the composition of transudates consist chiefly in variations in the proportion of proteids; and the chief difference between transudates and serous exudates is in the larger amount of albumin in the latter. The proportion of albumin in transudates ranges usually from 2 or 3 per cent down to small amounts, while in exudates it is usually from 3 to Digitized by Microsoft® PATHOLOGICAL FLUIDS. 223 8 per cent. The specific gravity of transudates is usually 1. 005 to 1.015, that of exudates 1.018 to 1.030; as the variations in specific gravity depend chiefly on variations in the amount of albumin, the specific gravity may be taken in general as an in- dex of the proportion of albumin and as a distinguishing charac- teristic between transudates and exudates. Microscopically transudates may exhibit a few leucocytes and endothelium cells, sometimes showing fatty degeneration. Cho- lesterin crystals sometimes appear. On standing a fibrinous nubecula similar in appearance to that of urine sometimes forms. Amcebse have been seen in ascites fluid. At times transudates may be tinged reddish or brown with blood or transformed blood pigment, or green with bile pigment. Cyst fluids vary widely in cjiaracter according to the condi- tions which produce them, being serous, mucoid, colloid, fatty, etc. The fluid is obtainable by aspiration. Retention cysts originate from an accumulation of secreted fluid in a glandular cavity whose outlet has become occluded. The fluid in such cysts partakes to a certain degree of the characters of the corresponding normal secretion of the gland, and the identiflcation of the origin and character of the cyst depends in general on the detection in the fluid of the specific gland cells or secreta. Thus, the presence of trypsin, shown by its power of digesting coagulated albumin in alkaline media, would indicate a pancreatic cyst; urinary ingredients "and renal cells would ap- pear in hydronephrosis ; mucinous material in cysts of mucous glands, colloid material in goitrous cysts, milk or its products in galactocele, fatty matter in sebaceous cysts ; hair, teeth, sebaceous matter, etc., in dermoid cysts. In the course of time, however, the gland substance may be entirely destroyed and the retained fluid may lose the specific glandular ingredients. Ovarian Cyst Fluid. — The recognition of this fluid may be of importance in diagnosticating between ascites and ovarian cysts. The fluid from these cysts is usually clear, colorless, yellowish, or brownish ; the presence of blood elements gives a red or brown color. The fluid varies from a thin, limpid consistency to a thick, viscid, and gelatinous consistency, and ranges in specific gravity from 1.007 to 1.026; the variations in consistency and specific gravity depend chiefly on the presence of colloid, mucoid, and proteid material in varying amount. Metalbumin or paralbu- Digitized by Microsoft® 224 A MANUAL OF CLINICAL LABORATORY METHODS. mill is a characteristic proteid ingredient of most ovarian cyst fluids, as distinguished from other cysts. Microscopically, ovarian cyst fluid may exhibit leucocytes, epithelium cells (rounded, elongated, ciliated, or squamous), Drysdale's corpus- cles, cholesterin crystals, red blood corpuscles, rounded colloid bodies. The epithelium cells may be fatty or granular, and their character indicates the nature of the cyst walls. Drys- dale's corpuscles are rounded, non-nucleated, granular cells about the size of leucocytes ; the protoplasm between the granules is clear and transparent; the granules are distinctly outlined, insoluble in acetic acid, and insoluble or incompletely soluble in ether; Drysdale's corpuscles are regarded as very characteristic of ovarian cysts, and are perhaps freed nuclei. In dermoid ovarian cysts dermoid elements are present. The chief distinguishing characters of ovarian cyst fluid as compared with ascites fluid are the colloid consistency, the pres- ence of columnar or ciliated cells and of Drysdale's corpuscles. The distinction cannot always, however, be made. Exudation cysts arise from excessive effusion into normal cavi- ties, as in hydrocele. Sydrocele fluid in its characters frequently approaches those of inflammatory exudates, containing a large proportion of pro- teids and often with a high specific gravity, 1.015 and upward. The fluid is usually serous, limpid, clear, amber or yellow in color ; frequently great numbers of large macroscopic glistening plates of cholesterin are present, and sometimes the fluid is dark- colored and cloudy from the presence of transformed blood ele- ments. A very few cases of chylous hydrocele or chylocele have been observed, in which the sac contained a milky chylous fluid. Disintegration cysts contain fluid varying in character, result- ing from necrotic liquefaction of solid tissues. Parasitic cysts are produced by parasites, such as echinococci, cysticerci, trichinae, and others. Most varieties contain little besides the parasitic forms, but echinococcus or hydatid cysts also contain a large amount of fluid. Hydatid fluid is clear and serous, with a speciflc gravity of 1.006 to 1.010, and contains very little or no albumin; micro- scopically, the detection of shreds of cyst-membrane, scolices, or hooklets is diagnostic. Inflammatory exudates, produced by inflammatory processes. Digitized by Microsoft® PATHOLOGICAL FLUIDS. 225 may be serous, fibrinous, sero-flbrinous ; when associated with hemorrhagic or suppurative processes the product becomes bloody or purulent, and when involving a mucous surface mu- cinous elements are introduced. Serous exudates, best exemplified by the effusion in serous pleuritis and blisters, are usually limpid, clear, colorless or yel- lowish, containing 3 to 8 per cent of proteids, and have a specific gravity of 1.018 to 1.030; they sometimes coagulate on standing. They resemble dropsical transudates, except in their higher specific gravity, their greater proteid content, and their tendency to coagulation. Microscopically the centrifuged sediment ex- hibits a few leucocytes and endothelial cells, usually fatty. Bacteria may be demonstrable by staining, cultures, or animal inoculation. Fibrinous exudates, consisting of white, somewhat adhesive, fibrinous coagula, are produced in acute plastic inflammations, especially of the peritoneum and pleura. Fibrin has rather characteristic staining properties, by which it may be distin- guished ; it is oxyphile, and stains with acid stains, while mucin is basophile. After fixing with alcoholic mercuric-chloride solu- tion (5 per cent) fibrin stains red with the triple stain (mucin green). Fibrin retains a blue stain in the Weigert-Gram method, which is a$ follows : Fix and harden in alcohol. Stain five to fifteen minutes in a strong solution of gentian violet in anilin- water. Wash in .6-per-cent sodium-chloride solution. Dry with filter-paper on a slide or cover-glass. Transfer to Gram's iodine solution two or three minutes. Dry with filter- paper. Decolorize sufficiently in anilin oil 2 parts, xylol 1 part. Wash in xylol and mount. 8ero-fibrinov^ exudates, as from peritonitis, pleuritis, arthritis, consist of a serous exudate in which are suspended fibrinous coagula and particles. Cancerous exudates, such as may be produced in malignant dis- ease of the pleura or peritoneum, have as a basis a serous exu- date usually containing blood and cells or fragments of tissue from the neoplasm. The fiuid usually has a large proteid con- tent (3 to 6 per cent) and' a high specific gravity (over 1.020). Blood, fresh or transformed, is often present. Abundant cells or fragments of tissue, sarcomatous or carcinomatous, may be present in thp fluid and aid in diagnosis ; but the identification 15 Digitized by Microsoft® 226 A MANUAL OF CLINICAL LABORATORY METHODS. of isolated cells as sarcoma or carcinoma cells is always a diffi- cult and uncertain matter. Sometimes the free cells undergo fatty degeneration and give the fluid a chyliform or fatty char- acter. Hemorrhagic Effusions. — Blood may become mingled with and add its characters to any form of transudate or exudate, in con- sequence of traumatic or ulcerative conditions, etc. If fresh, the color will be bright-red ; if stale, the decomposed and transformed blood pigment produces a brown or dark color. The presence of fibrin factors causes some tendency to clotting. The tests for blood will be positive, and microscopically red blood corpuscles may appear. Bloody effusions in the pleura, peritoneum, or tunica vaginalis are especially due to malignant disease, trau- matism, or tuberculosis of the part. Chylous Effusions. — In numerous instances the fluid withdrawn from cases of ascites, and in a very few instances effusions into the pleura (chylothorax), pericardium, and tunica vaginalis (chylocele), have been found to be white, opaque, and milky — true chylous fluid. Microscopically, fat particles are seen sus- pended in a state of very fine granular division, with a few red blood corpuscles, leucocytes, and endothelium cells. On stand- ing, a creamy layer rises to the top, and a gelatinous coagulum settles to the bottom. The reaction is alkaline or neutral, spe- cific gravity 1.007 to 1.023, total solids 5 to 12 per cent, proteids up to 6 per cent. The effusion is caused by a leakage of chyle into the serous cavity involved, either from rupture of lymphatic vessels, or obstruction of the thoracic duct, or filariasis. Blood may also be present. Fatty effusions (also called chyliform, chyloid, adipose, oily, and lacteal effusions) resemble true chylous effusions, but the fat is not so finely divided, occurring in larger droplets or coalescing in masses of fluid oil. These effusions are produced by fatty de- generation of the cells in the fluid, usually endothelium cells or carcinoma cells, less often leucocytes or fibrin. Pus is the specific product of suppuration. Pure typical pus is produced by uncomplicated suppuration in the solid tissues, as in acute abscesses, and exhibits characteristic features. When the suppurative process is associated with serous or ca- tarrhal inflammation, hemorrhage, or necrotic processes, the resulting product is of a mixed type, containing pus mixed with Digitized by Microsoft® PATHOLOGICAL FLUIDS. 