CORNELL UNIVERSITY. THE THE GIFT OF ROSWELL P- FLOWER FOR THE USE OF THE N. Y. STATE VETERINARY COLLEGE 1897 CpHNELL UNIVERSITY UBRARY 3 1924 104 224 872 Cornell University Library The original of tiiis book is in tine Cornell University Library. There are no known copyright restrictions in the United States on the use of the text. http://www.archive.org/details/cu31924104224872 PATHOLOGICAL TECHNIQUE A Practical Manual for Workers in Pathological Histology and Bacteriology ^^^^^=^:^^=^= including ■ Directions for the Performance of Autopsies and for Clinical Diagnosis by Laboratory Methods BY FRANK BURR MALLORY, A.M., M.D. Associate Professor of Pathology, Harvard University Medical' School ! First Assistant Visiting Pathologist to the Boston City Hospital ; Ai:. Pathologist to the Children's Hospital / . % • J*: M. m < t '%i4is,^^j^. ^ V . Fig. 36. — Pneumococci with capsules in a cover-glass preparation from sputum stained by Gram's method and Bismarck brown ; X 2000 (V^^right and Brown). This is composed of a mucin-like substance. It may be seen usually in cover-glass preparations stained by the ordi- nary methods, especially if the preparations be examined in water-mounting. I40 PATHOLOGICAL TECHNIQUE Stained by Gram's method. Not motile. Glycerin Agar-agar. — Feeble growth of very minute gray- ish colonies. Bouillon. — Clouded faintly. Litmus-milk. — Sometimes turned pink and coagulated. Growth on other culture-media is very feeble. The organ- FlG. 37, — DiplococcTis pneumoniae ; cover-glass preparation from the heart's blood of a rabbit; X 1000 (Frankel and Pfeiffer). ism dies out rapidly in cultures. To keep it viable it should be transplanted every forty-eight hours. Pathogenesis. — The pneumococcus is very pathogenic for mice and rabbits, less so for guinea-pigs. Subcutaneous inoculation with virulent cultures causes the death of mice in from twenty-four to thirty-six hours, and of rabbits in from thirty-six to forty-eight hours, with septi- cemia. This infection is the " sputum-septicemia " of Sternberg. At the autopsy there will be found in the blood everywhere the characteristic encapsulated lancet-shaped organisms, usu- BACTERIOLOGICAL METHODS. I4I ally in pairs (Fig. 37). Great variation in the virulence of the organism is observed. In some cases no effect will be produced by the inoculation ; in others a more or less exten- sive fibrino-purulent exudation will be produced about the point of inoculation, and the animal will survive for a con- siderable length of time or recover. Inoculation into the ear-vein or peritoneal cavity of a rabbit will sometimes cause a rapidly fatal septicemia, when subcutaneous inoculation with the same culture will only cause a local reaction. The viru- lence of the pneumococcus is quickly lessened by cultivation. Occurrence. — The pneumococcus may be demonstrated in the pulmonary exudate of practically all cases of genuine lobar or croupous pneumonia. At autopsies on cases of this disease it may be found in large numbers in the consolidated lung, and sometimes in smaller numbers in the blood of other internal organs. Cultures from the lung may some- times show the presence of other bacteria in addition to the pneumococcus, but these are to be regarded as either second- ary infections or contaminations from the smaller bronchi. The pneumococci in the pneumonic exudate die in large numbers after a time, and in cases near resolution numerous capsules may be found in cover-glass preparations from the lung in which it is impossible to demonstrate the organism by staining methods. The pneumococcus is also frequently found in broncho- pneumonia, acute peri- and endo-carditis, acute pleuritis and empyema, acute purulent meningitis, and in otitis media. In cases of pneumonia and bronchitis it may be present in the sputum in large numbers. It has been observed in cases of peritonitis, of synovitis, of osteomyehtis, and of abscess-forma- tion in various .situations. At autopsies on individuals dead of these conditions it may be frequently found, by means of cultures and animal inoculations, generally distributed throughout the internal organs in variable numbers. It is also often present in the mouth and in the saliva of healthy individuals. Diagnosis. — If the pneumococcus be present in very small numbers in pathological material, the quickest and most 142 PATHOLOGICAL TECHNIQUE. certain method of demonstrating its presence is the inocula- tion of a mouse with some of the material (see page 119). This is also the best way to prove the identity of the organ- ism. The pneumococcus can usually be identified in exudates, blood, tissues, or sputum by examination of cover-glass preparations alone, by reason of its peculiar morphology and its possession of a capsule. The capsule can be seen in most instances in cover-glass preparations, stained in the usual manner, if they be examined in water-mount. The capsules appear as a hyaline material usually with definite outlines surrounding the paired organisms. If it be desired to stain the capsules the following methods may be used. Their working is somewhat uncertain. Methods of Staining the Capsule of the Pneumococcus. — Welch's Method. — This method depends upon the fact that acetic acid precipitates the mucin-like substance of which the capsule is composed, and that the precipitated material of the capsule is not soluble in a 2 per cent, solution of sodium chlorid. Therefore, any necessary washing of the preparation is to be done in the solution of sodium chlorid, and the mount is to be made in the same solution or in balsam. The method is as follows : 1. Cover the preparation with glacial acetic acid for a few seconds. 2. Drain off and replace (without washing in water) with aniline-gentian-violet solution. The staining solution is to be repeatedly added to the surface of the cover-glass until all of the acid is replaced. 3. Wash in a 2 per cent, solution of sodium chlorid and mount in the same. W. H. Smith's Method. — This has been found particu- larly useful in demonstrating the pneumococcus in the -Sputum. The sputum or other material should be fresh. The cover-glasses should be spread as thinly as possible and fixed by passing three times through the flame in the usual manner. BACTERIOLOGICAL METHODS. I43 1. Stain in aniline-gentian-violet solution for a few seconds, gently warming until the staining fluid steams. 2. Wash in water. 3. Cover with Gram's solution of iodin for thirty seconds. 4. Wash with 95 per cent, alcohol until the color ceases to come out. 5. Wash with ether for a few seconds. (To remove fat.) 6. Wash in absolute alcohol for a few seconds. 7. Stain one to two minutes in a saturated aqueous solu- tion of eosin. 8. Wash with absolute alcohol for a few seconds. 9. Clear with xylol. 10. Mount in balsam. The pneumococcus is stained blue-black, while the capsule is stained pink. This method gives beautiful preparations. With the following modification it has been used by Smith as a routine stain for sputum. The advantage of this modifica- tion is that influenza bacilli and other bacteria which do not stain by Gram's method are clearly brought out, as are also eosinophilic leucocytes. This modification consists in wash- ing the preparation with Loffler's alkaline methylene-blue solution just after it has been stained with eosin, as described above, and then, after the excess of eosin has been removed by the methylene-blue, steaming the methylene-blue solu- tion for a few seconds while on the cover-glass. The prepa- ration is then washed in water, rinsed with alcohol, cleared with xylol, and mounted in balsam. Streptococcus Capsulatus. — This seems to be the best name to apply to a capsule-bearing bacterium resembHng both the pneumococcus and the streptococcus pyogenes in morphology, but differing definitely from them in cultural and other peculiarities. It has been found chiefly in lobar pneumonia, but occurs in other inflammatory processes and probably has been sometimes mistaken for the pneumococcus or the streptococcus pyogenes. The lack of general recog- nition of this micro-organism as a separate and distinct species vitiates, to some extent, the value of statistical studies of infections with the streptococcus and the pneumococcus. 144 PATHOLOGICAL TECHNIQUE. The best description of the streptococcus capsulatus is that of Oscar Richardson/ based upon observations on 4 cases of lobar pneumonia in the laboratory of the Massa- chusetts General Hospital. Richardson points out the fol- lowing chief characteristics by which it may be distinguished from the pneumococcus and the streptococcus pyogenes : Fig. 38. Fig. 39. Fig. 40. Fig. '^. — Pneumococcus ; blood-serum culture. Fig. 39,— Streptococcus capsulatus ; blood-serum culture. Fig. 40. — Streptococcus capsulatus ; glucose-agar stab culture (Oscar Rich- ardson; photos by L. S. Brown), 1. The capsules persist in cultures (see Fig. 41). 2. On the surface of coagulated blood-setum its colonies are flat, colorless, viscid, mucus-like, of irregular outline, and may attain a diameter of 2 or 3 mm. They may become confluent and form large patches of mucus-like material (see Fig. 39)- 3. In glucose-agar stab, adju.sted to a reaction of 0.5, there is growth all along the line of inoculation, from which, in 1 " Pseudopneumococci in Lobar Pneumonia," Journal of, Boston Society of Medical Sciences, vol. v. , No. 2, p. 499. BACTERIOLOGICAL METHODS. 145 places, fusiform or hemispherical masses of growth extend into the surrounding medium in a vertical plane, apparently occupying clefts in the medium (Fig. 40). It is very im- portant for the development of these characteristic appear- ances that the glucose-agar be known to have at the time of inoculation a reaction very close to that above indicated. • m % '5 '. '^i; > ■^ f \ • • • ', '1' ~: * ^ » u Fig. 41. — Streptococcus capsulatus from culture (Oscar Richardson; photo by L. S. Brown). Gonococcus." — Morphology . — Cocci of medium size, coin- posed usually of two hemispheres separated by a narrow unstained interval. Sometimes two of these pairs of hemi- spheres are joined together in the manner of " tetrads," or groups of four, showing evidence that division occurs in two directions at right angles to each other (Fig. 42). Decolor- ized by Gram's method. The gonococcus will not grow satisfactorily upon any of the culture-media ordinarily employed, but requires special media for its cultivation. The colonics on suitable culture-media appear after eigh- teen to twenty-four hours as minute, grayish, translucent points. Later they may attain a diameter of 2 mm. Under ' Neisser : Centralbl. f. ..»«! 1 Fig. 52. — Diphtheria bacilli from a culture on blood-serum, stained by Loffler's methylene-blue solution, showing long and irregularly shaped forms of the bacillus, as well as the irregularity of staining ; X 2000 (Wright and Brown). , tion of the media rapidly becomes acid, but changes to alka- line after a variable length of time. Potato. — Growth not visible to the naked eye. The bacil- lus grows, however, to a certain extent, and usually assumes very atypical and irregular forms (involution forms). Agar-agar and Gelatin. — The growth on these media is slower and more feeble than upon blood-serum (Fig. 53). It presents nothing remarkable. Pathogenesis. — Subcutaneous inoculations of guinea-pigs BACTERIOLOGICAL METHODS. 1 59 are fatal in thirty-six to seventy-two hours in the case of virulent cultures. The lesions produced consist usually of edema, hemorrhage, and fibrino-purulent exudation about the point of inoculation in the subcutaneous tissue, hemor- rhagic enlargement of the lymphatic glands, congestion and Y- edema of the lungs, hemorrhages into the suprarenal cap- FlG. 53. — Bacillus diphtherias; agar-agar culture (photograph by Dr. Henry Koplik). sules, and less frequently necrosis of the liver and pleural effusions. Histological examination of the lymph-glands shows marked " fragmentation " of the nuclei of the cells, giving rise to numerous deeply staining globules of chroma- tin scattered throughout them. The bacilli are ordinarily found only about the point of inoculation, but cultures from the various organs will sometimes show the presence of the bacilli in some of them. l6o PATHOLOGICAL TECHNIQUE. Toxin-production. — The effects produced by infection with the bacillus diphtherise are due to the action of a so-called toxalbumin or " toxin " which the organism manufactures in its growth. The poisonous substance is produced in cultures. Its presence may be demonstrated by inoculating an animal with a small quantity of the filtrate, obtained by passing a bouillon culture some weeks old through an unglazed porcelain filtering apparatus, by which all the bacteria are removed from the fluid. The "toxin" is contained in solution in the filtrate. If this be fairly rich in "toxin," the injection of -^ c.c. subcutane- ously into a guinea-pig should lead to the death of the animal in three or four days with the various lesions above described. The local reaction, however, is not so marked as in the case of inocu- lation with the bacilli. With the ordinary bouillon the produc- tion of a great amount of " toxin " by the growth of the diph- theria bacilli in it is very uncertain. Theobald Smith has recently shown that this uncertainty is due to the presence of variable amounts of muscle-sugar from the meat used in the preparation of the bouillon, and that this substance prevents the accumulation of toxin. He has found that that bouillon yields the most toxin which has the least muscle-sugar in it. He prepares such bouillon as follows : " Beef infusion, prepared either by extracting in the cold or at 60° C., is inoculated in the evening with a rich fluid culture of some acid-producing bacterium (I use temporarily B. coli) and placed in the thermostat. Early next morning the infusion, covered with a thin layer of broth, is boiled, filtered, pepton and salt added, and the neutralization and sterilization carried on as usual." This bouillon is placed in two 500 c.c. Erlenmeyer flasks, 250 c.c. in each flask. In these, cultures are made and kept for at least eight days in the incubator. After this time a fair amount of toxin may be assumed to have developed, and the contents of the flask are then filtered through a porcelain cylinder. A filtrate is to be regarded as containing a reasonable amount of toxin if ^ c.c, injected subcutaneously, kills a medium-sized guinea-pig in three days. The filtrate containing the "toxin" can be preserved by the addition of 0.5 per cent, pure carbolic acid. Occurrence. — The bacillus diphtheriae occurs in the local lesions in all cases of true diphtheria, in rhinitis fibrinosa, and in many cases of the milder forms of acute inflammation of the air-passages. It may persist in the mucous mem- brane of the throat and nose long after convalescence has been established. In fatal cases of diphtheria the organism is nearly always present in the lungs, and it may be often found by culture- methods more or less generally distributed in compara- BACTERIOLOGICAL METHODS. l6l tively small numbers throughout the internal organs. In the majority of diphtheria autopsies an invasion of the blood- stream by the streptococcus pyogenes, and sometimes by other bacteria, may be demonstrated by cultures. The ba- cillus may also be found in company with other bacteria in ulcerated or excoriated surfaces on the skin, as well as in other suppurative processes, in individuals affected with diphtheria, and on the soiled linen of the patient. The in- fection of wounds with the bacillus diphtheriae has also been observed without coincident diphtheria. Diagnosis. — The bacteriological diagnosis of infection with the bacillus diphtheriae depends upon the characteristic morphology and peculiarities of staining, as well as rapidity of growth, which this organism presents when cultivated upon coagulated blood-serum. The identification by direct cover-glass examination of the exudate is very uncertain. The method is as follows : A blood-serum culture-tube is inoculated with a small amount of the material from the mucous membrane affected, and is placed in the incubator twelve to eighteen hours. After this length of time the re- sulting growth is examined by cover-glass preparations stained either with Loffler's methylene-blue solution or by one of the special methods given below. The bacillus diphtheriae, if present, may then be recog- nized and differentiated from other bacteria present in the preparation by its characteristic morphology and peculiarity of staining, described on page 157. The gross appearances of the culture present little that is characteristic, as a rule, and the main reliance is to be placed on the microscopic ex- amination. Early in the infection the greater part of the growth may be made up of the specific bacilli, but toward convalescence they fall into the minority. The ordinary forms of agar-agar culture are not suitable for use in the bacteriological diagnosis of diphtheria, owing to the com- parative feebleness of the growth of the organism on these media, and because of the fact that its microscopic appear- ances when cultivated on such media are not sufificiently characteristic. 11 l62 PATHOLOGICAL TECHNIQUE. The material for culture is very conveniently obtained by means of sterilized cotton swabs. In collecting this material the swab is removed from its test-tube and touched to the affected areas of the mucous membrane of either the nose or throat. It is then to be gently rubbed over the surface of a blood-serum culture-tube, or it may be replaced in the test- tube and the inoculation of the culture-tube made later in the laboratory. In the latter case the inoculation should be made within an hour or two after the material has been col- lected, the infected swab meanwhile being prevented from drying by firmly replacing the cotton plug. In cases with membrane-formation the greatest number of bacilli are on the surface or in the upper layer of the membrane, and the swab should therefore be touched to these portions rather than to the tissue beneath. Special Methods of Staining the Bacillus Diphtherise. — Owing to the fact that the bacillus diphtheriae may be recognized by its pecuUar morphology and characteristic staining in cover- glass preparations from its growth upon certain culture-media, as already pointed out, various special staining methods have been devised for accentuating and rendering more striking to the eye the peculiar deeply stained points and granules in the bodies of the individual bacilli, which have been referred to as of great im- portance in the identification of the organism. These special methods of staining are said to be of great advan- tage in cases where only a few specific bacilli may be suspected to be present among a large number of other bacteria. N'eisser' s Method. — i. Stain for one to three seconds in a solu- tion which is made as follows : i gram of methylene-blue (Grue- bler), in powder, is dissolved in 20 c.c. of 96 per cent, alcohol. To this add 950 c.c. of distilled water and 50 c.c. of glacial acetic acid, and filter. 2. Wash in water. 3. Stain for three to five seconds in a solution of vesuvin (Bismarck brown), made by dissolving 2 grams of the dye (in powder) in 1000 c.c. of boiling distilled water. 4. Wash in water, and mount. The diphtheria bacilli stained by this method appear as pale brown rods bearing bluish-black granules, usually of oval shape and of a diameter somewhat greater than the rod. The majority of the bacilli show a granule at each end or at only one end, but not rarely three granules are present, one being near the middle of the rod. More granules than these are exceptional (see Fig. 54). The bacilli must have been grown on Loffler's blood-serum BACTERIOLOGICAL METHODS. 1 63 medium, coagulated at 100° C. , and the culture must be at least nine hours and not more than twenty-four hours old. Hunf s Method. — i. Stain in saturated aqueous solution of methylene-blue one minute without heating. 2. Wash in water. 3. Cover with aqueous solution of tannic acid, 10 per cent., for Ten seconds. 4. Wash in water. 5. Stain in saturated aqueous solution of methyl-orange one minute, without heating. 6. Wash in water. 7. Dry, and mount in balsam. % - ■-.■« -w • • • • * •• «r . • • .••' ' • • 'S7j3^H % • •• , • • • •' • • • , • . >'^ • . • • t % ^ • • « « .* • ,» • - . '..-. ■ •••# ■ * , % # • • . . •^ « ■ • « *■ . • • " • .' • » • «•• .' .