227 serous exudate, mucus, blood, and disintegrated tissue, forming sero-purulent, muco-purulent, sanguino-purulent exudates, etc. Pure pus may, however, be generated from serous or mucous surfaces. Typical and pure pus, such as is found in acute abscesses, consists essentially of a crowded mass of leucocytes suspended in a fluid (pus serum), together with the bacteria or other parasites causing the suppuration, and with more or less granular, amor- phous, and degenerative materials. The color ranges from gray- ish to light-yellow or greenish. The bacillus pyocyaneus pro- duces bright-green pus, the admixture of blood gives a brown color. The consistency is creamy, varying from a quite fluid con- dition to a thick, viscid, semi-solid mass. The odor is disagree- able and may be very foul and repulsive. The reaction is al- kaline, the specific gravity 1.025 to 1.040. After standing or centrifugalizing, the corpuscles settle, leaving the pus serum supernatant. The pus serum is a solution of about 8 to 10 per cent of solids, which are chiefly proteids, with a small amount of fatty material. The leucocytes of pus are almost exclusively of the polynuclear neutrophile variety; an occasional large mononu- clear leucocyte is present, while in gonorrhoeal pus and asthmatic sputum eosinophile leucocytes are often comparatively abundant. Amorphous granular or flaky material, fat particles, etc., are seen in greater or less amount, especially in abscesses of long standing. Crystals of cholesterin, fatty acids, or triple phos- phate may appear in pus from chronic abscesses, and red blood corpuscles or hsematoidin may appear when there has been an admixture of blood. Fragments of necrotic tissue are sometimes present. In pus from circumscribed abscesses ordinarily only the bac- teria causing the suppuration are present, while in pus from localities exposed to contamination adventitious bacteria may be abundant. The pyogenic bacteria that occur in pus are numer- ous, the most common forms being the staphylococci and strep- tococci, less common being the bacillus pyocyaneus, bacillus coli communis, gonococcus, micrococcus lanceolatus, tubercle bacil- lus, and numerous others. Actinomycetes, streptothrix, blasto- mycetes, the amoeba coli, psorosperms, filarige, trematodes and their ova, and other fungous and animal parasites are at times Digitized by Microsoft® 228 A MANUAL OF CLINICAL LABORATORY METHODS. found in pus. Theoretically but scarcely in practice pus may be generated by irritants independently of bacterial action. Pus from chronic or old abscesses varies considerably in some features from the typical pus of acute suppurations. The leuco- cytes are frequently fatty or otherwise degenerated, diminished in number, and at times almost absent ; they often do not take stains in the normal way. There may also be an abundance of amorphous cheesy material, which seems to take the place of leucocytes. The bacteria are sometimes diminished in number, and the pus may even become sterile. Crystalline, granular, and fatty material may be abimdant. In exudates of mixed type, the pus is mixed in various pro- portions with serous, mucinous, and other elements. The amount of pus present is proportionate to the quantity of leucocytes, which may be estimated from the amount of sediment thrown down by the -centrifuge or from the appearance of microscopical specimens. Examination of Pus. — The color, odor, consistency, and general appearance are obvious to simple inspection. Sedimentation or centrifugation affords an idea of the quantity of leucocytes pres- ent. Examination of fresh unstained pus under the microscope reveals leucocytes, crystals, amorphous material, amoebae, filariae, fungi, or other parasites. The leucocytes can be studied by the same methods of fixing and staining (triple stain, etc.) as are em]ilOyed in the case of the blood. The chief clinical impor- tance of pus ordinarily depends on the kinds of bacteria present. The determination of the bacteria in pus is accomplished by staining methods or by the usual cultural methods or animal inoculation. The methods of staining pus are similar to those of staining sputum. By means of the platinum loop a drop or two of the fluid to be examined is spread out in a thin layer on a slide or cover-glass and allowed to dry ; or a drop of the pus is squeezed out into a thin, even layer between two cover-glasses, which are then slid apart and dried, leaving a thin film on each cover. In prepara,tion for staining, the cover-glass spreads are "fixed" by flaming or passing them three times through a Bunsen or alcohol flame at moderate speed and brief intervals. The films are then stained for a few Seconds or minutes by covering them with or Heating them on the staining fluid employed. For staining most Digitized by Microsoft® PATHOLOGICAL FLUIDS. 229 bacteria Loeffler's metliyleue blue, dilute carbol-fucbsin, gentian violet, and Gram's metbod are tbe best. For tbe tubercle bacil- lus Gabbet's method (page 130) is employed. For the gonococ- cus Loeffler's methylene blue or Gram's method is used. Special methods may be employed for special purposes. Catarrhal Exudates. — The products of inflammatory processes affecting mucous membranes correspond to exudates in general, but are modified by the introduction of special elements, notably mucin, epithelial cells, and many adventitious bacteria. Spu- tum, a typical catarrhal discharge, has been considered in detail ; the discharges from other mucous surfaces are in general similar to sputum. The exudate varies according to the form of inflam- mation. In the initial stage of catarrhs (as in acute rhinitis) the exudate may be purely serous, thin, clear, and limpid ; ordinar- ily,- catarrhal exudates contain much mucin, affording purely mucous discharges or mixed muco-serous or muco-purulent fluids ; in the presence of suppurative processes a proportionate amount of pus is introduced into the exudate, or even pure pus is produced. Foul putrefactive products and gases are generated in necrotic, putrid, or stagnant cases. A fibrinous exudate in the form of a pseudo -membrane is produced on mucous mem- branes by the diphtheria bacillus, streptococci, staphylococci, micrococcus lanceolatus, or in debilitated conditions. In myco- tic catarrhs, as in thrush, a fungous growth develops on the mucous surface. Catarrhal exudates therefore consist of serous, mucoid, puru- lent, bloody, putrid, or fibrinous elements in pure form or mixed in variable proportions. The color varies from colorless, grayish, yellowish, greenish- yellow, brown, rarely bright-green or other color. The consist- ency, homogeneity, and odor vary according to circumstances, much as in the case of sputum. Leucocytes or red corpuscles are present in proportion to the extent of suppuration or bleeding. Epithelium cells from the mucous membrane involved are present, either the surface cells of the part or the deeper germinal cells immaturely and prema- turely cast oft ; they are usually more or less swollen, altered, or degenerated. Bacteria of many varieties and in enormous num- bers are often present, though in some cases they may be limited to the variety causing the trouble. Digitized by Microsoft® 230 A MANUAL OF CLINICAL LABORATORY METHODS. The purpose of the examination of catarrhal exudates is partly to determine as to the presence of leucocytes, blood cor- puscles, or epithelium, but ordinarily especially to determine the bacteria causing the inflammation in the case under investiga- tion. For this purpose the usual methods of staining and micro- scopical examination, cultural methods, etc., are employed as in the case of sputum or pus. Special methods are employed for special micro-organisms, as for tubercle bacilli, gonococci, and diphtheria bacilli. Granulation Exudates. — The initial effusion from wounds con- sists of blood and lymph from severed vessels. If infection, in- flammation, or suppuration subsequently develops, the corre- sponding inflammatory or purulent exudates are generated in abundance. Sterile and uninfected granulating wound and ulcer surfaces yield a scanty amount of whitish fluid, of a creamy or sticky consistency, odorless, or nearly so, and entirely free from bacteria; microscopically numerous leucocytes with amorphous matter are seen. Necrotic Fluid. — The fluid materials produced by the liquefac- tive disintegration of necrotic tissue vary in their ultimate fate and character. If isolated by solid tissue walls and not infected, cyst-formations may result, with fluid or gelatinous contents. If, as is frequently the case, infection occurs, foul putrid suppura- tion results, with exceedingly repulsive discharges, as in pul- monary gangrene. Frequently in operation wounds through a thick panniculus adiposus (especially for hernia) that are sterile and infected throughout, necrosis of the fatty subcutaneous tissues occurs, with the production of a fluid that much resembles pus and therefore gives rise to needless alarm. This form of necrotic fluid is creamy, dark-colored, and contains leucocytes, amor- phous material, and sometimes masses of necrotic tissue; bac- teria are absent, and the condition is not in any way the result of infection. Digitized by Microsoft® X. CALCULI. Scarcely any part of the body, either solid tissues or hollow viscera or ducts, is exempt from the formation of hard calculous bodies. The calculi of the gall bladder and urinary organs are much the commonest and most important clinically. In special situations calculi may be composed of special substances, as cholesterin and bile pigments in the gall bladder, uric acid, cal- cium oxalate, etc., in the urinary organs. The generality of cal- culi are composed of insoluble earthy salts mingled with organic matter; of the earthy material calcium phosphate ordinarily greatly preponderates, with calcium carbonate, ammonio-mag- nesic phosphate, magnesium phosphate, magnesium carbonate, and calcium sulphate in amounts varying from zero or traces up to considerable proportions; cholesterin or fatty matter is sometimes present. Most but not all calculi are formed by the deposition of solid material on a nucleus or matrix of organic matter or of some foreign body, as fibrin or mucus, inspissated secretions, bacterial or fungous growths, caseous or necrotic tis- sue, or a fragment of another calculus. Frequently calculi con- sist of concentric lamellae which sometimes differ from one an- other in composition. The size and form vary greatly; in number they may be single or multiple ; when a number are in contact, the apposed surfaces are smooth and facetted. Calculi can be considered both with reference to their situ- ation and their composition. In varicose veins and vascular tumors concretions (phlebo- liths) occasionally occur, composed of the usual calcareous and earthy deposits mingled with fibrin. Calculous deposits, chiefly calcium phosphate, occur rarely in the thoracic duct, receptacu- lum chyli, and lymphatic glands, in the latter especially when caseous. Salivary concretions (sialoliths) of the usual calcareous com- position occur occasionally in the submaxillary gland or its duct, less often in the sublingual or parotid, rarely in the small mucous Digitized by Microsoft® 232 A MANUAL OF CLINICAL LABORATORY METHODS. glands of the palate, and are similar in composition to the tartar of the teeth. In the tonsils calculi sometimes form from inspissation and calcification of the contents of obstructed crypts. Calculi are rare in the stomach, being formed chiefly by an aggregation of insoluble ingested substances, as lime and mag- nesia salts, salol, fatty material, resinous substances, etc. Aega- gropili consist of hair, ingested by the mouth and matted to- gether in balls; they occur in the oesophagus, stomach, or intestine, and are commonest in herbivora, but are rarely found in man. Enteroliths, or intestinal calculi, are formed either (a) from insoluble ingesta like salol, lime or magnesia salts, or fat, (6) by the deposition of calcareous matter on some food particle (like seeds) or a bit of hard fsecal matter as a nucleus, or (e) most commonly by the inspissation of faeces and infiltration with earthy salts (coproliths). They are formed chiefly in the ap- pendix, caecum, or saccular pockets of the colon or rectum. They often contain large amounts of triple phosphates. Calculi of the usual calcareous composition sometimes form in the ducts of the pancreas and may attain large size. Biliary calculi are frequently observed. They vary in num- ber from one to hundreds or even thousands, and in size from that of the distended gall bladder to fine granules. They are composed of the ingredients of the bile, mixed in varying pro- portions, sometimes one, sometimes another substance prepon- derating. The chief materials of which gall stones are formed are cholesterin, bile pigments, earthy phosphates and carbonates, and occasionally biliary or fatty acids. Traces of metallic sub- stances are sometimes present. Most gall stones contain 70 to 90 per cent of cholesterin, and some are almost pure cholesterin. Some of them have a nucleus of bacteria or other particles, and some are laminated and heterogeneous in composition. The cholesterin and pigment calculi are rather soft, the calcareous stones harder and firmer. The bile pigment present, according to its amount and kind, gives gall stones a brown, or exception- ally a black, green, blue, or yellowish-red color. Cholesterin and calcareous concretions nearly free from .bile pigment maybe white or gray. Occasionally fibrinous concretions occur in the peritoneal cav- Digitized by Microsoft® CALCULI. 