< .. • ./ ». • * " • * • • . ' • -|H -. • * . • % • ., • # • • • • • • • • • . . Ji Fig. 54- — Diphtheria bacilli from blood-serum culture stained according to Neisser's method ; X 2000 (Wright and Brown). By this method the granules, etc. , are dark blue or almost black, and stand out very sharply against the light-green coloring of the body of the bacillus (see Fig. 55). The solution of methyl-orange should be freshly prepared, for it deteriorates in a few days. Bacillus of Typhoid Fever.' — Synonyms: Bacillus typhi abdominalis ; Bacillus typhosus ; Typhoid bacillus. 'Eberth: Virckow's Arch. f. Path. Anat, 'BA. 81, 1880; Bd. 83, 1881; Gaffky : Mitth. a. d. Kais. Gesundheitsamte, Bd. 2, 1884. 164 PATHOLOGICAL TECHNIQUE. Blood-serum. — Round, grayish, viscid-looking colonies, which may attain a diameter of 2 mm. after forty-eight hours in the incubator. ^, ^ Fig. 55.— Diphtheria bacilli from blood-serum culture stained according to Hunt's method ; x 2000 (Wright and Brown). Morphology. — Medium-sized bacilli with rounded ends, generally short (Fig. 56), but sometimes long or thread-Hke, V /■ "^.^^ :% Fig. 56. — Typhoid bacilli from a bouillon culture, showing characteristic irregu- larity in staining and variability in length ; X 2000 (Wright and Brown). and frequently showing faintly-stained, sharply-defined areas in their protoplasm (Figs. 56 and 57). BACTERIOLOGICAL METHODS. 165 Gelatin Slant. — Broad translucent streak with wavy, irregular margins. The gelatin is not liquefied. Growth is w """^^ . "^ - « fh ^ 1 • i ^ % « •^ Fig. 57. — Typhoid bacilli from a culture on potato, showing unstained areas in the bacilli and polar granules ; X 2000 (Wright and Brown). slower than that of the bacillus coli communis in the same medium. An isolated colony, slightly magnified, on gelatin, is shown in Fig. 58. Fig. 58. — Bacillus of typhoid fever : superficial colony two days old, as seen upon the surface of a gelatin plate ; x 20 (Heim). Glucose-gelatin Stab. — Growth all along the line of inocu- lation in the form of confluent spherical grayish colonies, and o\\ the surface about the point of entrance of the platinum l66 PATHOLOGICAL TECHNIQUE. wire in the form of a circular translucent grayish layer. No production of gas-bubbles. No liquefaction. Glucose Agar-agar. — Growth similar to that in the pre- ceding. No gas-formation. Litmus-milk. — No visible change. Potato. — Growth occurs, but it is usually invisible. Fig. 59- — Typhoid bacilli, from a culture on agar-agar, showing flagella, from a preparation stained by Dr. Hugh Williams; x 2000 (Wright and Brown). Dunham's Peptone Solution. — No indol-production — i. e. no red color appearing in the twenty-four- to forty-eight- hour cultures after the addition of 5 drops of concentrated sulphuric acid, c. p., and i cubic centimeter of a solution of sodium nitrite, i : 10,000. Motility. — Very marked. BACTERIOLOGICAL METHODS. 1 67 Flagella (Fig. 59) may be demonstrated by the special methods of staining described elsewhere. Decolorized by Gram's method. Does not form spores. Bouillon. — Clouded, with the formation of some sediment. The clouding of the medium is not so marked as in the case of the bacillus coli communis. In general, the growth of the typhoid organism is not so vigorous on culture-media as is the growth of the bacillus coli communis. When to a bouillon culture a small quantity of the blood- serum of a typhoid-fever patient is added, the bacilli lose their motility and aggregate in clumps (" serum reaction "). Pathogenesis. — The inoculation of animals is usually with- out results if moderate quantities of the organism are used. Sometimes, however, death occurs apparently from the effects of the toxic material injected. Occurrence. — Found in the spleen in large numbers at autopsies in typhoid fever. Its presence may also be demon- strated in the intestinal lesions, rose spots, mesenteric lymph- glands, liver, bile, kidneys, urine, and blood of the heart. As a rule, the number of bacilli found in the liver, kidneys, and blood of the heart is small. In the bile they may be numerous and may persist in it for a long period of time after the disease has subsided. In some cases the urine con- tains enormous numbers of the bacilli. The typhoid bacillus may also occur in the suppurative sequelae of typhoid fever, especially those involving bones. In these conditions, however, it may be accompanied by the pyogenic cocci. Occurs in contaminated water. DiflPerential Diagnosis between the Bacillus of Typhoid Fever and the Bacillus Coli Communis. — The most im- portant points of difference between these two organisms are as follows, and to distinguish with certainty between them it is necessary that attention be paid to all of them : Motility. — The typhoid bacillus is actively motile, the bacillus coh communis not motile or exceptionally motile. Potato Cultures. — The typhoid bacillus usually grows in- visibly, the bacillus coli communis as a dirty, slimy layer. l68 PATHOLOGICAL TECHNIQUE. Gas-production in Media Containing Glucose. — The bacillus coli communis produces gas, the typhoid bacillus does not. Litmus-milk Cultures. — The bacillus coli communis changes the blue color of the medium to a pink color and usually coagulates the milk. The typhoid bacillus produces no visible change. Indol-production. — The bacillus coli communis produces indol, the typhoid bacillus does not. Serum or Clump Reaction. — The typhoid bacillus shows the clump reaction, while the bacillus coli communis does not. As it is not always possible to have a typhoid serum at hand by which to determine whether this reaction is present, a stock of dried blood from a typhoid case, con- tained in filter-paper, may be kept ready for use. That this is quite practicable has been clearly shown by Dr. Mark W. Richardson. The blood may be obtained from the heart at the autopsy of a typhoid-fever case by soaking a piece of filter-paper with it. This is allowed to dry, and then is cut into pieces about i cm. square. When it is desired to make the test, one of these pieces is extracted with ten or fifteen drops of water, and a drop of this extract is mixed with a drop of an eighteen- to twenty-four-hour bouillon culture on a slide, covered with a cover-glass, and examined with the high-power dry lens. Dr. Richardson has found that the blood under these conditions retains for months its " clumping " power- with reference to the typhoid bacillus. Other differences are — the production of a red color in litmus-lactose agar-agar by the bacillus coli communis, and no change in color of this medium by the typhoid bacillus, and the slower and less vigorous growth of the typhoid bacillus in culture-media. The Blood-serum Reaction in Typhoid Fever. — A few drops of the blood of a suspected case of typhoid fever are collected in a small test-tube, either from the finger or the ear. After clotting has taken place, transfer a drop of the serum by means of a medicine-dropper to forty drops of a recent bouillon culture of the typhoid bacillus. After mixing, place a drop of the mixture on a slide, cover it BACTERIOLOGICAL METHODS. 1 69 with a cover-glass, and examine with a high-power dry- objective. If the bacilli are seen to be motionless and to be agglom- erated in clumps within twenty minutes, the reaction is to be regarded as present and typical. If the clumping occurs within thirty minutes, but free bacilli are still moving, the reaction is to be regarded as doubtful. The reaction is present, as a rule, only after the first week of typhoid fever. The slide and cover-glass should be sterilized in a flame J " . r'" , 'S .'(^- '■ w •-V';^^^' JH ■m--:^. M TMV >-;/- / ^H / ^ ft \\'' ."^ v.l ^ \ "t.'*' '/, V J H ..m Fig. 60. — Showing the clumping of typhoid bacilli in the serum-reaction. Wet preparation, not stained. At one point a crenated red blood-corpuscle is seen (Wright and Brown). aftfer the test is completed. The bouillon culture used should be grown at room-temperature and should not be more than four or five days old. Before carrying out the test it is well to assure one's self that the bacteria are actively motile by examining a drop of the culture as above indicated. The stock cultures of the typhoid bacilli are best made on agar-agar. The reaction may also be obtained from the dried blood. A few drops of the blood may be collected on a glass slide or a piece of paper and allowed to dry. It may then be brought to the laboratory, where as much of the dried blood as would correspond to a drop is scraped from the slide into a small test-tube, containing forty drops of a bouillon-culture of the typhoid bacillus, or as much of the paper as may be I/O PATHOLOGICAL TECHNIQUE. assumed to contain one drop of blood is placed in forty drops of the bouillon culture and allowed to soak for a few minutes therein. Microscopical examination is then made with the mixture thus obtained as above indicated. Cultivation of the Typhoid Bacillus from the Blood during Life. — The method of doing this is indicated on page 98. It is important that at least 10 c.c. of the blood should be obtained and that this be mixed with a large excess of sterile bouillon — say i or 2 c.c. to 200 or 400 c.c. of bouillon in flasks. Cultivation of the Typhoid Bacillus from Rose Spots. — - Dr. Mark W. Richardson, of Boston, uses the following method : The skin is first washed with corrosive sublimate and then with alcohol and ether. The skin about the spot is then frozen with ethyl chlorid and a small crucial incision made in it. The wound is immediately scraped with a very small curette, and with the material so obtained a bouillon tube is inoculated. It is not to be expected that the bacillus will develop in every culture so made. Cultivation of the Typhoid Bacillus from the Feces. — Various culture-media have been devised upon which colonies of typhoid bacilli will grow with appearances dis- tinguishing them from colonies of colon bacilH and other bacteria. Of these, we here describe but two. In all cases the colonies supposed to be those of the typhoid bacillus should be proved by transplants in various media and the bacteria shown to conform to the requirements indicated on pages 167 and 168. Medium of Hiss} — This is composed of agar-agar 1 5 gm. ; gelatin, 15 gm. ; Liebig's extract of meat, 5 gm. ; sodium chlorid, 5 gm. ; dextrose, 10 gm. ; distilled water, looo c.c. The agar-agar is dissolved in water by boiling for thirty to forty-five minutes. The other ingredients, except the dex- trose, are then added. When dissolved, the mixture is cleared with the whites of two eggs and filtered through absorbent cotton. The sugar is added to the filtered mix- ture. The medium thus prepared is then run into tubes — ^ Journal of Medical Research, June, 1502, vol. viii., No. I, pp. 148-167. BACTERIOLOGICAL METHODS. I71 10 c.c. in each tube — and sterilized at 100° C. in the usual manner on three successive days. Make an emulsion of one or more loopsful of the feces in sterile bouillon and from this make five or six plate-cultures with the medium, using one to five loopsful of the emulsion to each plate. The plates are to be examined after twelve to eighteen hours in the incubator. The colonies of typhoid bacilli below the surface of the medium are of irregular shape and present filamentous or thread-like processes extending into the medium. The colo- nies of colon baciUi beneath the surface of the medium are larger, denser, and darker, and do not present the filament- ous processes. The surface colonies of the typhoid bacillus are smaller, less dense, thinner, and more homogeneous than are the colonies of colon bacilli. Medium of v. Drigalski and Conradi} — {a) Preparation of agar : i kilo of finely ground beef is mixed with two liters of water and allowed to stand until the next day. The re- sulting meat infusion is boiled for an hour, filtered, and to it are added 20 gm. of peptone (Witte), 20 gm. neutrose, 10 gm. sodium chlorid, and the whole boiled for another hour and filtered. To the filtrate 60 gm. of agar-agar are added and the mixture boiled for three hours (or one hour in the auto- clave), is made faintly alkaline to litmus, is again filtered, and is boiled one-half hour more. [b^ Litmus solution : litmus solution (according to Kubel and Tiemann), 260 c.c, boil ten minutes, add 30 gm. chemi- cally pure lactose, and boil for fifteen minutes. {c) Add the hot litmus-lactose solution to the fluid agar. Shake well and readjust, if necessary, to a weak alkaline reaction. Next add 4 c.c. of a hot sterile solution of 10 per cent, anhydrous sodium carbonate and 20 c.c. of a freshly prepared solution of o.i gm. of crystal violet (B. Hochst) in 100 c.c. of warm sterile distilled water. This medium should be stored in flasks of about 200 c.c. capacity in order to avoid protracted heating when it is de- sired to melt it. When used, the medium is poured into ^ Zeitschrift fur Hygiene und Infectionskrankheiten, 1902, Bd. 39, p. 283. 172 PATHOLOGICAL TECHNIQUE. round glass plates 15 to 20 cm. in diameter in sufficient amount to form a layer at least 2 mm. thick and is allowed to solidify. Its surface is then inoculated with a suspen- sion of the fecal material, applied by means of a glass rod bent in a certain form. Obvious means are to be taken to secure such a distribution of the bacteria that a good separation of the colonies will result. After inoculation the medium is to remain uncovered in the plates until the sur- face is dry. After twenty-four hours in the incubator the colonies of the typhoid bacillus are blue in color and smaller than the colonies of colon bacilli, which are red and opaque. Colo- nies of other bacteria may be blue, but they are generally larger. Bacillus Coli Communis.^ — Synonyms : Bacterium coli commune ; Colon bacillus. r V / / v. V f' ^ •" •• f ■ / 3 ^ ^/ ' \ . \« 1 " '— ' Fig. 61. — Bacilli coli communis from a bouillon culture, showing the irregu- larity of staining of the bacillus ; X 2000 (Wright and Brown). Blood-serum. — Rounded, grayish-white, slightly elevated, viscid-looking colonies, which may attain a diameter of 3 mm. after twenty-four hours in the incubator. Morphology. — A medium-sized bacillus with rounded ends, 1 Escherich : Forischr. d. Median, 1885, Nos. 16, 17. BACTERIOLOGICAL METHODS. 1 73 often short or even coccus-like, but may grow in long forms. Faintly staining, sharply defined areas are present in the pro- toplasm (Fig. 61). Gelatin Slant. — Grayish translucent strip with wavy mar- gins. The gelatin is not liquefied. Growth is more rapid than in the case of the typhoid bacillus. A single colony on a gelatin plate is shown in Fig. 62. Glucose-gelatin Stab. — Growth along the line of stab in the form of confluent spherical colonies, and on the surface about the point of entrance of the needle as a thin gray Fig. 62. — Bacillus coli communis: superficial colony two days old upon a gelatin plate ; X 21 (Heim). circular layer. Gas-bubbles are produced in the gelatin from fermentation of the glucose. The gelatin is not liquefied. Glucose-agar-agar Stab. — Growth essentially the same as in the preceding, except that the gas-formation is more marked. Litmus-milk. — Turned pink and usually coagulated. Potato. — Dirty grayish or brownish, viscid-looking layer. Dunham's Peptone Solution. — Marked indol-production. This is shown by the appearance of a red color in the cul- ture after the addition of 5 drops of concentrated sulphuric acid, c. p., and i c.c. of a i : 10,000 solution of sodium nitrite. The culture in the peptone solution should have 174 PATHOLOGICAL TECHNIQUE. been at least twenty-four hours — or, better, forty-eight hours — in the incubator before the test is made. Motility. — Usually not motile, but some varieties show sluggish independent movement. Flagella. — May be demonstrated by the special methods of staining. They are less numerous than in the case of the typhoid bacillus. Decolorized by Gram's method. Bouillon. — Markedly clouded, with formation of a sedi- ment. The clouding is more marked than in the case of the typhoid bacillus. Lactose-litmus-agar-agar Slant. — Growth has a pink color, and the blue color of the medium is changed to red. Pathogenesis. — " Its virulence as tested upon animals is variable, but is generally manifest only after inoculation of large doses, which kill by intoxication rather than infection " (Welch). The lesions produced are not sufficiently characteristic to be detailed here. Occurrence. — Occurs constantly in the intestinal canal, and is widely distributed in the external world. " The colon bacillus is a frequent invader of the internal organs in all sorts of diseases, especially when there are in- testinal lesions. It manifests no evident pathogenic action in most of these cases, and is then without clinical signifi- cance. It occurs frequently associated with other bacteria in infected wounds and other inflammations of exposed sur- faces. Here also it does not usually appear to cause serious disturbance. The fact that the colon bacillus is so common and widely distributed, and found so often as a harmless in- vader, should lead to much caution in interpreting the sig- nificance of its presence when it occurs in definite lesions. There is no doubt, however, that it may be pathogenic for man. It plays an important role in inflammations of the urinary tract and biliary passages ; also, but usually with less independence, in peritonitis and appendicitis. " The list of diseases in which it may be found is a very long one, and includes inflammations in all organs and parts BACTERIOLOGICAL METHODS. 1 75 of the body. In general its pathogenic properties are of a mild character. One of its leading roles is to invade terri- tory already occupied by other bacteria or previously damaged. It may be concerned in the production of gall- stones, in the interior of which it has been found by the writer with great frequency " (Welch, Dennis's System of Surgery, vol. i.). The bacillus above described is to be regarded as a type of a group of bacilli constitudng the so-called "colon group" of bacilli. These present certain quantitative differences among themselves which are not quite sufficient to characterize them as distinct species. Welch regards as belonging to this group the Bacillus pyogenes fmtidus, distinguished by the stinking odor of its cultures, and the Bacillus lactis aerogenes, which is characterized chiefly by its plumper form, its more energetic gas-production, its rapid coagu- lation in milk, and its denser growth in cultures. Theobald Smith ^ suggests that only those forms may be re- garded as typical members of the group which grow on gelatin in the form of delicate bluish or more opaque whitish expansions with irregular margin, which are actively motile when examined in the hanging-drop from young surface colonies taken from gela- tin plates, which coagulate milk within a few days; grow upon potato either as a rich pale or brownish -yellow deposit, or merely as a glistening, barely recognizable layer, and which give a dis- tinct indol-reaction. Their behavior in the fermentation-tube' must conform to the following scheme : ' Amer. Jour. Med. Sci., Sept., 1895. '^ The fermentation-tube is a special form of culture-tube which may be obtained from dealers in bacteriolpgical supplies. The closed branch of the tube should be completely filled with culture-iluid, but no more fluid should be placed in the tube than can be conveniently held by the open branch of the tube, so that if gas be formed in the closed branch the culture-fluid will not be forced out of the apparatus. The bubbles which collect at the top of the closed branch, after heating during sterilization, should be removed by an appropriate tilting of the tube. Theobald Smith,* who was the first to demonstrate the great value of the fermentation-tube in bacteriology, thus describes the mode of its use : " The tubes are kept, after inoculation, in the thermostat at 37° C. A mark made on the sides of the closed branch at the end of every twenty-four hours with a glass pencil furnishes an approximate record of the rate of gas-produc- tion. Unless this is done it is impossible to know precisely when the formation of gas is at an end, and also whether or not the volume of gas has been dimin- ished by absorption. It is best to wait four or five days after the production * The Wilder Quarter-Century Book, Ithaca, 1893, pp. 186, 187. 176 PATHOLOGICAL TECHNIQUE. Variety a. One per cent, dextrose-bouillon (at 37" C). Total gas, approximately 1/2 ; H/CO^ approximately 2/1 ; reaction strongly acid. One per cent, lactose bouillon : As in dextrose-bouillon (with slight variation). One per cent, saccharose-bouillon : Gas-production slower than in the preceding, lasting from seven to fourteen days. Total gas finally about 2/3 ; H/CO2 nearly 3/2. The final reaction in the bulb may be slightly acid or alkaline, according to the rate of gas- production. Variety /5. The same in all respects excepting as to its behavior in saccharose-bouillon. Neither gas nor acids are formed in it. The Detection of the Bacillus Coli Communis in "Water. — The presence of bacilli of the ' ' colon group ' ' in water is to be has ceased before making »l final examination. This is done by noting the condition of the growth, the reaction of the fluid in the bulb,* and the maxi- mum quantity of gas produced. This is most easily done by laying directly on the tube a glass millimeter rule, and noting the tube length occupied by gas. The entire length of the closed branch is also noted, making due allowance for the upper convex extremity and the lower constriction. This mode of meas- urement is sufficient, since only comparative values are desired. For the same reason all barometric and thermometric corrections are omitted in these approxi- mate estimations. " The examination of the gas produced was limited to the determination of the quantity of carbon dioxid and of the explosive character of the gas remain- ing after the absorption of COj by sodium hydrate. These facts are deter- mined by the following simple manipulations : " The bulb is completely filled with a 2 per cent, solution of NaHO, and closed tightly with the thumb. The fluid is shaken thoroughly with the gas, and allowed to flow back and forth from bulb to closed branch and the reverse several times, to insure intimate contact of the COj with the alkali. Lastlv, before removing the thumb, all the gas is allowed to collect in the closed branch so that none may escape when the thumb is removed. If CO, was present, a partial vacuum in the closed branch causes the fluid to rise suddenly when the thumb is removed. After allowing the layer of foam to subside somewhat, the glass scale is again applied to the closed branch, and the amount of CO.; absorbed may thus be measured. In all cultures of this character thus far ex- amined the gas remaining was explosive in character, and probably hydrogen. The explosive character of this residue is easily demonstrated as follows : The cotton plug is replaced, and the gas in the closed branch allowed to flow into the bulb, and mix with the air there present. The plug is then removed, and a lighted match inserted into the mouth of the bulb. The intensity of the explosion varies with the quantity of air present in the bulb." * The reaction was noted by placing a drop of the fluid on delicate litmus- paper. The cultures were occasionally boiled to drive off any CO,. In no case did the reaction with the litmus-paper change. BACTERIOLOGICAL METHODS. I// regarded as good evidence of pollution with fecal material from man or animals. Theobald Smith's Method.' — A certain quantity of the water is added "to every one of ten fermentation-tubes containing a I per cent, dextrose-bouillon. This is added most easily by first diluting the water, so that i or 2 c.cm. are equivalent to the quantity which it is desired to add to each tube. Pipettes gradu- ated by drops are convenient, but not so accurate." "When gas begins to appear in the fermentation-tubes the amount accu- mulated at the end of each twenty-four hours should be marked with a glass pencil on the tube. From these tubes, which con- tain 50 to 60 per cent, of gas on the third day, and are very strongly acid, plates may be prepared to confirm the indications of B. colt. This, however, is not essential, for the writer has found as yet no species having these fermentative characters which is not one of the following : B. coli, B. lactis aerogenes, B. enteritidis, B. typhi murium, B. cholera suis. The three last- mentioned species are probably as rare in water as B. typhosus itself." Parat3T)lioid and Paracolon Bacilli. — Under these names certain observers have grouped various bacilli that they have found associated with inflammatory processes and with fevers clinically resembling typhoid. These bacilli have certain cultural peculiarities, like the typhoid bacillus and the colon bacillus, but they differ from them and from each other in certain cultural peculiarities which chiefly concern their fermentation of the various sugars, their effect upon litmus milk, their mode of growth on potato, and their indol production. At the present time it seems impossible to give a detailed description of the behavior of any one of them on the various culture-media that will hold good for another. They are not agglutinated by typhoid serum, but each of them is agglutinated by the serum of the patient from which it is derived. Bacillus Dysenteriae (Shiga). — This bacillus resembles the typhoid bacillus in morphology, but in general it is plumper and less frequently appears in filamentous forms. Involution forms quickly develop in glucose-agar cultures. It is decolorized by Gram's method, and does not form .spores. It is not motile. The bacillus grows in bouillon and on agar and gelatin, both in plate and tube cultures, 1 Amer. Jour. Med. Sci., Sept., 1895. 12 1,78 PATHOLOGICAL TECHNIQUE. with appearances very similar to those of the typhoid bacillus. It does not produce gas in media containing glucose or other sugars. Typical examples of the bacillus do not produce indol in peptone solution, but some strains have been found to do so. Litmus Milk. — During the first two or three days the milk becomes a pink color, but later becomes blue. It is never coagulated. Potato. — Growth, at first in the form of a moist, colorless, slimy, almost invisible layer, becoming, after two or three days in the incubator, of a yellowish to brownish tint with discoloration of the potato. Mannite Litmus Agar. — This medium consists of ordinary nutrient agar-agar containing i per cent, of mannite and i per cent, of a 5 per cent, aqueous solution of litmus. The agar-agar should have been made up with meat infusion free from muscle-sugar (see p. 74). In stab cultures on this medium the typical bacillus decolorizes the agar in the depths, but near the surface the original blue color remains unchanged. Some strains of the bacillus, however, change the color of the upper layers to red, as do typhoid and colon bacilli. Thus two varieties of the bacillus may be distin- guished with reference to their effect on this culture-medium. Both of these varieties have been isolated from the same dysenteric stools. The bacillus exhibits the agglutination reaction with the serum of dysenteric cases and with the serum of animals immunized against the bacillus. Pathogenesis. — The bacillus is pathogenic for the usual laboratory animals, especially for mice and guinea-pigs which may die in from twenty-four to forty-eight hours after subcutaneous or intraperitoneal inoculation. At the autopsy there may be found local inflammation at the seat of inocu- lation, ecchymoses of the serous membranes, serous or sero- hemorrhagic exudate in the pleural or peritoneal cavities, enlargement of the spleen, and hyperemia or hemorrhage in the intestinal walls. BACTERIOLOGICAL METHODS. 179 Occurrence. — In the stools, intestinal contents, and in the ulcerated mucous membrane of acute dysentery, whether sporadic or epidemic. It may be found in the mesenteric lymphatic glands, but is not found in the blood, in the spleen, or in other viscera. Method of Isolation of the Bacillus. — A small portion of mucus is to be broken up in a tube of sterile bouillon. From the suspension of bacilli thus obtained agar-agar plate-cultures are prepared in dilutions depending upon the number of bacilli observed in smear preparations of the mucus. After twenty-four hours in the incubator trans- plants are made from colonies resembling those of the typhoid bacillus into i per cent, glucose bouillon contained in tubes arranged as suggested by Durham for the demonstration of gas-formation. These tubes are prepared as follows : In an ordinary bacteriological test-tube containing about one-third more than usual of i per cent, glucose bouillon, there is placed a small test-tube mouth downward. The larger tube is then plugged with cotton, and is sterilized by steam in the usual way. After the sterilization it will be found that the air has been driven out of the inverted inner tube and has been replaced by the bouillon. When cool, the tube is ready for inoculation. If the bouillon at the bottom of the larger tube be inoculated with a bacterium capable of fermenting glucose with the production of gas, this property of the bacterium will manifest itself by the collection of gas in the upper part of the small inverted inner tube. As the dysentery bacillus does not produce gas from glucose, gas-formation in a tube is proof that the colony from which it was inoculated is not the dysentery bacillus. The bacteria in tubes in which no gas has developed are to be examined with reference to morphology and motility. If found to agree in morphology with the bacillus of dysen- tery and to be non-motile, then the agglutination test with dysentery serum may be applied to them immediately and they are to be transplanted on the various media to test their cultural peculiarities. i8o PATHOLOGICAL TECHNIQUE. The advantages of this method over that of first trans- planting the suspected colony into sugar-agar, as is usually done, are, first, that it enables the motility, agglutination re- action of the bacilli, and any gas-formation to be determined with the use of a single culture-tube ; and, second, that it dispenses with the use of glucose-agar tubes, which are much more troublesome to prepare than are the bouillon tubes. These considerations are of considerable importance when a number of cases are being examined, for at least six colonies should be tested in each case as above described. Bacillus Tuberculosis.' — Synonyms: Tubercle bacillus ; Bacillus of Koch. Blood-serum. — After three or four weeks in the incubator the colonies appear as dry, cream-colored, granular, slightly Fig. 63. — Branched tubercle bacilli from sputum ; X 2000 (Wright and Brown). elevated patches with irregular margins, i to 2 mm. in diam- eter. They may become confluent, to form a dense, dry, granular mass with irregular surface and of a creamy-white color. The growth is very friable, but coherent, and may be picked up in clumps on the platinum wire. The first generation from tissues is very slow in developing, but suc- ceeding generations grow more rapidly, and may form a wrinkled, dry, cream-colored membranous layer on the sur- face of the medium. Morphology. — Slender rods, usually shorter than when observed in sputum, and in fresh cultures staining homo- geneously ; in older cultures presenting a segmented or ' Koch: Berlin, klin. Wochenschr.,liio. 15, 1882; Mitth. u. d. Kais. Ge- sundheitsamte, Bd. 2, 1884. BACTERIOLOGICAL METHODS. l8l irregularly stained appearance. They frequently occur in pairs of short rods and in closely adhering clumps and strands. When once stained with fuchsin or gentian-violet they a"re not decolorized by treatment with Gabbet's solution or with a 20 per cent, solution of any of the mineral acids, followed by alcohol. In the sputum of pulmonary tuber- culosis the bacillus sometimes occurs in filaments which branch. On this account the organism is considered by many to belong to the group of the streptothrices (see p. 227). Stained by Gram's method. Not motile. Does not form spores. Glycerin Agar-agar Slant. — Growth similar to that on / blood-serum, but not so vigorous. By continued inoculation of this medium through a number of generations, however, the organism may eventually grow luxuriantly upon it. Glycerin Bouillon. — Growth on the surface in the form of floating patches or as a membrane similar in appearance to the growth on blood-serum. The growth sinks to the bottom from time to time. The glycerin-bouillon culture is best contained in Erlenmeyer flasks, filled to such a depth as to give a wide surface to the fluid and thus permit the access of plenty of oxygen to the growth. Potato. — The growth is not remarkable. Agar-agar or bouillon not containing glycerin is not suit- able for the cultivation of this bacillus. Pathogenesis. — The inoculation of guinea-pigs or rabbits by any method is followed by the development of general miliary tuberculosis. Guinea-pigs are most susceptible. These animals usually survive about two or three months, with marked emaciation. The lesions in the spleen and liver in the guinea-pig are characterized by extensive areas of necrosis not confined to the tubercular tissue, large parts of these organs being transformed into a firm yellow, opaque, friable material. Isolation of the Bacillus Tuberculosis from Tubercular Lesions. — The tubercular lesions in human tissues are not ordinarily favorable for the isolation of the bacillus, on 1 82 PATHOLOGICAL TECHNIQUE. ■ account of the frequent presence of other bacteria in them and because of the small number of tubercle bacilli usually present in tissue otherwise suitable. The best method of procedure is to inoculate a guinea-pig subcutaneously in the abdominal wall with tubercular material, and after four to six weeks, when the inguinal lymphatic glands have become enlarged, to kill the animal and make cultures on suitable media from tuberculous lymphatic glands. The object of killing the animal, rather than allowing it to die sponta- neously, is to secure fresh tissue and to avoid the chance of an invasion of the lesions by other bacteria. For the cultivation of tubercle bacilli from tubercular lesions, M. Dorset highly recommends his egg-medium, which is prepared as follows : Fresh eggs are broken under aseptic precautions into a wide-mouthed sterile flask, and the white and yolk mixed thoroughly. To every four eggs add 25 c.c. of sterile water. Any foam may be removed by straining the mixture through a sterile cloth. The mixture is then run into sterile tubes, — about 10 c.c. into each tube, — and slowly hardened in the form of " slants " in a blood-serum oven at a temperature of 73" to 76° C. This degree of temperature should be maintained for four or five hours on three successive days. On the first two days the temperature is maintained at 73" C, and on the third day at 76° C. Just before inoculating the medium three or four drops of sterile distilled water should be added to each tube to supply the moisture required for the satisfactory development of the tubercle bacillus. After inoculation the tube should be placed in the incu- bator at 38° C. in an inclined position, so that the surface of the medium may keep moist. Colonies first become visi- ble after seven or eight days in the incubator. • A number of tubes are to be inoculated, say three or four, from each of the two or three glands, a large quantity of material being spread upon the surface of each tube. Great care is to be exercised to avoid contamination with other bacteria in preparing these cultures. The culture-tubes used BACTERIOLOGICAL METHODS. 183 should contain freshly prepared moist medium, and imme- diately after inoculation should be sealed air-tight to prevent evaporation. This may conveniently be done by first cutting off the projecting portion of the cotton stopper and insert- ing a cork into the mouth of the tube in such a way as to push the cotton stopper before it. In order to prevent the invasion of fungi from the cotton, the neck of the tube should be heated in the Bunsen flame until the cotton begins to brown before inserting the cork, which should also be charred in the Bunsen flame before in- sertion. The tubes may also be sealed with wax or paraffin or covered with small rubber caps. Wolbach and Ernst ' have noted the following chief points of difference between tubercle bacilli from human and from bovine lesions when cultivated on Dorset's egg medium. The human cultures as compared with the bovine cultures grow more profusely and the membranous layer that is formed is more nodular, drier looking, less translucent, more adherent to the medium, harder, and more difficult to break up with the platinum wire. The human bacilli generally show a slightly greater variation in length and thickness than do the bovine bacilli. Occurrence. — In tubercular lesions generally and in the sputum of pulmonary phthisis, in the urine in many cases of genito-urinary tuberculosis, and in the feces in intestinal tuberculosis. The tuberculosis of cattle and of birds is due to different varieties of this bacillus. Does not multiply outside of the body except in cultures. May occur on the surface of objects contaminated with the excreta of tuberculous individuals or in the dust of places inhabited by such individuals. Diagnosis. — For clinical purposes the tubercle bacillus may be identified in cover-glass preparations by means of the special methods of staining, which depend upon the fact that the bacillus tuberculosis, when once thoroughly stained with an aniline dye, does not give up its stain in the presence of acids, as nearly all other bacteria do. The bacillus tuber- ^ Journal of Medical Research, December, 1903, vol. x.. No. 3, p. 313. 1 84 PATHOLOG/CAL TECHNIQUE. culosis may therefore be identified even among a mixture of other bacteria by this property, taken in connection with its morphology, in most of the routine work of the pathological laboratory. Practically, the only other bacilli with which it may be confounded are the bacillus of leprosy and the smegma bacillus, both of which, when stained, resist the decolorizing action of acid. It may be differentiated from the smegma bacillus by the fact that it is not decolorized by alcohol (95 per cent.) after the usual treatment with acid, while the smegma bacillus is decolorized under these circum- stances. As a rule, the differential test with alcohol need only be applied in the examination of urine and the material derived from about the external genitalia, especially in the case of females. The differentiation from the bacillus of leprosy by certain quantitative differences in staining reactions has been at- tempted, but it is very unsati-sfactory, and it is doubtful if there is as yet any reliable method of distinguishing between these two organisms, considered by themselves. :^xaminatiou of Sputum for Tubercle Bacilli. — The morning sputum should be taken for examination. Select one of the dense, grayish- white particles, and with the aid of small pointed forceps or the platinum wire rub it over the surface of a cover-glass, breaking it up as much as possible. The material should be spread in a very thin layer. The preparation is next to be " fixed " in the ordinary way described for cover-glass preparations (see p. 92), and is then to be treated as follows : I. Stain in carbol-fuchsin solution, steaming for one minute over the Bunsen flame, with the staining solution thoroughly covering all the surface of the cover-glass. None of the surface of the cover-glass should be allowed to become dry by evaporation, as this causes a precipitate to form, but more of the staining fluid should be added from time to time to keep it completely covered as evaporation occurs. The object of the heating is thoroughly to impregnate the bacilli with the dye. BACTERIOLOGICAL METHODS. 185 2. Wash in water. 3. Decolorize in a 30 per cent, aqueous solution of con- centrated nitric acid until the red color disappears. Do not allow the acid to act on the preparation longer than a few seconds. The solution should also be applied to the un- V ( f Fig. 64. — Tubercle bacilli in sputum (carbol-fuchsin and methylene-blue). charged side of the cover-glass to remove any dried stain which may have collected thereon. 4. Wash thoroughly in water. 5. Wash in 95 per cent, alcohol for thirty seconds. 