233 ity, either loose or attached by a pedicle. They have been ob- served larger than a billiard ball. They originate from the fatty omental appendages as nuclei, which become necrotic or calcified and then covered with proteid or fibrinous laminae. Lithopsedia also form calcified masses. Calculi of the nasal fossae and accessory sinuses (rhinoliths) are usually single, weighing from 2 to 10 grams. They consist mostly of calcium phosphate, with often notable proportions of calcium carbonate and magnesium phosphate. Most rhinoliths are formed on a nucleus of some foreign body, as seeds. Calculi rarely form in the bronchi (broncholiths) or pulmonary cavities, from deposition of calcareous material about a nucleus of some kind, as dried sputum, bits of tissue, etc. ; fatty bron- choliths have also been known. PnewmoUths are formed by the deposition of calcareous matter in the substance of the lung, most frequently in caseous tuberculous foci, but sometimes in healthy tissue. Calcareous deposits are sometimes laid down in the pleural membrane or free calculi may be formed in the pleu- ral cavity. Calculi in the skin occur rarely, resulting from inspissation and calcification of the contents of sebaceous cysts (seboliths). They are composed of fatty and calcareous material. They may also occur in the subcutaneous tissues. Urinary calculi may form in any portion of the urinary tract, renal parenchyma, renal sinus, ureter, bladder, or urethra. They vary widely in size, from small granules (gravel) up ; in one case a calculus weighed 1, 596 grams. They are usually single, but they may be multiple, up to several hundred in number. They are usually formed on a nucleus of inspissated mucus or pus, bacteria, epithelium cells, crystals, foreign bodies, etc. Sometimes they are laminated, and sometimes a calculus is com- posed of different substances in different parts. In composition urinary calculi present considerable variation. The commonest varieties are those composed of uric acid, urates, calcium oxalate, or phosphates, respectively ; rarely urinary calculi are composed of xanthin, indigo, cystin, calcium carbonate, fat or soap (uro- stealiths), cholesterin, or fibrin. Uric-acid calculi are the commonest form of primary urinary calculi, and frequently serve as nuclei on which large concretions are formed by deposition of phosphates or oxalate. They occur Digitized by Microsoft® 234 A MANUAL OF CLINICAL LABORATOEY METHODS. especially in children, old age, and the gouty, and may attain large size. They are usually formed in the kidney. Urate concretions may be developed in the kidneys of chil- dren. Urates do not often form the only ingredient of calculi, but are usually mixed with uric acid or calcium oxalate. Calcium-oxalate calculi are sometimes nearly pure, sometimes mixed with uric acid or urates, and are formed in acid urine similarly to uric-acid calculi. The phosphatic calculi are composed of calcium phosphate or ammonio-magnesic phosphate, usually mixed, but sometimes either one alone. Phosphatic concretions are formed especially in cystitis with ammoniacal urine, and hence chiefly originate in the bladder. They are usually formed on other calculi (uric acid or oxalate) as a nucleus, the latter setting up a cystitis that favors the precipitation of phosphates. They may attain large size. Cystin and calcium-carbonate urinary calculi are rare. Xan- thin, indigo, cholesteriu, and fatty or soapy concretions in the kidney or urinary passages have been noted only in a very few in- stances (one or two to a dozen or so each). Hardened masses of inspissated fibrin or blood clots have also been observed. In four or five cases calculi resembling urinary concretions have been found at the umbilicus, apparently formed in a patent urachus communicating with the bladder. Sebaceous concre- tions have also been found at the navel. Urethral calculi are usually formed in obstructed or traumatic conditions of the urethra. They may occur in any part of the canal from the meatus to the prostate, and usually consist of calcareous and phosphatic dep.osits on fragments of calculi from the bladder, or other foreign bodies. Calcium carbonate and uric acid may also occur in the deposits. They may be single or multiple, and have been observed of a weight as high as 780 grams. Perineal calculi resemble urethral calculi, and are formed in the perineal tissues under conditions of urethral obstruction or trauma similar to those giving rise to the latter. Preputial calculi are formed beneath the prepuce in cases of phimosis, and are composed of fatty material, fatty acids, cho- lesteriu, or epithelium, derived from inspissation of the smegma and impregnation with phosphates. They may be single or mul- tiple ; in one case two stones had a combined weight of 42 grams. Digitized by Microsoft® CALCULI. 235 Prostatic calculi are often found in the prostate of adults and old men, and are occasionally discharged with the urine. The glandular alveoli of the prostate normally contain numerous minute muco-calculous, round, concentrically marked bodies, which by accretion of calcareous and phosphatic material may attain macroscopic dimensions. They are ordinarily multiple. In one case the combined mass of calculi weighed 105 grams ; in another case a single concretion weighed 25 grams. Calcareous concretions have also been found in the seminal vesicles. Calculi rarely occur in the oviduct, uterus, and vagina. In the oviduct calcium- oxalate calculi have been found. In the uterus and vagina they are usually calcareous, originating from vesical calculi passed through fistulse, in vaginal cysts, as an incrusta- tion on hairpins, pessaries, or foreign bodies in the vagina, or otherwise. Calculi obstructing the nipple have been observed. In gout the deposits of urates in the articular tissues or joint cavities and elsewhere may assume a calculous character (chalk stones, tophi). In the brain or its meninges concretions are represented by the rare psammomata or tumors containing calcareous particles, also by calcified necrotic areas or tubercles. Calcareous concre- tions or granules (psammoma) have been noted in the auditory, facial, and optic nerves, especially the first-named, and also in the inner ear. Dacryoliths, or lachrymal calculi, have been observed in the lachrymal gland, the conjunctiva of the upper lid, the ducts of the Meibomian glands, the conjunctival cul-de-sac, the lower canaliculi, and the lachrymal passages. They consist of the usual calcareous and phosphatic ingredients, and in the lachry- mal passages frequently have a nucleus of leptothrix fungi. Characters and Tests of Calculi. In the examination of calculi, they should be sawed or broken in two to observe if they are uniform or heterogeneous in struc- ture. In the former case the sawdust may be used for analysis, in the latter fragments from the different portions may be pow- dered. The pulverized material is employed in testing as to solubility, the effect of heat, etc. In applying heat, a small portion of the powder is placed on platinum foil and heated to a Digitized by Microsoft® 236 A MANUAL OF CUNICAL LABORATORY METHODS. red or white heat in the Bunsen or alcohol flame. For certain purposes the blowpipe flame is used. Phosphatic calculi, composed of either calcium phosphate or ammonio-magnesic phosphate alone, or of both mixed, or mixed with other substances, are of the most general occurrence in the body. They are usually white or gray in color, with either a smooth or rough surface, rather soft, friable, and easily crushed, and have a stony fracture. Adherent or embedded crystals of ammonio-magnesic phosphate may appear as glistening points. The powdered material of which they are composed is insoluble in water and alkalies, readily soluble without effervescence in water acidulated with acetic or hydrochloric acid, and is precipi- tated from this solution' on making it alkaline with soda, potash, or ammonia. When heated on platinum foil to a red heat the powder does not melt, char, or burn, and suffers little or no diminution in amount. Ammonio-magnesic phosphate melts under the blowpipe flame, while calcium phosphate does not. On mixing powdered ammonio-magnesic phosphate with calcium or potassium hydrate and moistening, ammonia is evolved (es- pecially if heated), and may be recognized by its odor and its turning moistened red litmus paper, suspended near, to a blue color. Calcium-carbonate Calculi. — Calcium carbonate is frequently present in calculi mixed with calcareous or phosphatic material, but rarely is the sole or predominating constituent. When pure, the calculi are gray, smooth, and hard. On being touched with acid effervescence occurs ; on heating to a white heat the powder first turns black and then white again, calcium oxide being formed, which dissolves in water sparingly and in that solution gives an alkaline reaction. Cholesterin Calculi. — Cholesterin forms the bulk of most biliary calculi, or may constitute practically their entire substance ; it is frequently present in calculi from A'arious parts of the body, es- pecially in those derived largely from fatty material, as seboliths and preputial concretions; one or two instances of cholesterin urinary calculi are known. The color of cholesterin biliary cal- culi may be modified by the presence of bile pigment, and their consistency by the presence of calcareous salts. When in a state of approximate purity cholesterin concretions are white or gray, smooth, of waxy consistency, soft and easily crushed, and float Digitized by Microsoft® CALCULI. 