6. Wash in water and mount. The tubercle bacilli are stained red and the nuclei of cells and other bacteria are stained blue. Tubercle bacilli, when present in sputum in very small numbers, may sometimes be demonstrated by methods of sedimentation. A good means of sedimentation consists in heating the sputum in a test-tube either with boiling water or in the steam sterilizer for fifteen minutes. The heat coagulates the cells and albuminous constituents, which sink to the bottom, carrying with them the l86 PATHOLOGICAL TECHNIQUE. bacilli. The supernatant liquid may then be poured off and the sediment examined as above. Another method of treating the sputum when only a small number of bacilli may be present is as follows : Place 10-15 c.c. of the sputum in a wide-mouthed bottle of 100 c.c. capacity. Add 10 c.c. of water and 6 c.c. of liquefied carbolic-acid crystals. Close the flask with a rubber cork and shake for one minute. After shaking, fill the bottle with water and shake again. Then pour the contents of the bottle into a sedimenting glass, let stand for twelve to twenty-four hours, and examine the sediment. The cover-glass preparations of the sediment before staining are to be washed in ether or chloroform, and then in alcohol, or they may be washed in a mixture of alcohol and ether, equal parts. In a very few cases of gangrene of the lung bacilli like smegma bacilli have been found in the sputum. These may be mistaken for tubercle bacilli {j)ide ante). Tubercle Bacilli in Urine. — The sediment of the urine .should be examined. This may be rapidly thrown down by the centrifuge. With the sediment, cover-glass preparations are to be made and stained as described for sputum. Especial care should be taken to wash thoroughly in alcohol after the decolorization with acid, in order to decolorize any smegma bacilli that may be present. (See remarks on Diagnosis, p. 183.) Because smegma bacilli may be mistaken for tubercle bacilli and because the tubercle bacilli may be so few as to escape observation, the inoculation of a guinea-pig with the sediment is the better test for the presence of tuber- cle bacilli in the urine. For this purpose the urine should be collected in sterilized vessels and immediately centrifu- galized in sterilized tubes. The sediment is then to be injected subcutaneously into a guinea-pig with a sterilized syringe. (Seep. 116.) Surgical Tuberculosis. — The demonstration of the tuberculous nature of material removed at operations may be made by the histological examination, by the demonstra- tion of the tubercle bacilli on cover-glass preparations, as in sputum, and by the results of the inoculation of guinea-pigs with the material. Cultures are not ordinarily practicable. The examinatioji of cover-glass preparations is commonly of little value, owing to the small number of bacilli usually present. BACTERIOLOGICAL METHODS. 187 The histological examination may be often made very satis- factorily by the method of frozen sections described on p. 263. The inoculation of guinea-pigs is to be made subcutane- ously in the abdominal wall, either with a hypodermic syr- inge if the material is fluid, or, if it be in the form of tissue, by inserting a small piece beneath the skin. Material obtained on a swab may also be used for inoculation by introducing Fig. 65. — Bacillus of leprosy : section through a cutaneous nodule, showing the bacilli in the tissue ; X 750 (Wright and Brown). the infected swab beneath the skin and moving it back and forth a few times. If tubercle bacilli are present in the ma- terial, the animal will show enlargement of the inguinal lymphatic glands in about three weeks and will usually die of miliary tuberculosis in the course of six to ten weeks. If necessary, the glands in the inguinal region may be examined histologically after three weeks for the presence of tubercu- lar lesions, or examined by cover-glass preparations for tu- l88 PATHOLOGICAL TECHNIQUE. bercle bacilli. The discharges from sinuses, etc. may also be tested for the presence of tubercle bacilli as above indi- cated, the material being obtained on a " swab." I/Cprosy. — The bacillus of this disease shows essentially the same staining reactions as the bacillus tuberculosis. In sections of the lesions the baciUi are found in large numbers, mostly inside the tissue-cells (see Fig. 65). Spirillum of Asiatic Cholera (Comma Bacillus).'— Morphology (Figs. 66, 67), — In fresh cultures the organism appears usually as a slightly curved rod somewhat shorter & '^^'^; ^: <^T^-^ -^ ; ' >. Fig. 66. — Spirillum of Asiatic cholera, from a bouillon culture three weeks old, showing long and degenerate forms ; X looo (Frankel and Pfeiffer). than the tubercle bacillus, but much thicker. The curving of the rod varies, being very marked in some individuals and absent in others. Sometimes two rods are joined end to end with their convexity pointing in opposite directions, or moderately long, undulating threads may be found. It seems probable that the curved rods represent the segments of a spirillum, and hence the name of the organism. In cultures some days old degenerated and atypical forms are found (involution forms). The organism is motile, and a single flagellum is attached to the end of the rod. ^ Koch; Deutsche med. Wochenschr., 1884 and 1885. BACTERIOLOGICAL METHODS. 189 It is not stained by Gram's method. Colonies on Gelatin Plates (Fig. 68). — After twenty-four to forty-eight hours at a temperature of 20° to 22° C. the Fig. 67. — Spirillum of Asiatic cholera, showing the flagella ; x looo (GUnther). largest colonies will appear as masses of indefinite granular material lying in circular areas of liquefied gelatin in which granular shreds are scattered. Within the next twenty-four hours the areas of liquefaction increase, and the colonies ap- • • Fig. 68. — Developmental stages of colonies of the spirillum of Asiatic cholera at 20° to 22° C. on gelatin ; x about 75 diameters (Abbott) : a, after sixteen to eighteen hours ; b, after twenty-four to twenty-six hours ; c, after thirty-eight to forty hours ; d, after forty-eight to fifty hours ; e, after sixty-four to seventy hours. pear under the low power " as a dense granular mass sur- rounded by an area of liquefaction through which can be seen granular prolongations of the colony, usually extending ir- igO PATHOLOGICAL TECHNIQUE. regularly between the periphery and the central mass " (Ab- bott), while the margin of the liquefied area is marked by delicate radiating filaments closely packed together. The colonies on agar-agar plates are not characteristic. Growth is rapid. Gelatin Stab. — Growth all along the line of inoculation with Hquefaction at the surface in funnel form after forty- eight hours. The liquefaction proceeds in such a manner that the Hquefied area has a smaller diameter at the surface than immediately beneath, and, owing to the fact that the liquefied gelatin does not fill the cavity, a space is left be- tween the surface of the medium and the surface of the lique- fied gelatin so that the appearance of an air-bubble is pro- duced. Along the deeper portions of the line of inoculation the liquefaction is slow. Bouillon. — Diffusely clouded. A thin pellicle forms on the surface after a time. Litmus-milk. — Turned red and coagulated. Indol-production. — In cultures in Dunham's pepton solu- tion or in the pepton solution of Koch (2 per cent, pepton and I per cent, sodium chlorid) a rose-color is produced by the addition of sulphuric acid alone. (Concentrated c. p. acid is to be employed, as in the test for indol-production by the bacillus coli communis.) The production of the rose-color without the addition of the sodium nitrite shows that nitrites as well as indol are formed by the growth of the organism in the pepton solution. The reaction can be obtained in cultures which have been but eight hours in the incubator. Potato. — Thin, dry, grayish-white growth which does not spread over the surface. Pathogenesis. — The pathogenic effects of the cholera spirillum are best shown by the inoculation of guinea-pigs. There are two methods of inoculation, as follows : I. The Method of Pfeiffer. — Scrape from the surface of a fresh agar-agar culture as much of the growth as will adhere to a platinum wire bent into the form of a small loop. Sus- pend this amount of material in i c.c. of bouillon, and inject the suspension into the peritoneal cavity of a guinea-pig BACTERIOLOGICAL METHODS. 191 by means of a hypodermic syringe. With virulent cultures this inoculation soon produces a fall in the temperature of the animal, which continues and becomes more marked, death occurring in from twelve to twenty-four hours. At the autopsy of the animal a clear fluid will be found in the peritoneal cavity and in the thorax. 2. The Method of Koch. — This depends upon the fact that the animal may be infected through the alimentary canal, provided the acidity of the gastric juice be neutralized, this acidity being destructive to the cholera spirillum. A soft catheter is passed into the stomach of the animal through the mouth, and through this 5 c.c. of a 5 per cent, solution of sodium carbonate is injected. After ten or fifteen minutes 10 c.c. of a bouillon culture of the organism are in- jected through the catheter, and immediately afterward the animal receives subcutaneously i c.c. of the tincture of opium for every 200 grams of its body-weight. The object of this opium administration is to stop peristalsis, so that the organisms may be longer in contact with a given area of the mucous membrane of the intestine. The result of the inoculation first appears after about twenty-four hours. The animal then has no appetite and is listless. Later, paralysis of the hinder extremities appears, respiration is prolonged and weak, the heart-beats become feeble, and the body-tem- perature may become subnormal. Death usually occurs after the animal has been a few hours in this condition. At the autopsy the small intestine will be found to be in- jected and containing a flocculent colorless fluid in which comma bacilli are present in great numbers. Serum Reaction. — Cholera spirilla cease their motion, and aggregate together in clumps and masses when a bouillon culture of them is mixed with the serum of an animal immu- nized toward them. This phenomenon is called the serum reaction. Moreover, if cholera spirilla be placed in the peri- toneal cavity of an immunized guinea-pig, they will rapidly undergo disintegration, and will be destroyed. This is called Pfeiffer's phenomenon. Occurrence. — In the alvine dejections and in the intestinal 192 PATHOLOGICAL TECHNIQUE. contents of cholera patients (Fig. 69). It apparently only rarely invades the circulating blood. Its presence in the vomitus may sometimes be shown. It has been found in the water-supplies during epidemics. The cholera spirillum is the representative of a large group of spirilla, many of which may be found in river waters. According to Abbott and Bergey, the only trustworthy method of distinguishing some of these from the true chol- era spirillum is their failure to manifest a " clump reaction " with the serum of an animal immunized to infection with the true cholera spirillum. Fig. 69. — Cover-glass preparation of a mucous floccule in Asiatic cholera ; X 650 (Vierordt). Bacteriologioal Diagnosis.' — Because of the manifold chan- nels which are open for the dissemination of this disease it is of the utmost importance that its true nature should be recog- nized as quickly as possible, for with every moment of delay in its recognition opportunities for its spread are multiply- ing. It is essential, therefore, when employing bacteriological means in making the diagnosis to bear in mind those biolog- ical and morphological features of the organism that appear most quickly under artificial methods of cultivation, and which at the same time may be considered as characteristic of it — viz. its peculiar morphology and grouping ; the much greater rapidity of its growth over that of other bacteria with which it may be ' Abbott : Principles of Bacteriology. BACTERIOLOGICAL METHODS. 193 associated ; the characteristic appearance of its colonies on gela- tin plates and of its growth in stab-cultures in gelatin ; its property of producing indol and coincidently nitrites in from six to eight hours in pepton solution at 37° to 38° C. ; and its power of causing the death of guinea-pigs in from sixteen to twenty-four hours when introduced into the peritoneal cavity, death being preceded by symptoms of extreme toxemia, characterized by prostration and gradual and continuous falling in temperature of the animal's body. In a publication recently made by Koch ^ he called attention to a plan of procedure that is employed in this work in the Institute for Infectious Diseases in Berlin. In this scheme the points that have been enumerated are taken into account, and by its employ- ment the diagnosis can be established in the majority of cases of Asiatic cholera in from eighteen to twenty-two hours. In general, the steps to be taken and the points to be borne in mind are as follows. The material should be examined as early as possible after it has been passed : 1. Microscopic examination. From one of the small slimy particles that will be seen in the semifluid evacuation prepare a cover-slip preparation in the ordinary way and stain it. If, upon microscopic examination, only curved rods or curved rods greatly in excess of all other forms are present, the diagnosis of Asiatic cholera is more than likely correct ; and particularly is this true if these organisms are arranged in irregular linear groups, with the long axis of all the rods pointing in nearly the same direction ; that is to say, somewhat as minnows arrange themselves when swimming in schools up stream (Koch). In 1886, Weisser and Frank" expressed their opinion upon the value of microscopic examination in these cases in the following terms : (a) In the majority of cases microscopic examination is suf- ficient for the detection of the presence of the comma bacillus in the intestinal evacuations of cholera patients. (J?) Even in the most acute cases, running a very rapid course, the comma bacillus can always be found in the evacuations. ((t) In general the number of cholera spirilla present is greater the earlier death occurs ; when death is postponed and the disease continues for a longer period, their number is diminished. {d') Should the patient not die of cholera, but from some other disease, such as typhoid fever, that may be engrafted upon it, the comma bacilli may disappear entirely from the intestines. 2. With another slimy flake prepare a set of gelatin plates. Place them at a temperature of from 20° to 22° C, and at six- teen, twenty-two, and thirty-six hours observe the appearance of the colonies. Usually at about twenty-two hours the colonies of this organism can easily be identified by one familiar with them. 3. With another slimy flake start a culture in a tube of pepton ^ Zeitschrift f. Hygiene u. Infedionskrankheiten, Bd. 14, 1893. 2 Ibid., Bd. I, p. 397. 13 194 PATHOLOGICAL TECHNIQUE. solution — either the solution of Dunham or, as Koch proposes, a solution of double strength of that of Dunham (Witte's pepton is to be used, as it gives the best and most constant results). Place this at 37° to 38° C, and at the end of from six to eight hours prepare cover-slips from the upper layers (without shaking) and examine them microscopically. If comma bacilli are present and capable of multiplication, they will be found in this locality in almost pure culture. After doing this prepare a second pepton culture from the upper layer, also a set of gelatin plates, and with what remains make the test for indol by the addition of 10 drops of concentrated sulphuric acid for each 10 c.cm. of fluid con- tained in the tube. If comma bacilli are growing in the tube, the rose color characteristic of the presence of indol should appear. By following this plan "a bacteriologist who is familiar with the morphological and biological pecuharities of this organism should make a more than probable diagnosis at once by micro- scopic examination alone, and a positive diagnosis in from twenty to, at the most, twenty-four hours after beginning the examination" (Koch). There are certain doubtful cases in which the organisms are present in the intestinal canal in very small numbers, and micro- scopic examination is not, therefore, of so much assistance. In these cases plates of agar-agar, of gelatin, and cultures in the pep- ton solution should be made. The plates of agar-agar should not be prepared in the usual way, but the agar-agar should be poured into Petri dishes and al- lowed to solidify, after which one of the slimy particles may be smeared over its surface. The comma bacillus being markedly aerobic, develops very much more readily when its colonies are located upon the surface than when they are in the depths of the medium. A point to which Koch calls attention in connection with this step in the manipulation is the necessity for having the surface of the agar-agar free from the water that is squeezed from it when it solidifies, as the presence of the water interferes with the development of the colonies as isolated points and causes them to become confluent. To obviate this, he recommends that the agar-agar be poured into the plates and the water allowed to separate from the surface at the temperature of the incubator before they are used. It is wise, therefore, when one is liable to be called on for such work as this to keep a number of sterilized plates of agar-agar in the incubator ready for use, just as sterilized tubes of media are always ready and at hand. The advantage of using the agar plates is the higher temperature at which they can be kept, and consequently a more favorable condition for the development of the colonies. As soon as iso- lated colonies appear they should be examined microscopically for the presence of organisms having the morphology of the one for which we are seeking, and as soon as such is detected gelatin plates BACTERIOLOGICAL METHODS. 195 and cultures in pepton solution (for the indol reaction) should be made. The pepton cultures started from the original material should be examined microscopically from hour to hour after the sixth hour that they have been in the incubator. The material taken for examination should always come from near the surface of the fluid, and care should be taken not to shake the tube. As soon as comma bacilli are detected in anything like considerable numbers in the upper layers of the fluid, agar-agar plates and fresh pepton cultures should be made from them. The colo- nies will develop on the agar-agar plates at 37° C. in from ten to twelve hours to a size sufficient for recognition by microscopic examination, and from this examination an opinion can usually be given. This opinion should always be controlled by cultures in the pepton solution made from each of several single colonies, and finally the test for the presence or absence of indol in these cultures. In all doubtful cases in which only a few curved bacilli are pres- ent or in which irregularities in either the rate or mode of their development occurs, pure cultures should be obtained by the agar-plate method and the method of cultivation in pepton solu- tion as soon as possible, and their virulence tested upon animals. For this purpose cultures upon agar-agar from single colonies must be made. From the surface of one of such cultures a good- sized wire-loopful should be scraped, and this broken up in about I c.c. of bouillon, and the suspension thus made injected by means of a hypodermic syringe directly into the peritoneal cavity of a guinea-pig of about 350 to 400 grams weight. For larger animals more material should be used. If the material injected is from a fresh culture of the cholera organism, toxic symptoms at once begin to appear ; these have their most pronounced expression in the lowering of temperature, and if one follows this decline in temperature from time to time with the thermometer, it will be seen to be gradual and continuous from the time of injection to the death of the animal, which occurs in from eighteen to twenty- four hours after the operation. ' In general, this is the procedure employed at Berlin in the In- stitute for Infectious Disease under Koch's direction. Bacillus of Anthrax.^ — Blood-serum. — Irregularly rounded colonies, several mm. in diameter after twenty-four hours in the incubator. The colonies are grayish, finely granular, and have the appearance of being made up of a dense network of delicate fibrillse. The blood-serum is slowly liquefied. 1 Pfeiffer : loc. cit. 2 Pasteur: Bull. Acad. de. Mid., Paris, T. viii., 1879; Koch: F. Cohn's Beitr. 2. Biol. d. Pfl., Bd. 2, 1876. 196 PATHOLOGICAL TECHNIQUE. Morphology. — The organism grows in long segmented threads, the segments varying in length, but usually being two or three times as long as broad and having square or slightly concave ends. These segments represent the bacillus, which is among the largest of the bacteria (Fig. 70). Pathogenesis. — Mice, guinea-pigs, and rabbits inoculated subcutaneously die with a general invasion of the blood by the organism. Mice are most susceptible to the infection, dying in about twenty-four hours, while guinea-pigs and rabbits survive longer. Fig. 70. — Bacillus of anthrax : portion of 6 -; *}• . .'^. .•:'. Fig. 79. — Influenza bacilli from a culture on blood-agar ; X 2000 (Wright and Brown). Fig. 80. — Bacilli of influenza in a leucocyte in a cover-glass preparation from sputum. A pneumococcus also in the same leucocyte and other pneumococci free. The small size of the bacillus of influenza will be apparent by comparison with the pneumococci ; X 2000 (Wright and Brown). blood. The blood of man, rabbits, guinea-pigs, pigeons, or frogs will serve for this purpose, the best growth being BACTERIOLOGICAL METHODS. 207 obtained with pigeon's blood. The blood may be ob- tained from a needle-prick, and spread over the surface of the agar-agar by means of the platinum loop. The skin should be previously thoroughly washed with alcohol and ether, and the first drops of blood should not be used. Human blood is best obtained from the lobe of the ear Fig. 81. — Bacillus of influenza: colonies on blood agar-agar; low magniiying power (Pfeiffer). or from the finger. Tubes thus prepared are only rarely contaminated. Colonies. — After twenty-four hours in the incubator the colonies appear as minute colorless, glassy, transparent points resembling small drops of dew. They never attain any size, and do not become confluent. They are barely visible to the unpractised eye, and require a low magnifying power to be seen clearly. Under the low magnifying power they are translucent, homogeneous, not granular, and cir- cular in outline (Fig. 81). Decolorized by Gram's method. Not motile. Will not grow without oxygen. 2o8 PATHOLOGICAL TECHNIQUE. Pathogenesis. — The ordinary laboratory animals are not susceptible to infection with this organism. Occurrence. — Found in the exudate of the respiratory tract in influenza, frequently inside of leucocytes (Fig. 8o). It may be present in the small bronchi and in the exudate of broncho-pneumonia in this disease. It has been observed in purulent meningitis secondary to influenza. F. T. Lord/ working in the Laboratory of the Massachusetts General Hospital, found influenza bacilli in 60 of 100 unselected specimens of sputa repeatedly negative for tubercle bacilli. In 29 of these 60 cases the bacilli were present in great numbers. Eleven cases were of acute and 18 of chronic inflammation of the respiratory tract. In the chronic cases he demonstrated the per- sistence of influenza bacilli in the sputum for months or years. Lord believes that influenza bacilli are very commonly present in sputa apart from epidemics of influenza, and that chronic infec- tion with influenza bacilli is not infrequently mistaken clinically for tuberculosis. Diagnosis. — Microscopical examination of cover-glass preparations of the bronchial sputum shows very small, short, round-ended bacilli, often in very large numbers and frequently in the ,pus-celLs. These bacilli frequently occur in pairs and resemble pairs of cocci. Their ends may be more deeply stained than the central portions. For the staining of cover-glass preparations of the sputum Pfeiffer recommends that a very dilute carbol-fuchsin solution be applied for five to ten minutes. The cover-glass preparation is to be made from a distinctly purulent portion of the sputum. Staining with Loffler's methylene-blue solution also gives good results. See also W. H. Smith's method for staining the capsule of the pneumococcus, page 142. The bacillus of influenza may be cultivated from the sputum by breaking up a small portion of a distinctly puru- lent character in i or 2 c.c. of bouillon, and then spreading a platinum loopful of the suspension over the surface of a blood-agar-agar slant, which is then placed in the incubator. After eighteen to twenty-four hours the characteristic col- onies may be visible with the aid of a hand-lens. These ' Boston Medical and Surgical Journal, December 18, 1902. BACTERIOLOGICAL METHODS. 209 should not grow in ordinary media unless blood or hemo- globin be present, and should have the morphology of the bacillus of influenza. F. T. Lord obtains the best results by using as a culture-medium I part sterile horse-blood and 2 parts nutrient agar-agar in "slant" tubes. The blood is mixed with fluid agar -agar at 40° C. The colonies of the influenza bacilli may attain a diameter of more than a millimeter on this medium. The horse-blood may be easily obtained from any antitoxin plant and may be kept on hand in test-tubes for months without impairment of its utility for cultural purposes. Bacillus of Glanders (Bacillus '^isXi&i)}— Blood- serum. — Rounded, elevated, colorless, viscid-looking colonies, growing slowly and becoming well developed after thirty-six hours in the incubator. They may attain a diameter of 2 or 3 mm., and after a time they assume a brownish tint. Fig. 82. — Glanders bacilli from a young culture on potato ; X 2000 (Wright and Brown). Morphology. — Bacilli of medium size, variable in length, having round or conical ends, and frequently showing faintly stained areas in their protoplasm (Fig. 82). The larger forms of the bacillus are usually slightly bent or wavy in outline. Slight irregularities in shape may be observed. The mor- phology varies considerably on different culture-media. In cover-glass preparations from the lesions the bacilli * Loffler: Arbeitcn a. d. Kais, Gesundheitsamte, Bd. I, 1886. 14 210 PATHOLOGICAL TECHNIQUE. usually appear somewhat longer and thicker than the tuber- cle bacillus, and show numerous sharply defined, unstained or faintly stained areas in their protoplasm (Fig. 83). They have rounded or conical ends, and are sometimes slightly irregular in shape. As a rule, they are present in small numbers. If Loffler's methylene-blue solution is used for staining the cover-glass, it should be heated ; if carbol- fuchsin is used, it should be followed by a slight decolori- zation with 95 per cent, alcohol to better differentiate the bacilli. Potato. — After thirty-six hours in the incubator a rather thick, colorless, viscid-looking layer appears^ which soon Fig. 83. — Glanders bacilli in a cover-glass preparation from a lesion in a guinea-pig, showing the marked irregularity in the staining of the bacilli ; X 2000 (Wright and Brown). assumes a brownish tint and resembles honey in appearance. Later the brown color changes to a dark reddish-brown, and the growth becomes thicker and more opaque, while the potato takes on a dark-gray color. Pathogenesis. — When inoculated subcutaneously into guinea-pigs, the characteristic results are swelling and inflam- mation of the scrotum, appearing after a variable number of days, often after a week. The animals usually survive several weeks, with ulceration at the point of inoculation. The lesions produced consist in suppurative processes or abscess-formations in or about the testes, in the lymph-glands, in the anterior nares, about the joints, and in other situations, besides small grayish BACTERIOLOGICAL METHODS. 211 nodules or areas in the viscera — tiie so-called " glanders tubercles." The suprarenal capsules usually show red .areas, and they may be enlarged. On microscopical exami- nation the small nodules as well as the extensive suppura- tive areas will be found to be composed of necrotic material containing leucocytes and fragments of chromatin. The distribution and extent of the lesions vary with each animal, but the involvement of the testis or its membranes is prac- tically constant and pathognomonic of the bacillus of glan- ders. This involvement of the testis may consist, in early cases, in the presence of yellow foci in or about the tunica vaginalis, or in later cases the organ may show large yellow areas with purulent softening. Intraperitoneal inoculation with virulent cultures may be followed by death within forty-eight hours, with fibrinous exudate on the peritoneum in which minute grayish nodules are seen. The nodules are made up of a material which is apparently mainly dead or degenerated leucocytes and des- quamated peritoneal endothelium, together with many chro- matin fragments. In these acute cases also microscopical examination of the spleen and liver may show the presence of small nodules identical in structure with those seen in the more chronic cases. For the purpose of producing with cultures the characteristic lesions of the testis or its coverings it is better to inoculate the animal subcutaneously, for in the rapidly fatal intraperitoneal inoculations with virulent cultures these may not show any marked changes. The bacilli may be cultivated from the lesions, but not from the blood of the heart, in the chronic cases. They may be present in the blood of the heart, however, in small numbers in rapidly fatal infections following intraperitoneal inoculation. Field-mice may die from subcutaneous inoculation in about seventy-two hours. The most conspicuous lesion pro- duced is enlargement of the spleen, with the presence in it of minute grayish nodules. White mice are immune. Rabbits are not so susceptible as guinea-pigs to the infection. 212 PATHOLOGICAL TECHNIQUE. Decolorized by Gram's method. Not motile. Spore- formation not probable. Rate of growth is slow. Bouillon. — Diffusely clouded, with the formation of a viscid sediment. Litmus-milk. — Gradually turned red and coagulated. Agar-agar and Gelatin. — Growth not especially charac- teristic. Occurrence. — Found in the lesions of glanders and of farcy, and may invade the blood-stream in small numbers in acute cases of infection. Grows in the tissues in clumps or groups as well as scattered. In lesions on exposed sur- faces it may be accompanied by the pyogenic cocci. We have succeeded in demonstrating the presence of the bacillus in the sputum of a case of human glanders by inoculation of a guinea-pig with the sputum. Diagnosis. — In a case of suspected glanders the dis- charges from sinuses or ulcerated surfaces, or the contents of pustules, are to be examined for the presence of the bacillus of glanders by the usual methods. The material for examination may be collected on "' swabs." With this a guinea-pig is to be inoculated and cultures and cover-glass preparations are made. If the material be from sinuses or ulcerated surfaces, the isolation of the bacillus by cultures will be difficult, owing to the presence of other or- ganisms. The guinea-pig is to be inoculated in the peri- toneal cavity by introducing the infected swab into it through an incision in the abdominal wall, or by injecting about i c.c. of a suspension in bouillon of the suspected material into the peritoneal cavity with a hypodermic syringe. If the bacillus of glanders is present, the scrotum will usually show the characteristic swelling and inflammation in the course of three or four days, and death will occur after some weeks. In some cases the animal may die in thirty- six or forty-eight hours. In any case the characteristic lesions of glanders will be found as described elsewhere, and the bacillus may be isolated from them by cultures. The spleen will practically always yield glanders bacilli in pure culture even if no macroscopical lesion can be made out. BA CTERIOL O GICA L ME THODS. 213 In cultures the organism should show those character- istics of morphology, of culture, and of pathogenesis which have been described above. Bacillus of Chancroid (Bacillus of Ducrey). — In smears from the lesions the bacilli appear as short, round- ended rods, about 1.5 fi long and 0.5 fi thick, occurring characteristically but not always in chains. The middle portion of the rods does not stain so deeply as the ends. The bacilli are decolorized by Gram's method of staining, and are not motile. The following description of the cul- FlG. 84. — Bacillus of chancroid in smear preparation from pus (Lincoln Davis ; photo by L. S. Brown). tural peculiarities of the chancroid bacillus are based on the observations of Dr. Lincoln Davis in the Laboratory of the Massachusetts General Hospital.' The bacillus does not grow on the ordinary culture- media, but may be cultivated in blood or in media contain- ing one-third its volume of blood. It' is essential that the blood be fresh. In tubes containing blood, or a mixture of bouillon and blood, after twenty-four hours in the inctibator, the growth appears as whitish flocculi at the bottom of the tube. These flocculi are composed of tangled chains of the bacilli, the chains being often of extreme length. The 1 " Observations on the Distribution and Culture of the Chancroid Ba- cillus," Journal of Medical Research, vol. ix., p. 401. 214 PA THOLOGICAL TECHNIQUE. individual bacilli, as a rule, have the same morphology and staining reactions as in the smears from lesions, but occa- sionally long or even filamentous forms may be seen. On the surface of slant tubes, composed of a mixture of fresh blood and agar, the bacillus forms, after forty-eight hours in the incubator, small, rounded, grayish colonies difficult to pick up with the platinum wire, because they tend to glide before it. The bacilli from these colonies appear in smear preparations in short chains and singly. Involution forms are early apparent among the bacilli in all cultures. The bacillus dies out in cultures after about three days. Upon a monkey of the genus Macaccus, small ulcerations in the skin were produced by inoculation with cultures. - -X-:JU ^1 Fig. 8s. — Bacillus of chancroid from culture (Lincoln Davis ; photo by L. S. Brown). Method of Isolation from the Lesions. — Dr. Davis found that pure cultures were readily obtained by inoculating a small quantity of freshly drawn human blood in small tubes with material from the lesions, the fresh blood apparently inhibiting or destroying other bacteria. These small tubes containing blood are most easily prepared in a way devised by J. H. Wright. A small glass tube, about 5 or 6 cm. long BACTERIOLOGICAL METHODS. 21 5 and 4 or 5 mm. in internal diameter, is drawn out into a fine caliber at one end, and is then sterilized throughout in the gas-flame. When cool, the pointed extremity of the tube is immersed in the blood obtained from a needle-prick in the skin of the dorsum of the thumb near the nail, and then by- manipulation of the tube the blood is caused to flow into it. In this way a sufficient quantity — say, 0.2 to 0.5 c.c. — is easily collected in the tube, after which the pointed end is sealed in the flame and the tube is ready to be inoculated. The other end of the tube is plugged with cotton, which is impregnated with paraffin to prevent evaporation. The skin, before being pricked, is sufficiently cleansed by soap and water, followed by alcohol. A small tourniquet is applied about the base of the thumb, to increase the flow of blood from the needle-prick. Bacillus Proteus (Proteus Vulgaris). — Morphology. — Bacilli of very variable length, sometimes appearing like cocci or as filaments. Motile, being provided with terminal flagella. Does not stain by Gram's method. Colonies in Gelatiti. — Rapid growth with liquefaction of the gelatin. In a medium containing 5 per cent., instead of 10 per cent., of gelatin prolongations from the margins of the colonies may be formed. These may become separated from the mother colonies and form daughter colonies. Motions may be observed in these prolongations. Gelatin Stab. — Rapid liquefaction along the line of inocu- lation with cloudiness of the liquefied gelatin and a floccu- lent deposit. Agar-agar Slant. — Widely spreading, thin, moist, grayish- white layer. Potato. — Dirty white, moist layer. Litmus-milk. — Turned pink and slowly coagulated. Odor. — The cultures generally have a putrefactive odor. Pathogenesis. — Intravenous, intraperitoneal, or intramus- cular inoculations of rabbits may produce death in twenty- four to thirty-six hours after moderately large doses. Liq- uefied gelatin-cultures are said to be more virulent than 2l6 PATHOLOGICAL TECHNIQUE. bouillon cultures. Guinea-pigs seem to be less susceptible than rabbits to infection with this organism. Occurrence. — This bacillus and its varieties are among the most common and widely distributed putrefactive bacteria. It occurs in the intestinal contents. In pathological exam- inations it may be found in peritonitis and in abscesses, usually associated with other bacteria. It may also invade the circulating blood. The so-called " proteus group" includes several varieties of similar organisms — viz. the proteus vulgaris, the proteus mirabilis, and the proteus Zenkeri. The latter does not liquefy the gelatin, while the proteus mirabilis liquefies it slowly. Bacillus Mucosus Capsulatus.' — Blood-serum. — After twenty-four to thirty-six hours in the incubator the colonies ',^x'~y — .•;|'.^^^ Fig. 86. — Bacillus mucosus capsulatus, from the sputum of a pneumonia patient; x looo (Frankel and Pfeiffer). appear as translucent, colorless, rounded, convex elevations, resembling drops of mucus. If few in number, they may attain a diameter of 2-3 mm. They are viscid, and when touched with the platinum wire may be drawn out into ' Friedlander : Fortschritte tier Medicin, 1883, Bd. 1, S. 715 ; C. Fricke : Zeitschrift f. Hygiene ii. Infectionskrankheiten, Bd. 23, 1896. BACTERIOLOGICAL METHODS. 21/ threads. The water of condensation may become thick or viscid from the growth of the organism in it. Morphology. — Bacilli of moderate size, usually two or three times as long as broad, with rounded ends, occurring frequently in pairs and sometimes in long forms. Occasion- ally in cultures it shows a wide capsule. The capsule, how- ever, is best shown in cover-glass preparations from infected tissues (Figs. 86, 88). Pathogenesis. — White mice, rabbits, and guinea-pigs die from septicemia in a short time after inoculation, the capsule bacilli being present in the organs and blood of the heart in large numbers. Fig. 87. — Bacillus mucosus capsulatus, from a culture ; X 1000 (Wright and Brown). White mice die in twenty-four hours to three days. Rab- bits inoculated in the ear-vein and guinea-pigs inoculated in the peritoneal cavity may die within twenty-four hours. Subcutaneous inoculation of the animals last named leads only to local suppuration. The lesions produced by this organism consist in marked congestion of the super- 2l8 PATHOLOGICAL rECHNIQUE. ficial veins, hemorrhage into the lymphatic glands, and en- largement and softening of the spleen. In the guinea-pig a hemorrhagic condition of the supra-renal capsules is present, and in the peritoneal cavity there may be a small amount of clear, rather viscid fluid containing the bacilli in large numbers. The organs on microscopic examination may show pecu- liar areas in which the cells and nuclei are shrunken and in which the bacilli are aggregated. Fig. 88. — Bacillus mucosas capsulatus in blood; X looo (Frankel and Pfeiffer). Decolorized by Gram's method. Not motile. Does not form spores. Glucose Agar-agar Stab. — Growth along the line of inocu- lation, with the production of a few gas-bubbles in the medium. Bouillon. — Clouded with the formation of a thin pellicle. Potato. — Thin, colorless, viscid layer. Litmus-milk. — Turned red and coagulated. Gelatin. — Growth not remarkable. There apparently exists a number of varieties of aerobic capsu- lated bacilli differing from one another only in non-essential par- ticulars. The organism here described is to be taken as a type of a group of closely-related bacteria of which the bacillus pneu- monicE of Friedldnder is a v.ell-known member. BACTERIOLOGICAL METHODS. 219 Occurrence. — This organism or closely related forms may be met with in broncho- or lobular pneumonia and in inflam- matory conditions of the air-passages generally. It may also be present in the upper air-passages of healthy individ- uals. It has been observed in inflammations of the middle ear, in empyema, meningitis, endocarditis, peritonitis, and in pus-formations. In fatal infections the blood-stream may be found invaded by the organism. It is held by some bacteri- ologists that the members of this group may be the infective agents in genuine croupous pneumonia in rare instances. Representatives of this group have been found in the soil, in the air, and in contaminated water. Bacillus of Tetanus.' — This bacillus will not grow in the presence of oxygen. Fig. 89. — Tetanus bacilli showing flagella, from a preparation stained by Dr. Hugh Williams ; X 2000 (Wright and Brown). Morphology. — Slender rods with rounded ends, which may grow into long threads. In the incubator spores are rapidly formed. These are round, wider than the bacillus, and are situated at the end of the rod, giving the appearance of a drum-stick or a round-headed pin (Fig. 93). The colonies in anaerobic glucose-gelatin cultures appear after several days as small clumps of interlacing fibrillae from 1 Kitasato : Zeiischrift f. Hygiene u. Infectionskrankheiten, Bd. 7. 