237 in water. Cholesterin melts at 137° C. and at a little higher temperature burns with a yellow carbonaceous flame without leaving any residue. It is soluble in ether, chloroform, and hot alcohol. The addition of a drop of strong sulphuric acid to the chloroform solution of cholesterin produces a deep-red color. On allowing a drop of the hot alcoholic solution to cool on a slide the characteristic rhombic colorless plates, frequently with a corner lacking or with a side of an echelon or step-ladder form, crystallize out and may be recognized with the microscope. Bile-pigment Calculi,— Bile pigments (bilirubin, biliverdin, etc. ) enter into the formation of most biliary calculi to a greater or less extent, giving them a brown, dark-green, or yellow color. Occasionally biliary calculi are composed chiefly or entirely of bile pigment. Such calculi are hard, non-crystalline, sink in water, and yield the characteristic color reactions with nitroso- nitric acid ; when heated they do not melt or burn, but char and leave an ash. Bilirubin may be extracted with chloroform, in which the other bile pigments are insoluble. Fatty Calculi. — Fatty material, in the form of neutral fat, soaps, or fatty acids, occurs in many calculi in which there has been an inspissation of organic matter, as in seboliths and pre- putial concretions. Fat or fatty acids may enter into the forma- tion of biliary calculi, and rarely form the preponderating ingre- dient. Four or five cases are known of urostealiths, or urinary calculi composed of fatty or soapy material, sometimes mixed or covered with phosphates, and in two or three cases similar con- cretions have occurred in the stomach and bronchi. Urostea- liths are brown or yellow, soft and friable when fresh, hard and brittle when dry, softer again when warmed. The material is soluble in ether, saponifies with caustic alkalies ; when heated it burns with a yellow fiame and an odor of resin or shellac, and is entirely consumed. Uric-acid calculi are formed in the kidney and urinary organs. They are hard and brittle, concentrically laminated, brown or reddish, with smooth or tuberculated surfaces, and vary in size from small particles up to a weight of 150 grams. The powdered material yields the murexide reaction (page 191), is soluble in caustic alkali, and on being heated on platinum foil it is entirely or almost entirely consumed without flame, leaving only a trace of residue or none at all. Digitized by Microsoft® 238 A MANUAL OF CLINICAL LABORATORY METHODS. Urate calculi occur in the urinary organs and gouty joints. The gouty tophi consist of sodium urate. In the urinary calculi, the various urates, especially of ammonium, calcium, and sodium, may be present, usually associated with uric acid or calcium oxalate, though in children urates may form the predominating ingredient. Urate calculi are usually small, not over a couple of centimetres in diameter, of a light-yellow, brown, or gray color ; they are not so hard and dense as the uric-acid stones. Urates all yield the murexide reaction, and are soluble in hot water. Ammonium urate is completely consumed on heating. On mixing a portion of powdered ammonium urate with calcium or potassium hydrate and moistening, ammonia is evolved and may be recognized by its odor and by moist red litmus paper sus- pended over the mixture turning blue. The other urates leave a residue after heating. Sodium and potassium urates melt and leave a residue of carbonate which is alkaline when moistened ; sodium urate imparts the characteristic yellow color of sodium to the Bunsen or alcohol flame. Calcium and magnesium urate do not melt. Xanthin calculi have been found in the urine a few times. They are white to brown in color, of medium hardness, and range in size from that of a pea to that of a hen's egg. Uric acid may be mixed with the xanthin. The powder of xanthin calculi on being heated is entirely consumed, without flame. Xanthin does not yield the murexide reaction ; on dissolving a minute portion in a drop of nitric acid and carefully evaporat- ing, the residue turns bright-yellow and is insoluble in potas- sium-carbonate solution, but is soluble in potassium-hydrate solution with the formation of a reddish color. Calcium-oxalate calculi, nearly pure or mixed with uric acid or urates, are among the common urinary calculi, and have been also found in the female genitals. They are very hard and dense, dark-colored ; the larger forms have a rough, tuberculated surface ("mulberry calculi") ; sometimes they form small, round, seed-like bodies. On being heated, the powder first chars, and then becomes converted to a white residue, calcium carbonate, which effervesces with acid ; on still further heating the calcium carbonate is changed to calcium oxide, which affords an alkaline reaction when moistened. Digitized by Microsoft® CALCULI. 239 Indigo calculi, found in the kidney in three or four cases, have a dark-brown or blue-black color, and when drawn over paper leave a blue mark. On being heated a sooty odor is evolved, and the material sublimes ; on condensing the vapor on a glass slide and adding glycerin, blue prismatic crystals and granules are seen. The solution in sulphuric acid is brownish and then muddy blue ; on diluting and filtering a clear blue fluid is ob- tained. Cystin calculi, rarely found in the bladder, are usually of me- dium size, with finely granular or crystalline surface, rather soft, and of a pale-yellow or gray color. Cystin is soluble in am- monia, and on evaporation the characteristic hexagonal crystals are deposited. It is also soluble in soda, potash, and mineral (not vegetable) acids. On being heated, cystin burns with a blue flame, evolves a sulphurous odor, and is entirely consumed without yielding any residue. If dissolved in potassium-hydrate solution and boiled with lead acetate, a black precipitate of lead sulphate is formed. Calculi composed of animal matter, as fibrin, on being heated burn with a yellow flame, yield an odor of burnt horn, and leave little or no residue. They are largely soluble in potassium- hydrate solution. Microscopical examination may show epithe- lium, bacteria, etc., and in the case of coproliths fragments of food residue. Digitized by Microsoft® XI. PARASITES. The examination of parasites, animal and vegetable, fre- quently comes witMn the field of the clinical laboratory. The subject is an extensive one, and its mastery requires much spe- cial study and opportunities that are afforded to few. It is only the common parasitic forms with which the ordinary clinician has the opportunity to become familiar. Outside from common forms the subject can be only very briefly covered here. It should be remembered that of the animal and vegetable organ- isms found associated with the human body, only a portion are pathogenic and injurious, while others are adventitious, acci- dental, and innocuous. ANIMAL PARASITES. The animal parasites of man belong to the protozoa, the vermes, and the arthropoda. Protozoa. — The parasite of malaria (page 33) occurring in the blood, and the Amoeba coli (page 110), occurring in the colon in amoebic dysentery and in amoebic abscesses of the liver and lung, have been already considered. Other amoebtE have at times been observed in various fluids and parts of the body. Psorosperms and Coccidia. — These parasitic protozoa are com- mon in rabbits, very rare in man. They are oval forms 20 to 40 /J- long (Coccidium ovif orme) . At first the organism is granular throughout ; at a later stage of development the granular sub- stance collects in a ball in the centre of the cell, and still later it breaks up into four spores. In man the coccidia develop most often in the epithelium of the intestine or bile passages of the liver, causing intestinal erosions or hepatic neoplasms. They have also been observed in the human kidney, wall of the ureter, and in empyemic pus, and are perhaps associated with certain skin lesions (as Paget's disease, Darier's disease). Eelated protozoa (balbiania) have in one or two instances been found encysted in human muscle. Protozoan forms have been claimed to cause cancer, but the subject is not yet settled. Digitized by Microsoft® PARASITES. 241 Cercomonas. — This is a pear-shaped organism about 10 /i long, coming to a filamentary point at one end and with a flagellmn at the blunt end. It has been found in the intestine in diarrhceal conditions ; also in the liver and mouth. Trichomonas Vaginalis. — This is a pear-shaped parasite 15 to 30 iJ. long with three or four flagella on the blunt end and a longitudinal undulating membrane or comb of cilia. It is most frequently found in the vagina, where it is innocuous; it has also been observed in the bladder, associated with hsematuria, and a similar parasite has been found in the intestine and mouth. Megastoma Entericum. — This is a pyriform organism found a few times in the human intestine, 10 to 20 ij. long, with an oral depression at the blunt end, and four pairs of flagella. Balantidium coli is an oval, asymmetrical body, 70 to 100 ii long, with an oral and an anal structure, an elliptical nucleus, and two contractile vesicles. The surface is entirely covered with cilia. In Scandinavia it is common in the intestine, espe- cially in diarrhceal conditions, rarer in other countries. Vermes. — The worms parasitic in man belong to the classes of trematodes or flukes, cestodes or tapeworms, and nematodes or round worms. Trematodes, or flukes. — These -are mostly flat, fish-shaped worms infesting various parts of the body. Only two or three forms are common in man, in certain countries, the others being rare in man and all rare in America. Schistosoma Haematobium. — This is a bisexual fluke, 4 to 20 millimetres long, occurring chiefly in the veins of the pelvic vis- cera, bladder, rectum, and vagina. By the accumulation of the ova in the capillaries, their irritative effects, and rupture of the vessels into the neighboring cavities, marked local disturbances are produced, as local inflammations, ulcerations, nodular growths, hemorrhages (vesical, rectal, vaginal, etc.). The ova may also pass into other parts of the portal system, as the mesen- teric glands and liver, and have even been found in metastatic abscesses in the lungs. The ova and perhaps embryos occur, along with blood, in the urine, fseces, vaginal discharges, or pus of metastatic abscesses. The worm has been known to develop in the lungs, discharging ova into the sputum. Schistosoma in- fection is common in Egypt, and also occurs in South Africa and 16 Digitized by Microsoft® 242 A MANUAL OF CLINICAL LABORATORY METHODS. elsewhere; only two cases have been reported in the United States, both originating in Africa. Paragonimus Westermanii. — This fluke is a common parasite of the lung of man in eastern Asia, causing haemoptysis and other symptoms. It occurs rarely in the brain, liver, intestinal walls, and elsewhere. The ova appear in the sputum, and in the rare intestinal cases in the fseces. Fasciola Hepatica, or liver fluke. — This is common in the liver of animals, but is very rarely found in the liver of man, excep- tionally in the lungs or elsewhere. The ova, appear in the fseces. Dicrocoelium lanceatum has been reported in the human liver about six times. Opisthorchis felineus and OpisthorcMs sinensis cause human liver disease in Asia. Distoma eonjunctum has also been found in the liver. Fasciolopsis Buskii, Heterophyes hetero- phyes, and Amphistoma hominis have been observed in the human intestine. Monostoinulum lentis and Agamodistomum ophthal- mobium have been found in the eye. Cestodes, or tapeworms. — These are parasitic under two con- ditions, namely, as adult forms or strobilae occupying the intes- tine, and as larval forms developing in the solid viscera or tis- sues of the body. Most tapeworms found in man occur only in the adult form ; two (Tsenia echinococcus and Sparganum Man- soni) occur only in the larval' stage ; one (Taenia solium) occurs in both adult and larval stage. The following cestodes have been found in man, in the stage mentioned : Taenia saginata : adult form. Taenia solium: adult form in intestine; larval form (cysti- cercus cellulosae) in muscles, brain, eye, and elsewhere. Taenia echinococcus: larval form (hydatids) in liver, lungs, and elsewhere. Taenia conf usa : adult form. Hymenolepis murina : adult form. Hymenolepis diminuta : adult form. Dipylidium caninum : adult form. Davainea madagascariensis : adult form. Diplogonoporus grandis : adult form. Dibothriocephalus latus : adult form. Dibothriocephalus cordatus : adult form. Sparganum Mansoni: larval form in subperitoneal tissue. Of these only the Taenia saginata (page 111) is common in Digitized by Microsoft® PARASITES. 