1889. 220 PATHOLOGICAL TECHNIQUE. which delicate filaments radiate into the gelatin, which is slowly liquefied. The colonies in simple anaerobic glucose-agar plate cul- tures (see page 124) appear after twenty-four to forty-eight hours in the incubator, as groups and masses of long fila- ments radiating from a center (Fig. 92). V"/ Fig. 90. — Tetanus bacillus. Stab- culture in glucose-agar. In the upper layers of the medium the peculiar brownish coloration is shown. Fig. 91. — Bacillus of tetanus: six- days-old stab-culture in glucose-gelatin (Frankel and Pfeiffer). Pathogenesis. — Subcutaneous inoculation of mice at the root of the tail gives rise to tetanic symptoms in twenty- four hours, followed by death in two or three days. Guinea-pigs and rabbits are also susceptible to the infec- tion, the period of incubation in these animals being twenty- four to thirty hours in the former and two to three days in the latter animal, after subcutaneous inoculation. The symp- toms of tetanus appear first in the extremities nearest the BACTERIOL CICAL ME THODS. 221 point of inoculation. In mice the hind legs become rigidly extended backward. At the autopsy the bacillus is to be found only at the point of inoculation, and may be difficult or impossible to demonstrate there. Glucose-gelatin Stab.- — Growth along the line of inocula- tion, beginning 2 or 3 cm. below the surface, with delicate filaments radiating laterally into the gelatin (Fig. 91). Liq- uefaction and gas-production occur. In deep-stab cultures in glucose-agar faintly alkaline to litmus (see Fig. 90) growth appears first all along the line of inoculation to within about i cm. of the surface after about twenty-four hours in the incubator. Later, lateral out- growths extend into the medium from all along the line of ^;^:- * \ » '\; -oi^^^-^-' . .;,^1^^m|^^^;-v'^ '■ I* ' ', '<■' 1. f.- , ^^^^^ Fig. 92. — Colony of tetanus bacilli in anaerobic glucose-agar plate ; low mag- nifying power (Wright and Brown). inoculation below a point about i cm. below the surface. In the portion of the line of inoculation above this, growth is frequently observed up to the surface, but without lateral outgrowths. The growth eventually assumes the appear- ance of an inverted pine tree. A peculiar feature of the culture is the appearance of a brown pigmentation in the culture-medium in its upper layers in the form of a flat or cone-shaped zone. A small quantity of gas may be pro- duced. If the agar has a reaction of about i per cent, normal acidity to phenolphthalein (see p. 84) growth appears 222 PATHOLOGICAL TECHNIQUE. along the line of inoculation and spreads through the medium as a cloudiness extending to within a few millimeters of the surface. The employment of glucose culture-media not older than a week or so seems to be important for success in cultivating this organism. In the vegetative form the organism is sluggishly motile. It has numerous flagella. It is stained by Gram's method. Glucose-bouillon. — Growth appears first, after twenty-four to forty-eight hours, as a diffuse cloudiness. Later the fluid becomes clear, and a grayish sediment collects at the bottom of the tube. Only a small amount of gas is pro- duced. Occurrence. — Found in the soil, and often in the feces of herbivorous animals. In cases of tetanus the bacillus is to be found only in the wound or at the point of inoculation. It does not invade the blood-current. The bacillus of tetanus acts by the production of a " toxin " or " toxalbumin!' This is also produced in cultures. It may be demonstrated in the bacteria-free filtrate of bouillon cult- ures some days or weeks old. A very few drops of this fluid will give rise to fatal tetanus in a mouse. Method of Isolation. — Tetanus bacilli will grow in aero- bic culture if other bacteria are growing with them. Since tetanus wounds usually contain other bacteria, all that is necessary to obtain an impure culture of the tetanus bacillus is to inoculate an ordinary blood-serum culture-tube (see page 79) with material from the wound. After several days or a week in the incubator, if tetanus bacilli are present they can be recognized by cover-glass preparations from the growth in the tube by their morphology and spore- formation (see Fig. 93). There will also be a peculiar, stinking odor about the culture. The isolation of the tetanus bacillus is now to be proceeded with as follows: Mix a loopful of the mixed growth on blood-serum with a tube of sterile bouillon, and heat in a water-bath for at least fifteen minutes at 80° C, then make anaerobic cultures from this (see Anaerobic Methods, page 121), taking several loopfuls for inoculation. BACTERIOLOGICAL METHODS. 223 If other spore-bearing bacilli are present in the mixed culture in the blood-serum tube, it will be necessary to use some form of anaerobic culture on a solid medium in order to obtain separate colonies of the tetanus bacillus for further cultures. The bacillus may be isolated from wounds and from the Fig. 93. — Spore-bearing tetanus bacilli in an impure culture on blood-serum from a case of tetanus. In the bacillus on the extreme left the beginning of spore-formation is shown (Wright and Brown). soil by inoculation of mice subcutaneously, and proceeding as above described with material from the seat of inoculation. Bacillus Aerogenes Capsulatus.'— Will not grow in the presence of oxygen. Morphology. — Bacilli of about the thickness of the anthrax bacillus, variable in length, but usually 3 to 6// long. Ends rounded or square cut. Occurs singly, in pairs, in clumps, and sometimes in short chains, less frequently in threads and long chains. May show unstained spots or deeply staining granules in the protoplasm. Capsules may be frequently demonstrated in the specimens from the tissues, and sometimes in agar- agar cultures. Colonies in anaerobic cultures are grayish to brownish- white, with a central darker spot by transmitted light. In time they may attain a diameter of 2 to 3 mm. or more. Colonies in the depths are spherical or oval, sometimes pre- senting knob-like or feathery projections. Effects on Animal Tissues. — Not pathogenic for rabbits. 1 Welch and Flexner : Journal of Experim. Medicine, vo!. i. No. i, 1896. 224 PATHOLOGICAL TECHNIQUE. If a rabbit that has received 0.5 to i C;C. of a bouillon cuhure injected into the ear-vein be killed immediately after- ward and the body kept for twenty-four hours at a tempera- ture of 18° to 20° C, or for four to six hours at a tempera- ture of 30" to 35° C, the vessels and organs will be found to contain a great quantity of gas and large numbers of the bacilli. The organism multiplies post-mortem in the blood of the animal and produces the gas. This effect upon the tissues of the dead animal is characteristic of the bacillus. The subcutaneous inoculation of guinea-pigs with young cultures may produce fatal gas phlegmons. The hemor- rhagic fluid from the dead animal is virulent for other guinea- pigs, and may be virulent for rabbits. Gas-production is marked in agar-agar and gelatin cultures containing glucose. The gas produced burns with a blue flame and is odorless. Gelatin is liquefied slowly and to a limited extent. Glucose Bouillon. — Diffusely clouded at first, later becom- ing clearer, with an abundant whitish, more or less viscid sediment. Milk. — Coagulated, the clot being firm, retracted, and fur- rowed with the marks of gas-bubbles. Potato. — Growth thin, moist, and grayish-white, or it may not be visible. The bacillus is stained by Gram's method. It is not motile. Spore-formation has been observed by E. K. Dunham. The vitality of the organism depends upon the character of the culture-medium and the mode of cultivation. It sur- vives longer when cultivated by Buchner's method (see page 124) than when cultivated under hydrogen. Cul- tures on glucose media are shorter lived than those on plain media. Occurrence. — Occurs at autopsies in which gas-bubbles are present in the larger vessels, accompanied by the formation of numerous small cavities in the liver containing gas. It has been found also in emphysematous phlegmons, in puer- peral sepsis, in peritonitis, and in other conditions. BACTERIOLOGICAL METHODS. 225 Bacillus of Malignant Edema.'— This bacillus will not grow in the presence of oxygen. Morphology. — Rather large bacilli, sometimes growing into threads (Fig. 94), but occurring frequently in pairs, in which the proximal ends are square while the distal ends are rounded. Forms oval spores in the middle of the rod, which may give the rod a spindle or oval shape. The colonics in anaerobic glucose-gelatin cultures appear as spheres of cloudy liquefied gelatin marked by delicate radiating streaks. Gas-bubbles are formed in the mediun", (Fig- 95)- Fig. 94- — Bacillus of malignant edema from the edema fluid of a guinea-pig inoculated with garden-earth ; X looo (Frankel and Pfeiffer). In glucose-agar the colonies appear as hazy points made up of interlacing filaments and resembling very much the colonies of the tetanus bacillus. Pathogenesis. — Subcutaneous inoculation of mice, guinea- pigs, and rabbits is followed by death in from sixteen to forty-eight hours, depending upon the animal, mice being most susceptible. The typical lesions are extensive sub- cutaneous edema containing gas-bubbles and more or less blood, and enlargement of the spleen. The bacilli are found ' Liborius: Zeitschrift f. Hygiene u. Infectionskrankheiten, Bd. i, 1886. 15 226 PATHOLOGICAL TECHNIQUE. in the edema, in the viscera, and on the serous surfaces of the organs, but not in the blood of the heart if the exam- ination be made immediately after death, except sometimes in mice. The organism is not capable of multiplying in the living blood, owing to the presence of oxygen. In inoculat- ing subcutaneously a deep pocket should be made in the skin, and the material for inoculation introduced into the tissue as far away from the opening as possible. This is to prevent the access of too much oxygen to the organism. Slightly motile. Flagella may be demonstrated by special stain- ing methods. The bacilli in tissues are stained by Gram's method, but in cultures most of them are decolorized by it, probably because of rapid de- generative changes in them. Growth in anaerobic agar-agar and bouillon culture is good, but not characteristic. Occurrence. — Widely distributed in the soil and in putrefying sub- stances. Only a very few cases are on record of infection in man by this bacillus. In a paper by A. H. Gould ' will be found the account of a case in the Massachusetts General Hos- pital, a review of the literature, and a more extensive description of the bacillus than is here given. Actinomyces. — The proper clas.sification of the micro-organism known as actinomyces is somewhat uncertain. The published descriptions of various examples of it vary widely and have led to much confusion. It is probable that some of the micro-organisms cultivated ' " A Case of MaliTOaPt Edema," Annah of Surgery, October, 1903. Fig. 95- — Bacillus of malig nant edema; colonies growing in glucose-gelatin (Frankel and Pfeififei). BACTERIOLOGICAL METHODS. 227 and described as actinomyces were not such, but were con- taminations or secondary invaders of the tissues. Probably the best opinion to-day considers actinomyces to belong to a group of micro-organisms which, on account of their branching, are regarded as more highly developed than are the bacteria, but which are not so highly organ- ized as are the moulds or hyphomycetes. The members of this group, of which a number are known, are named streptothrices. While there is some objection to this name, it seems to us to be satisfactory. 1^ , ^'wim 1 ^B^^^' Si-"" ; -^ .--it*/^- /'■,«■-:'•-' ■■5<^ I ^Bffs- 'W'-'^ I JflM^fef' - '■ ' ■ I- "~ J Fic. 96. — Actinomyces granule crushed beneath a cover-glass, showing radial striations in the hyaline masses. Preparation not stained ; low mag- nifying power (Wright and Brown). . The organism appears in the pus from subacute or chronic suppurative lesions of the disease actinomycosis, as grayish or yellowish granules, usually less than i mm. in diameter. Sometimes these granules are aggregated in groups of two or three, and thus appear as lobulated larger granules. They are friable, and when gently crushed beneath a cover- glass and observed under the microscope, they are seen to have been broken up into hyaline rounded masses, at the margins of which, on close inspection, fine radial striations or filaments or hyaline club-shaped bodies, all closely set together, may be seen (Figs. 96, 97). The club-shaped 228 PATHOLOGICAL TECHNIQUE. bodies are variable in size, and are composed of a hyaline, refringent substance. The appearance of radial striation in the granule, when observed with the microscope, due to the presence and radial arrangement of these hyaline bodies, gave rise to the name " ray-fungus " for this parasite. Not all of the granules have these " clubs." In the granules obtained from the lesions in man they are much less fre- quently observed than in those obtained from the lesions in cattle. If a cover-glass preparation be made by breaking up one of the granules and staining with Gram's method, there will usually be found, upon examination with an oil-immersion lens, isolated and matted filaments, many of which may be Fig. 97. — A portion of an actinomyces granule crushed beneath a cover- glass, showing the "clubs." The preparation not stained; moderately higli magnifying power (Wriglit and Brown). seen to branch, in addition to longer and shorter fragments of filaments and fine detritus of the same (Fig. 98). The filaments are usually more or less wavy in their course, and are, as a rule, slightly thicker than the tubercle bacillus. Some of the filaments will be found to stain homogeneously ; others do not stain so deeply, and show numerous deeply staining points in their substance. If clubs are present in the granule, they also may be found scattered throughout the preparation. In sections of the tissues stained by Gram's method two chief forms of granules are found. In one of these forms the granule is seen to consist of filaments embedded in a hyaline substance, and usually arranged at the periphery in an indefinite radiate manner (Fig. 99). At the margin BACTERIOLOGICAL METHODS. 229 of the granule the filaments are usually much more numer- ous than in the central portions, where the hyaline material predominates. This hyaline material apparently consists of degenerate or dead filaments or their remains. The other form of granule seen in sections is distinguished by possessing at its margin a row of closely set radiating club- shaped bodies composed of hyaUne substance which does not stain by Gram's method (Fig. 100). These are the " clubs" previously mentioned, and they may occupy more- or less of the circumference of the granule. In certain Fig. 98. — Branching actinomyces filaments in a cover-glass preparation made from an actinomyces granule stained by Gram's method; X looo (Wright and Brown). instances a Gram-staining filament may be seen in the cen- tral portion of a club. The main mass of this form of granule is not essentially different from that of the first- mentioned form. The characteristics of both forms of granule may be found in some granules. The club-shaped bodies are to be regarded as products of degeneration of the marginal filaments. In some cases isolated or small groups of filaments may be found scattered among the pus-cells in the lesions. Cultures. — Actinomyces grows best in the incubator. On 230 PATHOLOGICAL TECHNIQUE. the surface of slant tubes of blood-serum and agar-agar the organism grows in larger and smaller grayish-white, shining, elevated colonies. If the colonies are few in number, they may attain a diameter of several millimeters, with highly elevated, irregular central portions and thin, wavy, marginal portions. If the colonies are very numerous, they may be confluent, and form a grayish granular layer. Growth on f • ~ Fig. 99. — Colony or granule of actinomyces in a section through a lesion, showing the Gram-stained filaments and hyaline material and also the pus-cells surrounding the colony (Wright and Brown). the surface takes place more rapidly, and with a develop- ment of more numerous colonies if the culture be kept under anaerobic conditions, the maximum of growth being obtained in about a week. In stab-cultures in sugar-agar the colonies appear as grayish granules along the line of inoculation. In bouillon the organism grows in the form of grayish- white, friable granules or clumps at the bottom of the tube. BACTERIOLOGICAL METHODS. 231 The maximum of growth is obtained in about four or five days under aerobic conditions. The culture-fluid is never clouded. The morphology of the organism in cultures is subject to considerable variation. In bouillon-cultures it grows in the form of longer or shorter filaments which sometimes may be seen to branch. Scattered along the filaments are sharply defined, deeply staining, oval or rounded areas occupying the whole diam- i^^ *V >*,- ,' .:, • '--1- \'^A«>* - i/ <'.^'^- \^ A i ■'.'V 1 Fig. icx). — Colony or granule of actinomyces in a section through a lesion, showing the peripheral arrangement of the " clubs." In several instances the central stained filaments in the " clubs " are seen ; x 750 (Wright and Brown). eter of a filament. Clubs are never observed in cultures, but sometimes round or oval swellings may be seen at the ends of the filaments in the older cultures. In cover-glass preparations from cultures on solid media the organism appears as longer and shorter rods, often more or less fusiform or club-shaped, and as slightly wavj^ fila- ments of variable length, sometimes segmented. Diagnosis. — The finding of the granules in suspected pus may be facilitated by spreading the pus on a slide. 232 PATHOLOGICAL TECHNIQUE. The identification of tiie organism is made certain only when the granules have been found to present the appear- ances described above after crushing under a cover-glass, and after cover-glass preparations made from them and stained by Gram's method show the branching filaments. Mycetoma (Madura Foot). — Mycetoma is a chronic inflammatory process, most commonly affecting the tissues of the foot, in which suppurative nodular swellings, sinus- Fk;. ioi. — Mycetoma. Black granule in a section from the lesions. The black rounded central mass represents the brownish hyaline refringent substance in which the fungus elements are embedded. Giant cells and granulation-tissue are shown (Wright and Brown). formation, and enlargement and distortion of the parts are prominent features. The bones may become involved and undergo a rarefying osteitis. The affection is characterized by the presence in the dis- eased tissue and in the discharge from sinuses, of peculiar granules, usually not more than one millimeter in diameter, but sometimes larger. These granules in certain cases are of a black color, of irregular shape, and are hard and rather brittle. In general they resemble grains of gunpowder. In other cases the granules are whitish, grayish, or yellowish in color, and are of a soft or cheesy consistency. They have BACTERIOLOGICAL METHODS. 233 been compared to fish-roe in appearance. A very few cases are also recorded in which the granules were of a red color. There is no good evidence to show that more than one kind of granules are ever present in any one case of the disease. l"^ Fig. 102. — Mycetoma; fungus elements from a black granule after maceration ; high magnifying power (Wright and Brown). On account of the fact that at least two very different kinds of granules are found associated with the lesions, two Fig. 103. — Mycetoma, showing the outgrowth of hyphce from one of the black granules after planting in fluid culture-medium (Wright and Brown). varieties or forms of the disease are recognized : the " mela- noid " or black variet)^ in which the granules are black, and the " ochroid " or pale variety, in which the granules are white to yellow in color. 234 PATHOLOGICAL TECHNIQUE. The pale variety is probably actinomycosis of the part, the " melanoid " or black variety is clearly not actinomycosis, and the parasite belongs to the hyphomycetes, a very dif- ferent class of organisms from that to which actinomyces belongs. The disease occurs frequently in India and some other sub-tropical countries. It is extremely rare in temperate climates. Wright has studied a case of the black variety of the dis- ease, and upon the results of this study ^ the following descrip- tion of the parasite is based : Fig. 104. — Mycetoma, showing details of the structure of the hyphre developed from the black granules (Wright and Brown). The black granules, characteristic of this variety of the disease, consist of a hard, rather brittle, amorphous, brownish or black substance in which a fungus mycelium is embedded (Fig. lOi). These fungus elements may be demonstrated by macerating the granules in a strong solution of alkali or sodium hyposulphite. This disintegrates the granule and dis- solves the brown embedding substance. The fungus ele- ments obtained from the granules by this maceration are shown in Fig. 102. ^ Journal of Experim. Medicine, 1898, vol. iii. Nos. 4 and $. BACTERIOLOGICAL METHODS. 235 The organism grows on ordinary culture-media about as rapidly as do the ordinary moulds of the laboratory. From a granule planted in bouillon numerous hyphae grow out into the fluid and may form eventually a whitish powder-puff-ball-like structure with the granule in the center (Fig. 103). The hyphae grow very long, are septate, have in places vacuoles, and form branches (see Fig. 104); on the surface of agar-agar the growth appears as a meshwork of widely spreading filaments of a grayish color. On potato it forms a dense, widely spreading, coherent membrane of velvety surface. In its central portions it is of a pale-brown color, but at the periphery it is white. A marked feature is the appearance of small spherical droplets of coffee-colored fluid on the surface of the growth after a time. It must be quite clear from the foregoing that the organ- ism belongs to the moulds or hyphomycetes. The formation of reproductive organs, such as .spore-bear- ing hyphse, was not observed. The results of animal inoculations were negative. Rabies (Hydrophobia).— The diagnosis of this disease from a pathological standpoint is usually made by the pro- duction of experimental rabies in a rabbit by intradural inoculation with material from the nervous system of the animal suspected to have died of it. The poison of the dis- ease is found in the brain, spinal cord, salivary glands, and pancreas. For purposes of inoculation a piece (i or 2 c.c.) of the medulla or brain, preferably the former, is rubbed up in a sterilized mortar with about 10 c.c. of sterihzed distilled water. The resulting fluid is filtered through absorbent cotton, and then through filter-paper, to remove tissue- shreds. Of the clear fluid thus obtained 4 or 5 drops are injected beneath the dura of a rabbit by means of a hypo- dermic syringe, the skull being trephined with a small tre- phine about 4 mm. in diameter. The most favorable place for opening the skull is at a point in the median line ju.st posterior to a line drawn through the middle of each eye. The symptoms of experimental rabies in the rabbit first 236 PATHOLOGICAL TECHNIQUE. manifest themselves after two weeks, never earlier, but they may not appear until later, not even until two months have passed. The first symptom is a weakness of the hind legs, followed by paralysis. The paretic condition soon extends to the fore legs, dyspnea appears, and death usually occurs in about three days after the onset of the symptoms. Para- lytic symptoms developing before two weeks are not due to infection with rabies, but to some other cause ; for instance, infection with the pneumococcus or other bacteria which may be present in the material inoculated. During the course of the disease the animal never appears stupid, with dull eyes, as in other infections, but remains " conscious," so to speak, until the last. It is claimed that the diagnosis of rabies may be made by means of microscopical examination of sections of the me- dulla and spinal cord and of the peripheral ganglia of the cerebrospinal and sympathetic nervous system. The lesions in the medulla and spinal cord consist in the accumulation of round cells in the perivascular spaces and about nerve- cells and central canal, together with degeneration of nerve- cells. The lesions in the peripheral ganglia are more im- portant and consist of degeneration of the ganglion-cells, together with a proliferation of the peculiar endothelial cells enveloping the ganglion-cells. The plexiform ganglia of the pneumogastric nerve are the best to examine. McCarthy and Ravenel ' state that they know of no other acute disease in dogs presenting these lesions, and they con- sider them to be 'pathognomonic of rabies in an animal that had manifested the clinical symptoms of the disease. In order that these characteristic lesions may develop and permit of a diagnosis being made, it is necessary that the suspected animal be allowed to die of the disease or be killed in its advanced stages. ^ Joiir. Amer. Med. Asscc, March 21, 1903. PART III. HISTOLOGICAL METHODS. Introduction. — The ideal function of the technique of pathological histology is so to fix tissues for microscopic examination that every tissue-element or pathological prod- uct is perfectly preserved with all its morphological and chemical properties intact, and so to stain tissues. that every tissue-element or pathological product can be readily differ- entiated from any other tissue-element or pathological prod- uct that resembles it. In certain respects only has this ideal yet been reached, but the number of differential stains is increasing yearly. In the following pages the various steps in the prepara- tion and staining of tissues have been arranged, so far as possible, in logical sequence. LABORATORY OUTFIT. Microscopes. — The most important laboratory instru- ment is the microscope. It should be, so far as means will permit, the best that skill can produce. Excellent micro- scopes are manufactured both abroad and in this country, but no make of microscope can be unconditionally recom- mended. Undoubtedly the best microscopes in every par- ticular and the most expensive are those manufactured by Zeiss. It is important for a beginner in microscopy, before buy- ing a microscope of any make, to have it carefully examined and its lenses tested at a pathological or other laboratory by some one skilled in its use. The continental form of stand of medium size is to be preferred to all others. The large stand is undesirable, because it is too heay and too high for 23r 238 PATHOLOGICAL TECHNIQUE. comfortable use. It should be furnished with rack and pin- ion, and with micrometer screw for coarse and fine adjust- ment, with a triple nose-piece, and with an Abbe condenser and iris diaphragm. The necessary objectives are a low and a high dry, and a -^-^ oil-immersion. Two eye-pieces, a low and a high, will be found sufficient for all ordinary purposes. The stands, oculars, and objectives of the Zeiss make generally used are the following : Stands, 11^, \Na, YSlb. Oculars, 2 and 4. Objectives, AA, D, and ^ oil-immersion. Or in the apochromatic series, Oculars, 4, 6, and 8. Objectives, 16.0, 8.0, 4.0, and oil-immersion 2.0 mm., apert. 1.30. Even if all these different parts cannot be purchased at the same time, it is important to buy a stand to which they afterward may be added, for the Hst includes only what every medical practitioner should have at his service for the proper examination of urine, sputum, blood, etc. The apochromatic lenses and compensation oculars are too expensive to come into general use. Fortunately, they are more important for photomicrography than for general microscopic work. The oil-immersion lens should always be cleaned after using by wiping off the oil with an old linen or silk hand- kerchief or with the fine tissue-paper now manufactured for that purpose. If the lens is sticky, moisten the cloth with benzol or xylol ; Leitz recommends alcohol. The same process can be used if necessary for the dry lenses, but it must be done quickly, so as not to soften the balsam in which the lenses are imbedded. Ordinarily a dry cloth is sufificient. In using the Abbe illuminating apparatus it is important to bear in mind that the best results are obtained, according to Zeiss, by employing the plain mirror, for the condenser is designed for parallel rays of light. The concave mirror is to be used only when some near object, such as the window- HISTOLOGICAL METHODS. 239 frame, is reflected into the field of vision or when artificial light is employed. A mechanical stage is now made which can be instantly- attached to any microscope. It is exceedingly useful for blood-counting or for searching carefully the whole surface of a stained cover-slip. For ordinary work it is undesirable. For microscopic work the best illumination is that obtained from a white cloud, although for some purposes the light which filters through a white curtain on which the sun is shining is very useful, especially with the highest powers of the microscope. When artificial light is necessary, the Welsbach burner, or, better still, the Edison electric lamp with ground-glass globe, will be found very satisfactory. The slight yellowish tint of the light can be corrected, if necessary, by means of a piece of blue glass inserted over the mirror or just below the object to be examined. For drawing, the Abbe camera lucida will be found ex- tremely useful and convenient. Freezing Microtome. — Freezing by means of the evaporation of ether, more rarely of rhigolene, is the method in general use. The process is both expensive and slow. A much cheaper and more rapid method of freezing was originated several years ago in the Sears Patho- logical Laboratory by Dr. S. J. Mixter, and has since been in constant use both here and in the hospitals in Boston. This method consists in the employment of compressed carbon-dioxid, which is found in commerce in iron cylinders containing each about twenty pounds of liquefied gas. It is commonly used for charging beer and soda-water. As a rule, the cylinders are loaned, so that it is necessary to pay for the contents only. The cylinder must be securely fastened in an upright position near the microtome, with its valve end below and with its escape-tube on a level with the entrance-tube into the freezing-box. The cap covering the escape-tube of the cylinder should have a small hole bored through it, and into this hole a small brass tube about 5 cm. long, with a fine bore, should be tightly driven. This permits the use of a 240 PATHOLOGICAL TECHNIQUE. smaller stream of gas than the escape-pipe of the cylinder would otherwise furnish. The same tap can be kept to use on ail future cylinders. The cylinder is connected with the microtome by means of a short piece of thick, strong rubber-tubing with small bore, so as to fit snugly over the escape-tube of the cylinder Fig. 105. — Freezing microtome. and the entrance-tube into the freezing-box. It is advisable to wire each end of the rubber-tubing around the tube if incloses. In order to obtain better leverage and more perfect con- trol over the escape of the gas than are needed for the purposes for which the cylinders are ordinarily used, it is necessary to lengthen to about 25 cm., in whatever way HISTOLOGICAL METHODS. 241 seems best, the handle of the key which opens the escape- valve. The first time the cylinder is used for freezing, a little water may escape, causing considerable sputtering. In freezing, the valve should be turned carefully, so that the gas may escape slowly and evenly. Tissues fixed by alcohol or any other reagent, except formaldehyde, must be washed in running water for some hours before they can be frozen. The freezing-box of the microtome should be stronger than when intended for the use of ether. The Bausch and Lomb table microtome. No. 3050, with freezing attachment No. 3082, is recommended. For cutting frozen sections the blade of a carpenter's plane, 2-| inches wide, mounted in a wooden handle (Fig. 106), will be found very serviceable and easy to sharpen. Fig. 106. — Knife for freezing microtome, mnde from the blade of a carpenter's plane. Directions for Cutting Frozen Sections. — Sections of 10 or 1 5 microns in thickness or as thin as good celloidin sec- tions, are readily obtained with most material by following these directions. The knife must be sharp and free from nicks. It must have a chisel edge, as shown in Fig. 106. It should be sharpened by grinding on a hone, and afterward by thor- oughly stropping on a razor-strop. Frequent stropping is just as neces.sary as in the case of the ordinary microtome knife. In cutting, grasp the knife by the thick wooden handle so that the end presses against the ball of the thumb and the palm of the hand, while the dorsum of the hand is upper- most ; then, with the wrist flexed and held against the chest, apply the edge of the knife to the glass ways of the micro- tome in such a manner that the edge, bevel side downward, 242 PATHOLOGICAL TECHNIQUE. is at right angles to the direction of the ways and the long axis of the knife at an angle of 45 degrees to their surface ; now, holding the knife and wrist rigidly in the positions just indicated, push the cutting-edge quickly forward along the ways through the specimen by moving the body forward from the waist, in the mean while pressing the cutting-edge steadily downward upon the ways with constant force. Thus a strong constant downward pressure of the edge upon the ways is maintained, and at the same time great steadiness and power are given to the cutting stroke, which are condi- tions that are very important for obtaining thin sections. With the fingers of the other hand manipulating the wheel of the microtome screw, a number of sections should be cut in quick succession in the maimer indicated without chang- ing the angle of the knife or the position of the hand and wrist above described, the edge of the knife on the back- ward movement being lifted from the ways only enough to clear the cut surface of the specimen. The sections will usually adhere to the knife, and a number of them may be allowed to collect thereon. They are removed from the knife by immersing it in water, in which they will float and flatten out, no matter how much wrinkled and compressed upon the knife they have been. The cutting of a number of sections in quick succession without pausing to remove each section from the knife seems to be necessary for obtain- ing the thinnest sections. The consistence of the frozen tissue is important. The specimen immediately after freezing will usually be too hard to cut without yielding sections that break over the edge of the knife, and are, therefore, to be rejected. If this happens, wait a few seconds and thereafter cut a section or two at short intervals until the specimen is found to have a con- sistence yielding satisfactory sections, whereupon a number of sections should be cut in quick succession as above de- scribed. The piece of tissue from which the sections are to be cut should not be thicker than 5 mm. Celloidin Microtome. — There are two types of celloidin microtomes — one in which the object is raised by a screw, HISTOLOGICAL METHODS. 243 a second in which the object is raised by being moved up an incHned plane. The first type of machine is the better, for two reasons : the screw affords greater accuracy in the even elevation of the object than is possible with an inclined Fig. 107. — Large laboratory microtome. plane, and the object remains at all times in the same rela- tive position with regard to the knife, so that an equally long sweep of the blade can be obtained for every section. An excellent instrument of this type is made by Bausch & Lomb (Fig. 107). For practical work it is much to be preferred to the elaborate Schiefferdecker-Becker microtome, designed for cutting sections under alcohol. A new and wholly original microtome, in which the knife remains fixed and is clamped at both ends, while the object- holder, which is raised by a screw, moves back and forth be- neath the knife, has recently been designed by Dr. C. S. Minot and is manufactured by Bausch & Lomb (Fig. 108). It is intended both for celloidin and for paraffin work. When but one instrument can be afforded, it is believed that this model will be found the most serviceable for both kinds of work. A drop-bottle on an elevated stand, with screw arrange- ment for regulating the amount of alcohol, is the most con- 244 PATHOLOGICAL TECHNIQUE. venient method for keeping the object and the knife wet while cutting ; 8o per cent, alcohol should be used. The Minot-Blake Microtome.' — This microtome is specially designed for cutting sections of i and 2 micra in thickness, but it may be adjusted to cut sections up to lO micra. The main sliding parts are designed to take advan- ' This microtome is made by the Buff Manufacturing Co., 506 Atlantic Ave., Boston, Mass. HISTOLOGICAL METHODS. 245 tage of the principle of three-point bearings, so as to avoid minute irregularities in their motions. The knife is provided with a brace to prevent the elasticity of the knife-edge from causing "skipping" when the machine is adjusted to i or 2 micra. Fig. 109. — Minot-Blake microtome. In operating this machine for thin sections the following important points should be borne in mind : I. The machine must be in accurate adjustment. The brace to the knife is to be so applied as not to produce a twisting strain upon it. The upper edge of the brace should he about -^-^ of an inch below the knife-edge. The knife is to be canted just enough to obtain clearance, and no more. 246 PATHOLOGICAL TECHNIQUE. 2. The knife-edge should be so well polished that, when observed with the microscope under a magnifying power of about 90 diameters, it appears as a straight line. 3. The specimen must be well embedded, and the paraffin block containing it, as well as the brass carrier to which it is attached, should be cooled in. ice-water immediately before cutting. The knife and its brace should be Hkewise cooled. The paraffin block is to be carefully trimmed as described elsewhere. Paraffin Microtome. — Although paraffin sections can be cut on a celloidin microtome, it is preferable to have an Fig. iio. — M^not wheel microtome. instrument designed for the purpose. The Minot wheel microtome manufactured by Bausch & Lomb (Fig. no) can be thoroughly recommended. Paraffin Bath. — The best bath for keeping paraffin at a constant temperature is a thermostat of suitable size with hot-water jacket, such as is used for growing cultures of bacteria. The paraffin is kept in it on shelves in glass dishes HISTOLOGICAL METHODS. 247 of various sizes. The advantages of this method over the old way of using copper cups set into the top of a water- bath are that the paraffin is kept absolutely free from dust, each worker can have his own set of dishes, and the smallest bits of tissue can be readily found in them, because they are transparent. A paraffin melting at 50° to 52" C. will be found convenient for use throughout the year if the laboratory is kept fairly warm in winter. Many workers, however, prefer one par- affin melting at 45° C. for winter, and another melting at 48° C. for summer. A preliminary bath of soft paraffin is wholly unneces- sary, and only prolongs the objectionable stage of heating. The regulator should register only one or two degrees above the melting-point of the paraffin. Paraffin should be melted and decanted or filtered before use, as it often contains foreign material. When hot it runs through an ordinary filter without trouble. A hot-water jacket to the funnel is not at all necessary. Vulcanised Fiber. — For mounting celloidin prepara- tions nothing is so poor as cork, although it has been in use for years. The chief objections to it are that it does not fur- nish a rigid support to the imbejlded object ; that, unless weighted, it floats in alcohol with the specimen downward ; and that it yields a coloring material which stains both the Fig. III. — Blocks of vulcanized fiber. alcohol and the specimen. Wood is not much better, although, of course, much firmer. Glass blocks have been proposed, and might do fairly well if there did not exist an ideal substance — viz. vulcanized fiber. This can be obtained in boards or strips, preferably |- or f inch in thickness and 248 PATHOLOGICAL TECHNIQUE. sav/n to any desired dimensions. It is perfectly rigid, is heavy enough to sink specimens to the bottom of the jar in an upright position, is unaffected by alcohol or water, is light red in color, so that it is easily written on with a lead pencil, gives off no coloring material, and is practically indestructible. Two or three parallel cuts, i to 2 mm. in depth, should Fig. 112. — Diagram of the direction of the movements in honing. be sawn into the upper surface of each block, so as to give the celloidin a firm hold. Knives. — The knives for both the celloidin and the paraffin microtomes should be heavy and not too long, so as Fig. 113. — ^Diagram of the direction of the movements in stropping. to afford as great rigidity as possibles they should be bicon- cavdlso that they may be easily sharpened. It is important that4very one who does much work in a pathological labor- atory should learn to sharpen his own knives. The requisite HISTOLOGICAL METHODS. 