243 this country. The other forms occur rarely or in distant coun- tries. Nematodes, or round worms. — These comprise a numerous and important class of parasites, of which the following are the chief well-established species found in man: Ascaris lumbricoides, common in the intestine, and occasionally found in adjacent cavities (page 112). Ascaris canis, occasionally found in the human intestine. Ascaris maritima, once found in the human intestine. Oxyuris vermicularis, a small, thread-like worm, common in the intestine (page 112). Dioctophyme renale (eustrongylus gigas) is a very large worm, 30 to 100 centimetres long, which in man has been found in the pelvis of the kidney in a few cases. The ova may appear in the urine. Strongylus longevaginatus has been found in the human lungs and stomach in two or three instances. Uncinaria duodenalis ( Ankylostoma duodenale) is an intestinal parasite causing extreme anaemia (page 112). Strongyloides intestinalis is an intestinal parasite causing diar- rhcea (page 113). Trichuris trichiura (Trichocephalus dispar) is a common but innocuous intestinal parasite (page 113). Trichinella Spiralis. — The adult forms of this worm, 1.5 to 3 millimetres long, occur in the intestine (page 113), where large numbers of living embryos are given off. These embryos make their way through the intestinal walls into the muscles of the body generally, where they form cysts (about .5 millimetre long) and produce severe disturbance. In the early stage of trichino- sis the worms may be found in the fseces; later the encysted trichinae may be found in portions of the muscle (preferably from the deltoid or biceps) removed for examination. Filaria Sanguinis Hominis. — The various forms of the filarise of the blood, namely, Filaria Bancrofti and its larval form Filaria nocturna, and the larval forms Filaria diurna, perstans, and De- marquaii, have been considered in connection with the blood (page 40). Filaria loa is not uncommon in Western Africa, occurring in the subcutaneous and subconjunctival tissue ; the Filaria diurna occurring in the blood is probably its larval form. Digitized by Microsoft® 244 A MANUAL OF CLINICAL LABORATORY METHODS. Other forms of filarite have iu isolated cases beeu noted in the human eye, mouth, respiratory passages, and elsewhere, as Filaria oculi, Filaria labialis, Filaria hominis oris, Filaria bronchialis, Filaria restiformis, Filaria lymphatica, Filaria volvulus, Filaria inermis, Filaria Magalhaesi, etc. Dracunculus medinensis, or Guinea worm, is a slender nema- tode sometimes reaching nearly a metre in length, that frequently produces subcutaneous abscesses in the tropical regions of Asia and Africa. Rhabditis Niellyi is an immature nematode allied to filaria observed in one case in cutaneous vesicles and the blood. Rhabditis pellio is a small nematode about a millimetre long that has been found iu the vagina and urine in two or three cases. Arthropoda. — The arthropoda parasitic in man belong to the classes arachnida, myriapoda, and insecta. These parasites in- flict bites or stings; inhabit the clothing, the surface, or the superficial cavities of the body; burrow into and beneath the skin ; or deposit their ova in the tissues or the superficial body cavities, where the larvse then develop. Arachnida, or spiders.— The most representative forms are as follows : Pentastoma twnioides is a mite common in the nasal cavity of dogs. It is occasionally found in the adult condition in the nasal fossa of man, where it occasions inflammation ; its larval form (Pentastoma denticulatum) has been often found in the human liver, less often in the lungs, kidney, spleen, and elsewhere, where it may not cause much trouble. An allied larval form, Pentastoma constrictum, has been seen in the human liver, lungs, and intestinal mucosa. Demodex fdlliculorum is common in the sebaceous glands about the face, and occasions little or no trouble. Sarcoptes scabiei, the itch mite, burrows under the skin, where it deposits the ova, and produces scabies. Other species of scabies infecting man are known. The ticl-s (Ixodes, argas) and the mites (leptus), including the chigger of the middle United States, attach themselves to or burrow into the skin, producing irritable sores. Some of the higher spiders and scorpions produce poisonous bites, rarely severe or dangerous. Digitized by Microsoft® PARASITES. 245 Myriapoda. — The centipedes may inflict poisonous bites, some- times severe or dangerous. Myriapoda are alleged to be capable of causing trouble by finding their way into the ear, nose, etc. Insecta. — Many varieties of insects are capable of inflicting painful and poisonous bites and stings, especially among the heteroptera, diptera, and hymenoptera. The pediculi (lice), cimex (bedbug), culicidse (mosquitoes), aphaniptera (fleas), are -well-known pests which irritate by their superficial bites. In another class of cases marked and even dangerous lesions are produced by the deposition of ova in the tissues or body cavities, and their subsequent development to the larval stage (maggots, etc.). In this manner the oestrus, flies (muscse), and especially the iMcUlia macillaria or screw-worm of the Southern States, are productive of abscesses, ulcers, inflammation, destruc- tion of tissues, etc. , especially in the nose or subcutaneous tissue. VEGETABLE PARASITES. The vegetable parasites of man are comprised in two broad classes, the Fungi and the Bacteria. The bacteria are of pre- eminent clinical importance, and the methods of their investiga- tion are briefly presented in the following chapter. Fungi. — The fungous parasites of man are much less common than the bacteria, though some of them produce serious lesions. Some of them distinctly produce lesions (pathogenic parasites), while others grow adventitiously in the tissues secondary to other conditions without themselves causing any trouble (sapro- phytic parasites). The growth of fungi in the tissues is often very different from their growth on media. Many of the patho- genic fungi, as the actinomyces and Oidium albicans, have not been definitely classified in the botanical system. Fungous growths in general consist of a vegetative portion, the mycelium, and a reproductive or spore-forming portion. The mycelium consists of a growth of fine filaments, interlacing, branching, or variously arranged. The spores are produced sometimes within the mycelial segments, sometimes in or on specialized structures. In many species and under propitious, conditions sexual spore-formation occurs from specially devel- oped structures. Fungous growths are examined and determined in the fresh condition, after staining, by cultivation on media, and by animal Digitized by Microsoft® 246 A MANUAL OF CLINICAL LABORATORY METHODS. inoculation. In fresh condition they may be examined by teas- ing them out in water or glycerin, by clearing away the adjacent material with liquor potassse, etc. They may be stained by the methods employed for bacteria, as Gram's stain, carbol-fuchsin, etc. ; many of them do not stain well. They may be cultivated on the ordinary bacterial media, as gelatin or agar, on moist ster- ilized bread, potato, etc. , or on special media ; many fungi grow better on acid than on-alkaline media. The chief varieties of fungi parasitic for man are as follows : Aspergillus. — Several species of this mould are parasitic and pathogenic for man, namely, Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, and Aspergillus nidulans. Aspergillus fumigatus is the commonest and most pathogenic, as it grows not only on free surfaces but is capable of producing serious le- sions of the internal viscera, while the others grow chiefly only on free surfaces. The commonest seat of growth of these fungi is the external auditory canal (otomycosis), where they form pseudo-membranes or plugs ; they have also been met with in the antrum of Highmore, the cornea, the anus, in the lungs as second- ary growths in tuberculosis and other wasting diseases, and else- where. In several reported cases Aspergillus fumigatus has caused primary pseudo-tubercular lesions in the lungs, and when its spores are injected into susceptible animals it is capable of producing disseminated and rapidly fatal lesions in the liver, kid- ney, lungs, and other viscera. The growth of these fungi in the ear consists of a luxuriant proliferation of mycelium, sporangia, and spores ; in the lungs and viscera the Aspergillus fumigatus grows chiefly as a mycelial network, rarely in ray form or with a tendency to sporulation. The mycelium of aspergillus consists of a network of branch- ing segmented filaments. The spores are developed on spherical, oval, or club-shaped bodies borne on a shaft or stalk projecting from the mycelium. From the surface of this spore-bearing head project minute branches (sterigmata), on which the spores are borne. Sclerotia, or dense rounded masses, are sometimes pro- duced. Aspergillus fumigatus : The growth is greenish, bluish, or grayish. The mycelial filaments are 2 or 3 /^ in diameter. The spore-bearing branches are short, with a shaft 5 a thick, a club- shaped head 8 to 20 ij. in diameter, bearing short unbranched Digitized by Microsoft® PARASITES. 247 projections or sterigmata about 6 a long, which are studded with smooth, rounded spores 3 to 4 //. in size. Sclerotia not observed. Aspergillus Jlavus : The growth is yellow or greenish. The spore-bearing head is borne on a shaft 4 millimetres long and 7 to 10 p. thick. The sterigmata are unbranched, often developed only on the upper half of the head. The spores are round, 5 to 7 ,a in size. The sclerotia are minute. Aspergillus niger : The growth is dark-brown. The spore- bearing head is spherical, up to 75^ in diameter, borne on a stalk up to 8 millimetres long and 10 to 15 /j- in diameter. The sterigmata are 20 to 100// long, branched at their extremities. The spores are very numerous, round, 3.5 to 5 /t in size. The sclerotia are about 1 millimetre in diameter. Eurotium glaucum and Eurotium repens are forms closely re- lated to aspergillus that have been found in some cases of oto- mycosis. They differ from aspergillus chiefly in their sexual sporulation structures, exhibiting perithecia instead of sclerotia. Mucor. — This genus somewhat resembles aspergillus macro- scopically and in its parasitic and pathogenic properties. The mycelial threads are little or not at all segmented. The sporan- gia or spore-bearing organs are rounded vesicles on a stalk; within the vesicles is an irregular mass of spores. A number of species parasitic in man have been observed, as Mucor corymM/er, Mucor septatus, the related Rhizomucor parasiticus, and other un- certain species. These fungi grow in the auditory canal, and have been observed a number of times in the lungs, usually in association with exhausting diseases like carcinoma. In one case a mucor caused a disseminated and fatal infection, produc- ing abscesses, ulcers, and other lesions in the pharynx, intestine, larynx, lung, and brain. The growth of mucor in the tissues may consist of both mycelium and spores. Verticillium Graphii and Penicillium minimum, forms somewhat similar to aspergillus, have been observed in cases of otomycosis. The various forms of tinea or ringworm are caused by two or three distinct kinds of fungi, the Microsporon Audouini, Tri- chophyton megalosporon endothrix, and Trichophyton mega- losporon ectothrix, beside occasional atypical related forms. To examine ringworm lesions for the fungi take a portion of the affected hair shaft or root, or scrape off a piece of the affected epidermis (previously if necessary macerated with a little liquor Digitized by Microsoft® 248 A MANUAL OF CLINICAL LABORATORY METHODS. potassse), and place it under the cover glass with a 5- to 20-per- cent solution of potassium hydrate ; examine with the microscope during and after the process of clearing. The fungi are easily grown on media. The ordinary staining methods (as Gram's) are not very satisfactory. Microsporon Audouini causes most of the cases of tinea tonsu- rans in children, and may produce lesions on the non-hairy skin differing from typical tinea circinata ; it does not affect the beard or nails or occur in adults. The growth on the hair consists of mycelium and spores. The spores are closely packed so as to form a layer on the outer surface of the hair^ beginning just above the bulb and extending upward ; they are round, with a double contour, and 2 to 3 /-t in diameter. The mycelial threads, about 2 iJL in diameter, slightly branching, are situated longitud- inally in the peripheral portion of the hair beneath the layer of spores. Just above the bulb the mycelium threads form a char- acteristic fringe projecting downward a little beyond the spores. The mycelial threads are divided into (usually) rather long seg- ments, each containing dark spots. Trichophyton megalosporon endothrix causes a considerable proportion of the cases of tinea tonsurans and tinea circinata ; it does not affect the beard or nails. It grows in the interior of the hair, in the form of longitudinal chains or rows of spores, some- times massed together so as to obscure the chain formation. The spores are quadrangular, oval, or rounded, 5 to 7 a in size. My- celium is not conspicuous, other than as longitudinal filaments breaking up into chains of spores. Trichophyton megalosporon ectothrix, derived from animals, is the cause of tinea sycosis, onychomycosis, a large proportion of cases of tinea circinata, and a few of those of tinea tonsurans. It may produce suppurative lesions. It grows chiefly as a sheath on the exterior of the hair root, perhaps invading the hair sub- stance to a slight extent (endo-ectothrix). Like the endothrix, it grows in longitudinal chains of spores 4 to 12 //. in diameter. Achorion Schoenleinii. — This is the fungus of favus, and has also been observed in the gastro-enteric tract. The crust of favus is composed almost entirely of this fungus, while the affected hairs may be extensively infiltrated with it. To ex- amine it, place a portion of the crust or affected hairs or epider- mis on a slide and cover it with a drop or two of liquor potassfe ; Digitized by Microsoft® PARASITES. 249 after a few minutes the fungus is ready for examination, when it may be covered with a cover-glass, or the potash solution may be removed with bibulous paper and replaced with glycerin. The fungus consists of a tangled network of branching my- celial threads mingled with large numbers of spores, all of a grayish or pale-greenish color. The mycelial threads are from 2 to 3 [J. in calibre ; some of them are simple and empty, others are divided into short segments and exhibit all stages of spore-for- mation. The spores vary in size from 2.3 to 5/ji, and also vary iQ shape, being round, oval, kidney -shaped, pyriform, dumb- bell form, etc. Several varieties of this species have been re- ported but not definitely established. Microsporon furfur grows in the horny layer of the epidermis, causing tinea versicolor. It appears as a luxuriant growth of spores, some single, many aggregated in clusters, in the midst of a mycelial network. The spores vary in size and shape, being round or oval, 3 to 8/* in size, very refractile. The mycelial threads are 1.5 to 4 ;a in diameter, straight or curved, and con- tain spores in places. To examine for the fungus scales scraped from the macules are placed under a cover-glass with liquor potassse. Microsporon minutissimum is the fungus of the skin disease erythrasma. It consists of a network of very fine mycelial threads divided into short segments, along with granular mate- rial. It is possibly a bacterium. Oidium Albicans, — The botanical place of this fungus is still uncertain, although numerous names and places have been as- signed to it. It grows most frequently on the mucous membrane of the mouth, forming the whitish membranes or aphthae of thrush. It also grows occasionally in the pharynx, oesophagus, vagina, vulva, and prepuce; it has been observed on the nip- ples of mothers nursing infected infants, and has in a very few cases been reported in the stomach, intestine, respiratory pas- sages, and lungs. It thus grows by predilection on mucous sur- faces lined with squamous epithelium. Zenker reported its oc- currence in the brain. The growth consists of segmented mycelial threads 2 to 6 /x in diameter, slightly granular, mingled with an abundance of clear, rounded or oval, spore-like (and also yeast-like) bodies 6 to 10 iJ- in size. These rounded bodies are partly free, partly grow Digitized by Microsoft® 250 A MANUAL OP CLINICAL LABORATORY METHODS. interposed between the long mycelial segments, partly grow out laterally from these segments in the form of buds or clusters. Actinomyces (ray fungus). — This fungus produces abscesses, chronic iniiammation, ulcers, tubercular growths, etc., in the parts infected; the lesions may occur in various parts of the body, especially the head and neck, tongue, lungs, abdominal vis- cera. The growth forms minute round, yellow granules, which are very characteristic of the pus or lesions. The centre of these bodies when fully developed consists of mingled mycelial fila- ments, minute spores, and granular material; outside this is a zone of filaments arranged radially; and next outside this are radiating club-shaped bodies, with their large ends outward ; the outermost portion consists of inflammatory cells. The fungus can be examined by crushing the granules under a cover-glass, or by hardening and sectioning the tissues affected. Specimens examined fresh show the clubs well, but not the mycelium. Addition of strong hydrochloric acid or liquor po- tassae, with the cover-glass then applied, shows the fungus well. Plardening in Miiller's fluid brings out the structure without further preparation. The parts of the fungus can also be de- monstrated by staining with haematoxylin and eosin, or by the ordinary bacterial stains. Gram's method shows the mycelium well. There are other fungous parasites and species closely related to ordinary actinomyces, among which is probably that causing Madura foot or mycetoma. Streptothrix. — This group embraces the "branching bacteria" and the actinomycetes. In a few instances pathogenic branching bacteria have been found, causing visceral abscesses, pseudo- tubercular lesions of the lungs, and other conditions. Saccharomycetes, blastomycetes, or budding fungi. — In this group the cellular, spore-like elements multiply by the growth of buds. The commonest and best-known representative of this group is the yeast fungus, or Saccharomyces cerevisise (Pig. 17). This consists of oval bodies, variable in size, 3 to 10 /* long, occurring singly or with two, three, or four united together in chains. They multiply by budding, and buds may be seen projecting from parent cells in all stages of development, from minute out- growths to a size equal to that of the parent. This organism has Digitized by Microsoft® PARASITES. 251 the power of producing the alcoholic fermentation of sugar. Yeast fungi are usually present in the stomach contents (at least after bread diet), frequently in the faeces, sometimes in the urine (especially in diabetics), producing no other evil effects than occasional fermentation. Fungi of the saccharomyces type (blastomycetes) have been found parasitic and pathogenic in a few cases, causing chronic inflammation and ulceration of the skin, endometritis, and ab- scesses and other lesions in the bones and viscera. lu cultures of these pathogenic yeasts on media mycelial threads may appear as well as the oval bodies. In the affected tissues only the budding oval forms are seen ; they stand out conspicuously on treatment of unstained sections with liquor potassse. Some of the parasites reported in cancer are of the saccha- romyces type, but their etiological relationship is not established. Leptothrix. — This is commonly classed among the bacteria and consists of a luxuriant growth of large, rod-like segments or ba- cilli growing end to end in long filaments. It is very frequently found in the mouth (Leptothrix buccalis), also in other situa- tions, and has little or no pathogenic power. In one case of puerperal septicaemia leptothrix was said to have been found in the blood during life. Digitized by Microsoft® XII. CLINICAL BACTERIOLOGY. The procedures employed in the deteinii nation of the pres- ence and kind of bacteria, in moj'Ijid conditions may be broadly classed as cultural methods, microscojvical methods, and animal inoculation. Sterilization.— .Alethitds of sierilization lie at the basis of bac- teriological investigation. Sterilization may be accomplished by means of dry heat, moist heat (boiling, steam, superheated steam) , chemical antiseptics, and filtration through porcelain. Fig. 35.— Hot-air Oven tor Sferilization. (Lentz & Sons.) Dry heat : Metal instruments may t)e sterilized by heating them a few moments in a Bunsen or alcohol flame; this injures steel, but for certain purposes it is convenient to have an old pair of scissors, kni^-es, etc., available for quick sterilization in this way. The platinum wire and loop are sterilized by heating the wire to a white heat, and then passing the glass handle a few times to and fro in the flame; platinum suffers no injury by this treatment. Glassware, as test tubes and Petri plates, may be Digitized by Microsoft® CLINICAL BACTERIOLOGY. 253 sterilized by heating in a hot-air oven, a large sheet-iron oven heated by a large gas flame beneath (Fig. 35), at a temperature of from 150° to 200° C. Boiling in water (or 1-per-cent sodium-carbonate solution) for five to fifteen minutes is a useful means of sterilizing instru- ments or glassware. Water and most solutions may be sterilized by boiling. Sterilization by steam is applicable to instruments, glassware, culture media, etc. Arnold's steam sterilizer is one of the most convenient for the purpose (Fig. 36). The objects to be steril- ized are placed in the steam chamber for one-half to one hour or more while the steam is gene- rated from below; the tempera- ture thus applied is slightly over 100° C, at the- ordinary atmos- pheric pressure. DiscontinuouH stenUzation is a method employed for culture media. The material is heated in the steam sterilizer once each day for three successive days, fif- teen to thirty minutes each time. Between the sterilizations the fig. 36.- culture medium is kept at a tem- perature of 20° to 37° C. in order to encourage the development of spores resistant to heat. Sterilization by steam under pressure, or superheated, is ac- complished by an apparatus called the autoclav ; one exposure of fifteen minutes under a pressure of one extra atmosphere (giving a temperature of 122 ° C. ) , is sufficient. Except that ster- ilization is completed in less time, the method ofi'ers no great advantages for laboratory purposes over the ordinary cheaper steam sterilizer. Antiseptics: Mercury bichloride in .1-per-cent solution is a useful antiseptic for the skin, glassware, or for saturating cloths used for temporarily wrapping organs from autopsy. Phenol in 2- to 5-per-cent solution is useful for sterilizing instruments, syringes, etc. Traces of chemical antiseptics may, if desired. -Arnold Steam Sterilizer. Castle & Company.) Digitized by Microsoft® 254 A MANUAL OP CLINICAL LABORATORY METHODS. be removed after sterilization by rinsing with clean sterile water. When it is desired to study the products of bacterial growth without the alterations which the use of heat or chemicals would produce, the bacteria may be removed by filtering the fluid con- taining the growth through a porous, unglazed porcelain filter, as Chamberland's filter. A. CXTLTTTRAL METHODS. Culture Media. — Artificial media of various kinds, fluid and solid, are employed for observing the mode of growth and chemi- cal products of bacteria. The chief culture media employed are bouillon, gelatin, agar, blood serum, milk, potato. Preparation of Culture Media. — These media in general are put up in test tubes in which they are safe against contamination. The best size for the tubes is about 15 by 1.8 centimetres. The tubes should be thoroughly cleaned with soap and water, or with the bichro- mate solution (page 5) if necessary. Old tubes containing material once used are first boiled to destroy the germs and dissolve out the media. Into the mouth of each tube is inserted a plug of non-absorbent cotton two or three centimetres long ; these plugs (unless wet) prevent the entrance of bacteria or spores of moulds, and it is Culture^ Tubes. 'IbIiuscS important that they be kept dry and un- * ^^^■'^ mixed with the culture media. It is cus- tomary to sterilize the empty tubes, thus plugged, before intro- ducing the culture media, either in the hot-air oven or steam sterilizer; this preliminary sterilization may, however, be dis- pensed with. For convenience in handling large numbers of tubes they are placed upright in wire baskets (Fig. 37), a layer of cotton in the bottom of which prevents breakage. Introduction of the various culture media into the empty tubes is much facilitated and expedited by the following, ar- rangement : One end of a short rubber tube is attached to a fun- nel and into the other end is inserted a short glass tube or nozzle drawn to a point. A pinchcock on the rubber tube enables it to be opened or closed at will. The funnel is placed upright in Digitized by Microsoft® CLINICAL BACTERIOLOGY. 255 a support and into it is poured a quantity of liquid or liquefied culture media. The cotton plugs are removed from the tubes, one by one, the tube is held beneath the funnel, the pinchcoek is held open long enough to allow 5 to 10 cubic centimetres of the medium to flow into the tube, and the cotton plugs are then re- placed. After depositing the requisite amount of culture media in the duly plugged tubes, they are sterilized, either in the ordinary steam sterilizer one-half hour each day for three days, or in the autoclav. Culture media if kept a long time before use are apt to dry out and become useless and wasted. This may be partly ob- viated by placing air-tight rubber caps (sold for the purpose) or tying oiled silk over the ends of the tubes. Or a quantity of the media may be kept in bulk in flasks, kept closed air-tight by oiled silk ; from these flasks tubes may be filled at intervals as required. Bouillon. — This is a fluid culture medium, useful for many purposes. By the introduction of special substances it is em- ployed for special purposes ; and gelatin and agar media consist practically of bouillon to which enough gelatin or agar has been added to make the material solid at ordinary temperatures. Bouillon consists 'essentially of a watery infusion of the princi- ples of lean meat ; to which it is customary to add ^ per cent of sodium chloride, 1 per cent of peptone, and sufficient alkali to make the fluid neutral or slightly alkaline. The best peptone for bacteriological purposes is Witte's dry peptone. Meat bouillon : Bouillon prepared directly from meat is prob- ably rather more nutritious and ef&cient than that made with meat extract, but is more troublesome to prepare. It is made as follows: 500 grams of lean beef or mutton free from tendon, fat, etc., is chopped fine and soaked in one litre of water in an ice box for twenty-four hours. At the end of that time all the liquid is pressed or squeezed out of the meat through a piece of muslin. In the fluid thus obtained 10 grams of peptone and 5 grams of pure sodium chloride are dissolved, and the whole is boiled in an enamelled iron pan until the albumin is entirely pre- cipitated. During the boiling the fluid is carefully and exactly neutralized or made slightly alkaline by adding a small amount of a strong solution of sodium hydrate or sodium carbonate, until the proper reaction, as shown by litmus paper, is attained. Digitized by Microsoft® 256 A MANUAL OF CLINICAL LABORATORY METHODS. At the conclusion of the boiling, sufficient water is added to make the amount up to a litre, the fluid is filtered, introduced into the tubes (or a flask for storage), and sterilized in the steam sterilizer by the discontinuous method. Meat-extract bouillon : The commercial meat extracts, as Lie- big's or Armour's, are often used in the preparation of bouillon, being easier to use and for most purposes as efficient as the meat bouillon. About 3 grams of meat extract (Liebig's), 5 grams of sodium chloride, and 10 grams of peptone are boiled in a quan- tity of water, made neutral or slightly alkaline, water is added to make 1 litre, and the material then filtered, tubed, and sterilized. Glucose-bouillon is the same as ordinary bouillon, with either one or two per cent of glucose added. This medium is used in testing the fermentative power of bacteria, and also where the added nutritive action of glucose is desirable. Other sugars, as lactose, sucrose, may be used instead of glu- cose, to test fermentative action on them. A few drops of a strong aqueous solution of crude litmus may be added, coloring the bouillon blue, so that the formation of acid by the bacterial growth may be directly shown by reddening of the fluid. For some purposes slightly acid bouillon, or bouillon made to a defi- nite alkaline or acid strength by titration with phenolphthalein, is used. Gelatin. — This consists of bouillon to which 10 per cent of gelatin has been added. It is solid below about 25° C, liquid above that temperature ; it cannot be used in the culture oven, and in summer cannot be easily managed. It is employed for isolating bacteria in plate cultures and for testing the liquefying action of bacteria. IsTutrient gelatin may be prepared with either meat bouillon or meat-extract bouillon. To a litre of the boiling bouillon con- taining . 5 per cent of sodium chloride and 1 per cent of peptone, add 100 grams (per litre) of the best gelatin (as the French "gold-label" brand), and stir constantly until well dissolved. Neutralize in the usual manner, boil vigorously for ten to fifteen minutes (until albumin is entirely precipitated), and add water enough to make one litre. If upon trial it is now found that the material will filter per- fectly clear, it may be filtered without further treatment ; but if it does not filter clear, it should be clarified, as follows : Cool the Digitized by Microsoft® CLINICAL BAGTEEIOLOGY. 257 liquid below 60° C. and mix with it an egg which has been thor- oughly beaten with 50 or 100 cubic centimetres of water. Then boil the mixture for five or ten minutes, without stirring, so that the egg albumen will coagulate in large flakes and collect the fine suspended particles. The large coagula may be removed with a skimmer, but breaking them up into fine particles should be avoided. The material should after this second boiling be made up to a litre. Filtration, the next step, presents some difficulties. Two or three thicknesses of filter-paper should be used, being folded into ridges to increase the filtering surface, or a wire filtering frame being employed. The paper is thoroughly moistened, and a por- tion of the melted gelatin poured in carefully or allowed to flow gently down a glass rod. As the fluid cools, filtration is apt to be retarded, but may be again promoted by adding another por- tion of the material hot and fresh from the flame. By thus add- ing the hot gelatin in successive portions filtration can be usually accomplished at the room temperature ; but if difficulty is ex- perienced, the filter, supported on a tripod with the flask con- taining the filtrate beneath, may be placed in the steam sterilizer until filtration is complete. The gelatin is then introduced into tubes, sterilized in the steamer fifteen minutes daily for three days, and then set aside in an upright position to allow the gelatin to harden, a depth of about 5 centimetres being in each tube. Gelatin should not be heated longer than necessary, as excessive heating lowers its solid- ification point. After sterilization the gelatin media should re- main clear. As in the case of bouillon, special modifications of gelatin may be made, as the addition of 1 or 2 per cent of glucose, litmus, etc. Agar. — This is one of the most useful bacteriological culture media, and is employed for a great variety of purposes. It con- sists of bouillon to which is added 1.5 per cent of agar-agar, an Oriental alga rich in vegetable gelatin. This culture medium solidifies at about 40° C. ; it can therefore be employed for grow- ing bacteria at body temperature, and bacteria can be introduced into it while melted without injuring their vitality. To prepare a litre of nutrient agar, to about two litres of water add 3 grams of meat extract, 5 grams of sodium chloride, 17 Digitized by Microsoft® 258 A MANUAL OF CLINICAL LABORATORY METHODS. 10 grams of peptone, and 15 grams of agar-agar fibres cut into fine pieces ; tliese ingredients are best added and dissolved sepa- rately. If meat bouillon is preferred, dilute the meat infusion about twice, salt, peptone, and agar being added in the same amounts. Bring the mixture to a boil in an enamelled iron pan, and boil it vigorously for half an hour or until the excess of water is evaporated. Stir the mixture until the ingredients are dissolved, and afterward neutralize in the usual manner. From time to time remove with a skimmer the scum that forms. Pro- longed and vigorous boiling is necessary to obtain a medium clear of precipitate. It is well to have a mark on the side of the pan to show the level of one litre. After boiling a sufficient time clarify the liquid with an egg, if necessary, in the same manner as with gelatin. The amount being brought by the addition of water to one litre, filter exactly as in the case of gelatin. Introduce into tubes, sterilize thirty minutes daily for three days, and after the third sterilization lay the tubes in an inclined position until the agar hardens in the form of a slant extending from the bottom of the tubes nearly to the cotton plugs. If a precipitate appears after sterilization the medium must be again melted, boiled, and filtered. Glucose agar is the same as plain agar with 1 or 2 per cent of glucose added. Other sugars may be used instead, and litmus may be added. Lactose-litmus-agar (lactose 2 or 3 per cent) is sometimes used. Glycerin agar contains 6 per cent of glycerin, which is added after the agar is filtered, just before the material is tubed and sterilized. Blood Serum. — The coagulated serum of the blood of cattle (and of the human subject when obtainable by venesection, etc.), affords a very nutritious culture medium, especially use- ful for certain bacteria that do not grow well on other media, and hence well adapted to post-mortem bacteriological examinations. It is thus prepared : At the slaughter-house a tall glass jar is filled with blood fiowing from the freshly cut carotid artery. Eigid asepsis is unnecessary. The jar is set aside for a few minutes to allow firm clotting to take place, and it is then placed in an ice box for from twenty-four to forty-eight hours for the clot to contract and the serum to separate ; during this period the jar should be handled or shaken as little as possible, to avoid disar- ranging the clot. The clear, straw-colored serum is then care- Digitized by Microsoft® CLINICAL BACTEKIOLOGY. 259 fully decanted or drawn off wltli a pipette, and introduced into plugged test tubes, 5 to 10 cubic centimetres in each. The tubes are then placed in a hot-air oven or special coagulating oven in an inclined position, so that the serum forms a slanting surface from the bottom to the upper part of the tube. The temperature of the oven is brought up to about 90° C. and maintained there until the serum in the tubes is firmly coagulated throughout; ' constant attention will be required to see that the temperature does not reach the boiling-point, since in that case the serum will bubble and its surface when coagulated will be rough and broken instead of smooth. After being thoroughly hardened, the tubes may be placed upright, and are sterilized in the steam sterilizer in the usual way. The application of tight rubber caps until the tubes are used will prevent evaporation and preserve them longer in good condition. Loeffler^s blood serum, for the cultivation of the diphtheria ba- cillus, is prepared by mixing 3 parts of beef -blood serum with 1 part of glucose (1 per cent) bouillon. The mixture is tubed, coagulated, and sterilized as is plain blood serum. Milk is used chiefly to test the acid-forming and casein- coagulating properties of bacteria. As usually employed litmus is added to reveal directly the reaction. To fresh milk, with the cream removed, is added a small amount of strong aqueous lit- mus solution to color it light blue ; it is then placed in test tubes, a few cubic centimetres in each, and sterilized by the discontin- uous method. Potato is sometimes employed as a culture medium to aid in differentiating certain bacteria. The potatoes are washed and pared; cylindrical pieces of sufficient size to fit into the test tubes are cut out or punched out, and each cylinder is divided into two parts by a longitudinal oblique cut ; one part is placed in each tube, so that a surface slanting upward is presented. The piece of potato should be raised slightly above the bottom of the test tube to leave a space for surplus water to collect; this may be accomplished by placing a short piece of glass rod or tube in the bottom of the tube to hold up the potato, or by the use of special tubes with a constriction near the lower end. Inoculating Culture Media.— For inoculating or planting upon the culture media the bacteria desired to be grown, the "plati- Digitized by Microsoft® 260 A MANUAL OF CLINICAL LABORATORY METHODS. num needle" and the "platinum loop" are used (Pig. 38). These consist of a piece of platinum wire attached to one end of a glass rod as a handle. The glass rod serving as a handle should be about 6 millimetres in diameter and 15 to 18 centimetres long. To attach the wire, heat the end of the rod to redness, push one end of the wire into the soft glass for a short distance, and allow it to harden. The platinum wire should be about ^ mil- - ^ limetre in diameter and 4 to 7 centimetres long. The " needle " has the wire straight, the " loop " has a loop 1 or " oese " about 2 millimetres in diameter at the end. i Just before making inoculations with the needle or loop it is sterilized by heating it to a white heat in the flame, and passing the glass rod a few times to and fro in the flame ; before use about ten seconds should be allowed to elapse, to allow the wire to cool sufficiently not to kill the germs picked up. The loop or the end of the needle is then touched to the bacterial growth or material to be inoculated ; the wire is passed into a culture tube, the plug being temporarily removed and held by its outer end be- tween two fingers, and the material adherent to the loop or needle is deposited in or on the surface of the culture medium, avoiding contact with the sides I of the tube, after which the plug is replaced. Before 1 inoculating them, it is well to heat the upper half J U of the culture tubes and flame the plugs. After in- oculation, tubes should be labelled for identifica- tion. Inoculated tubes should be kept upright, which is conveniently accomplished by standing them in tin boxes or ordinary drinking-glasses with a layer of cotton in the bottom. In transplanting a growth from one tube to a fresh tube, the two are held upright side by side in the left hand, and their plugs removed and held by their outer ends between the sides of the disengaged fingers of the left hand. The platinum wire, held in the right hand, is sterilized, passed into the fertile tube, and a small portion of the growth taken on the end of the wire ; the wire is then withdrawn, passed into the fresh tube, and this in- oculated, after which the plugs are replaced. FIG. 38.— Plati- num Inocu- lating Needle and Loop, in fla£S bandies Digitized by Microsoft® CLINICAL BACTERIOLOGY. 261 In inoculating liquid media (bouillon, milk), the loop or needle is passed into the fluid and stirred around to dislodge the bacteria and mix them with the culture medium. Inoculations on the surface of solid media (agar slants, blood serum, potato) are made by drawing the loop or point of the needle, charged with bacterial material, over the surface. Streak cultures are those made by drawing the end of the charged needle over the surface of the medium in a straight line, so that the resulting bac- terial growth develops in a line or streak. Stab cultures are made in the depths of solid media, usually gelatin, by plunging the needle, with its end charged with bacteria, for a few centimetres in a straight line into the medium and then withdrawing it along the same line. Occasionally the culture tubes are inoculated otherwise than with the platinum wire, as by rubbing bits of tissue, held in sterile forceps, over the surface of solid media, or by dropping pieces of tissue or of ligatures, dressings, etc., whose sterility is in question, into liquid media. Swabs made of cotton twisted on the end of a wire, kept in a plugged test tube and so sterilized, are sometimes used for making inoculations, as in examining for diphtheria bacilli. Growth of Cultures. — After inoculation, a variable time is re- quired for the development of colonies of bacteria, usually from one to three days. Proper conditions of temperature, access of oxygen, etc. , must be afforded to obtain the optimum growth. As to temperature, cultures are ordinarily grown either at the room temperature, or in an incubating or culture oven at a tem- perature of 37° C, or body heat. Most pathogenic germs grow better or more rapidly at 37° C. Gelatin cultures can be grown only at room temperature, below 25° C. ; other media can be used at any temperature. Culture ovens (Fig. 39) are specially constructed for the cul- tivation of bacteria. These ovens have jacketed walls, and are surrounded with a space filled with water. Heat is applied by a small gas flame beneath, provided with an automatic cut-off which stops the flow of gas should the flame be accidentally ex- tinguished. By means of an adjustable regulator, or "thermo- stat," the flow of gas is so controlled that any desired temper- ature can be constantly maintained. A thermometer passing through the top of the apparatus registers the temperature. Digitized by Microsoft® 262 A MANUAL OF CLINICAL LABORATORY METHODS. Some bacteria {aerobic) grow only (obligate aerobic) or best (facultative) iu the ijreseuce of oxygen ; others (anaerobic) grow only (obligate anaerobic) Ji^-^ioiL or best (facultative) when fi'ee oxj'geu is excluded. As ordinarily grown cult- ures ai'e aerobic ; anaero- bic cultures require spe- cial methods (see below). Isolation of Bacteria in Pure Culture. — ^^^lcll dif- ferent kinds of bacteria are mixed or supposed to l)e mixed together in any juateifal to be in-\'esti- gated, it is in general nec- essary to separate or iso- late each kind in pure culture in order to inves- tigate or determine the various kinds present. In some cases bacteria (Eiraer & pau be isolated by animal inoculation, or by the use of special culture media or conditions which favor the gro-n-th of some A'arieties at the expense of others. The isolation of bac- teria is usually, however, accomplished by "plate cultures," also by