249 skill is not difficult to acquire, and the time spent'in learning is fully compensated for by the ability always to have a sharp knife when it is wanted. For honing a knife either a fine water-stone or a gl»ss plate with diamantine and Vienna chalk may be used. I In honing, the edge of the knife is for- ward and the motion is from heel to toe. The knife should always be turned on its back, and the pressure on it should be at all times rather light. In stropping, the movement is reversed. The back of the knife necessarily precedes the edge, and the motion is from toe to heel. I The direction of the movements in honing and stropping iybest illustrated by the diagrams (Figs. 112, 113). Running; water for washing out specimens which have been fixed in Flemming and other solutions is most easily Fig. 114. — Large laboratory sink, showing adjoining shelf and arrangement for running water, supplied by having a water-pipe, furnished with numerous cocks 5—10 cm. apart, run horizontally over a slightly slop- ing shelf adjoining the sink. Attached to each cock is a rubber tube, with a glass tube in the end of it long enough to reach to the bottom of the jar (Fig. 114). By this arrange- 250 PATHOLOGICAL TECHNIQUE. ment the imoiint of water supplied to each specimen can be easily regulated. Slides should be of colorless glass with ground edges. The English form, measuring i X 3 inches (76 X 26 mm.), is to be preferred for ordinary use. Occasionally broader slides are needed. Thick slides are preferable to thin ones; the latter are so light that they are easily lifted by the oil- immersion lens ; they also seem to warp when heated to attach paraffin sections to them. Cover-slips should be square or oblong according to the shape of the specimen. Most dry lenses are adjusted for cover-glasses measuring 16 or 17// in thickness, so that if possible no cover-slips ranging outside of 15 to i8;« should be used. With an oil-immersion the exact thickness is not quite so important. Slides and cover-slips are cleaned by dipping in alcohol and wiping dry with a soft crash towel or old linen handker- chief To clean old slide preparations, heat them until the bal- sam softens so that cover-slips and slides can be drawn apart. The slides and cover-slips are then treated separately with nitric acid. A 10 per cent, solution is usually sufficient, but occasionally the strong acid will be found necessary. Some workers prefer equal parts of alcohol and hydrochloric acid ; still others the following mixture : Bichromate of potassium, 2 parts ; Sulphuric acid, 3 Water, 25 " A thorough washing in running water, followed by alco- hol, completes the process. Alkalies are not so good for cleaning purposes, because they attack the glass. Staining Dishes. — Watch-glasses are not satisfactory on account of their instability. Concave dishes with flat bottoms are much better for ordinary use, and can be ob- tained of several patterns. They should be large enough to hold 25 c.c. of fluid. The Syracuse solid watch-glasses are very good dishes of this shape. HISTOLOGICAL METHODS. 251- Stender dishes of various sizes will be found useful for many purposes. The "New Practical Staining Dish" (No. 16,618) manu- factured by the Bausch and Lomb Optical Co. is very useful for staining at once a number of paraffin sections. Fig. 115. — New practical staining disti. Oblong rectangular Petri dishes are very convenient for staining preparations mounted on the slide. For staining at once a large number of sections for class purposes, the Hobb's Tea Infuscr has been found very useful. If set in a small lemonade cup, but little stain is required. Large concave dishes holding looo c.c. will be found the most convenient for holding frozen sections of fresh tissue, because a slide can be dipped into them and under the sections. Metal Instruments. — Spatulas of different sizes are r Fig. 116. — Spatula. needed. They should be thin, smooth, and large enough, so that a section will not curl over the edge (Fig. 1 16). The best instrument for transferring sections under all cir- 252 PATHOLOGICAL TECHNIQUE. cumstances is a piece of platinum wire mounted in an ordi- nary screw needle-holder. It is pliable and can be bent to any shape, will not break like a glass needle when dropped, and is not affected by acids. Ladies' hat-pins form a cheap but serviceable substitute. They are readily bent to any desired shape by heating. Forceps, scissors, scalpels, and many other instruments required in microscopical work do not need any special mention. Bottles. — For cover-slip work and for staining on the slide dropping-bottles will be found extremely convenient. The patent T. K. pattern of 50 c.c. capacity, with flat top, is probably the best form and size. EXAMINATION OF FRESH MATERIAL. Fresh tissues may be examined either in teased prepara- tions or in sections. Teased preparations are made by cutting out a very small bit of the tissue in question and dividing it as finely as pos- sible, by means of two sharp, clean needles, on a slide in a drop or two of some indifferent fluid, such as the normal salt solution. Teased preparations are often made, for in- stance, of the heart-muscle when fatty degeneration is sus- pected. If the tissue is soft, the cells are easily obtained by simply scraping the cut surface with the edge of the knife. Sections of fresh tissues can be made with a razor or with a double knife, but much the better way, at least for general diagnostic purposes, is to use frozen sections, which can be very quickly and perfectly made with the freezing microtome. The fresh sections are put into salt solution in a glass dish large enough to permit of a slide being dipped into it, so that a section can be floated and spread out evenly on its surface. The slide is then carefully raised, the excess of fluid wiped off, and a cover-slip put on. If it is desired to stain the section, a few drops of Loffler's methylene-blue solution are poured over it after it is spread HISTOLOGICAL METHODS. 253 evenly on the slide. In a few seconds the coloring fluid is thoroughly washed off with salt solution, a cover-slip put on, and the section examined in the salt solution. If sections of fresh tissues are put directly into a staining fluid in the ordi- nary manner, they pUcker up and do not stain evenly. Sections of fresh tissue may be fixed, stained, cleared, and mounted in balsam by a slight modification of the method for frozen sections given on p. 263. This modification con- sists in covering the section with 95 per cent, or absolute alcohol after it has been spread out evenly on the slide as described in the method referred to. The alcohol is to be dropped on the section carefully from a drop bottle, in order to avoid folding. After thirty seconds the alcohol is drained off and the section flattened out on the slide with blotting- paper and further treated according to the method above mentioned. If the section is not treated with alcohol before blotting, it will adhere to the blotting-paper and not to the slide. Fresh preparations are often treated with chemicals for various purposes. Of these chemicals, acetic acid is the. most generally useful in pathological work. It shrinks the nuclei and renders their outlines more distinct. It swells connective tissue, making it more transparent, so that the elastic fibers which are unaffected stand out distinctly. It precipitates mucin and dissolves or renders invisible the albuminous granules so abundantly present in the proto- plasm in the cloudy swelling of various organs in disease. Its main use as" a reagent for fresh tissues is to demonstrate fat and to differentiate that substance from albuminous gran- ules. Acetic acid is ordinarily used in a i to 2 per cent, aqueous solution, a few drops of which are placed at one edge of the cover-slip, and then drawn beneath it by placing a piece of filter-paper on the opposite side. If in a hurr}^ however, stronger solutions, or even glacial acetic acid, may be used. Other reagents are of less importance, but are occasionally used. Osmic acid is sometimes employed in a i per cent, aque- 254 PATHOLOGICAL TECHNIQUE. ous solution to demonstrate fat, which it stains brown to black. An alcoholic solution of Scharlach R. or Sudan III. is being used more and more for the same purpose. It stains fat orange to red. Hydrochloric acid in a 3 to 5 per cent, solution is used to demonstrate calcification. Phosphate of lime is simply dis- solved, while from carbonate of lime bubbles of carbon- dioxid (COj) are set free. Indiflferent Fluids. — Fresh tissues are usually examined in normal salt solution — a y^^- per cent, solution of common salt in water. It has the advantage over water that tissues do not swell up so much in it, blood-globules are unaffected, and the finer structures are better preserved. A very few drops of Lugol's solution added to the stock-bottle of salt solution will be found u.seful in preventing the growth of mould. Serous fluids, such as hydrocele fluid, are occasionally used. Artificial serum is made by adding i part of egg- albumin to 9 parts of normal salt solution. Macerating fluids are little used in pathology. Occa- sionally, however, when tissues are tough, so that they cannot be readily teased apart, they are macerated in certain fluids which dissolve the substances that hold the different ele- ments together. The reagents most commonly used are the following : 1 . Ranvier's one-third alcohol is made by taking i part of 96 per cent, alcohol and 2 parts of water ; twenty-four hours are usually enough. 2. Very dilute solutions of chromic acid are recommended — TTO' t° TriT °f ' P^"" cent. 3. jj per cent. Caustic Potash. — Tissues are macerated in a few minutes to one hour : they must be examined in the same fluid, because the cells are destroyed if the solution is weakened. Bxaminatiou of Fluids. — Small fragments of tissue should be picked out with forceps. If much blood is ad- herent, wash the tissue well in salt solution. When the cell- HISTOLOGICAL METHODS. 255 ular elements are few in number, they are obtained with a pipette, just as in urine-work, after allowing them to settle at the bottom of the glass. A centrifugal machine will be found of great service when the sediment is slight. INJECTIONS. Injections are not much used in pathology. The process is an art that requires much patience and considerable ex- perience. The purpose of an injection is to render vessels and vessel-walls more visible than under ordinary circum- stances. Transparent, deeply colored fluid mixtures are used, which will become hard in the vessels. Some injec- tion-masses are employed cold, others warm. The warm injection-masses contain gelatin, and are much more trouble- some to use, but give much the more perfect results. For coloring the mass carmine is the best material, because it is a permanent color. The instruments required are cannulas of various sizes aryi a syringe, or, better still, a constant-pressure apparatus. When a warm injection-mass is used, the bottle containing the mass must be placed in a water-bath and kept at a tem- perature of about 45° C. The organ or animal to be in- jected must likewise be placed in a water-bath of the same temperature. It is very important that in connecting the end of the tube carrying the injection-mass with the cannula inserted in the vessel no air-bubbles shall enter. When blood-vessels are to be injected, it is advisable to wash them out first with normal salt solution. Cold Injection-masses. — i. Blue Coloring Mass. Soluble Berlin blue, i ; Distilled water, 20. 2. Carmine Injection-mass (Kollmann). — Dissolve i gram of carmine in i c.c. of strong ammonia plus a little water ; 256 PATHOLOGICAL TECHNIQUE. dilute with 20 c.c. of glycerin. To this solution add i gram of common salt (NaCl) dissolved in 30 c.c. of glycerin. To the whole soltition add an equal quantity of water. Fischer has obtained good results by washing out the vessels in the usual way with physiological salt solution, or, better still, with a fibrin-dissolving fluid such as a freshly filtered 8 per cent, solution of nitrate or sulphate of sodium and then injecting good fresh milk. Fix the tissues for at least twenty-four hours in a 10 per cent, solution of formalin plus 2 per cent, of acetic acid. Cut frozen sections and stain with Scharlach R. Counterstaiii in alum-hematoxylin. Mount in glycerin. The fat can also be stained with osmium tetraoxid. The capillaries are outlined by the fat emulsion stained red or black. Warm Injection-masses. — i. Berlin Blue. — Warm the solution of Berlin blue given above, and add it, with con- tinual stirring, to an equal quantity of a warm, concentrated solution of gelatin prepared as follows : Allow clean sheets of the best French gelatin to swell up for one to two hours at room-temperature in double the quantity of water. Then dissolve them by warming gently over a water-bath. Filter tke combined solution through flannel. 2. Carmine-gelatin Mass. — This is by all means the best injection-mass to use, because it is permanent, but it is very difficult to prepare. Dissolve 2 to 2.5 grams of be.st carmine in about 15 c.c. of water, to which just enough ammonia is added, drop by drop, to effect the solution. Filter the fluid obtained, and add it, with continual stirring, to a filtered warm, concen- trated solution of gelatin (prepared as above) over the water- bath. Then add acetic acid slowly until the color changes to a bright-red shade. The exact amount desired is when the solution loses its ammoniacal odor and has a pecuHar sweetish aroma free from acid. Examined under the micro- scope, no granular precipitate of carmine should appear. If too much acetic acid has been added, so that the carmine is precipitated, the mass must be thrown away and a new lot prepared. Organs which have been injected with a cold mass are placed directly in 80 per cent, alcohol. After a few hours HISTOLOGICAL METHODS. 257 they are to be cut up into pieces that are not too small. After a warm injection-mass the organ or animal is placed firstin cold water to hasten the solidification of the gelatin, and then transferred to 80 per cent, alcohol. Masses already prepared for injecting cold or warm can be obtained from Gruebler. FIXING REAGENTS. The various reagents used for the preservation of fresh tissues possess the properties of penetrating, killing, fixing, hardening, and preserving in different degrees. Of these properties " fixing " is the most important, and to a certain extent implies or includes the others. The term " fixative " has been used more particularly, perhaps, for reagents which preserve faithfully the various changes of the nucleus in karyomitosis. In a broader sense, however, it refers to the faithful preservation of any tissue-element or pathological product, and of the chemical properties peculiar to that ele- ment or product. A good fixative is a reagent that pene- trates and kills tissues quickly, preserves the tissue-elements, and particularly the nuclei, faithfully in the condition in which they are at the moment when the reagent acts on them, and hardens or so affects them that they will not be altered by the various after-steps of staining, clearing, and mounting. Most fixatives are mixtures of different reagents so combined that all the desirable properties may be present in as large a degree as possible. The choice of the proper fixing reagent for a given tissue is often difficult, and must depend largely on the nature of the pathological lesions present or suspected, and on the purposes for which the tissue is preserved. For diag- nosis, for general bacteriological study of tissues, and for many valuable and important chemical reactions alcohol is to be preferred to any other reagent. Although by no means an ideal fixative, it will be found exceedingly useful. For finer histological study it is important to preserve 17 258 PATHOLOGICAL TECHNIQUE. valuable tissue in some more perfect fixative than alcohol. Zenker's and to some extent Orth's fluid will be found the most generally useful. Flemming's solution is especially to be recommended for the study of renal lesions with fatty degeneration. For general pathological work, aside from the nervous .system, these four fixatives will be found the most valuable. It is strongly advised that in all important cases pieces of tissue be hardened both in alcohol and in Zenker's fluid — in alcohol for bacteria and for chemical reactions; in the other for bacteria, nuclear figures, blood, and general histo- logical study. For special investigations other fixatives are sometimes desirable. Tissues fixed in alcohol or in a solution of formaldehyde may remain as long as desirable in those fluids. Tissues hardened in most of the other fixatives must be transferred, after thorough washing in water, to alcohol for preservation. It is usually recommended to pass the .specimens through graded alcohols, either through 30, 60, 90, and 96 per cent., or through 50, 70, and 96 per cent., allowing them to remain from a few hours to a day in each strength. For most purposes it will be found sufficient to transfer the specimens directly from water to alcohol of 70 to 80 per cent, in which they may remain until it is desired to imbed them. Alcohol extracts chrome salts from tissues hardened in solutions of them. As these salts are precipitated in the alcohol under the action of light, it is desirable, although by no means necessary, to keep all such specimens in the dark. Alcoliol. — The .strength of alcohol ordinarily used in laboratories is 95-96 per cent. Absolute alcohol is much more expensive. Tissues hardened in either of these strengths shrink a great deal. The exposed surface be- comes extremely hard, and the outer layers of the cells of tissues, like a rabbit's kidney, for example, are as shrunken and flattened as though dried in the air. It is only inside of this hard casing, where the alcohol has penetrated more HISTOLOGICAL METHODS. 259 slowly and has been somewhat diluted by the fluid of the tissue, that the cells are better preserved. Moreover, this extreme hardening of the surface hinders the penetration of the alcohol into the deeper parts. Tissue which is to be hardened in absolute or 95 per cent, alcohol should be cut into thin pieces, preferably not over \ cm. thick. The volume of alcohol used for hardening should be fifteen to twenty times as great as the specimen, and should be changed after three or four hours. The tissue should be kept in the upper part of the alcohol by means of absorbent cotton, or the jar may be frequently inverted and the alcohol thus kept of even strength. The advantages of strong alcohol, 95 per cent, and abso- lute, are that the tissue is more quickly fixed than with weaker strength, and that at the same time it is made quite hard — a quality more necessary formerly than now when tis- sues are so generally embedded. Tissues hardened in strong alcohol should later be transferred to 80 per cent, alcohol for preservation, or the staining properties will gradually become impaired. For general purposes it will be found better to place tissues at first into 80 per cent, alcohol, which should be replaced in two to four hours by 95 per cent, alcohol. In this way less shrinkage is caused and the surface of the tissues is not made so hard. Tissues which have been fixed in Zenker's and other fluids should, after thorough washing in running water, be placed directly in 80 per cent, alcohol for further preservation. Change the alcohol occasionally as it becomes cloudy. Zenker's Fluid. — Bichromate of potassium, 2.5 grams ; Sulphate of sodium, I gram ; Corrosive sublimate. 5 grams ; Glacial acetic acid, 5 c.c. ; Water, ad 100 Dissolve the corrosive sublimate and the bichromate of potassium in the water with the aid of heat. 26o PATHOLOGICAL fECHNIQUE. The solution is practically Miiller's fluid saturated with corrosive sublimate, plus 5 per cent, of glacial acetic acid. It is advisable not to add the acetic acid to the stock solu- tion, but only in the proper proportion to' the part taken for hardening pieces of tissue, because the .acid evaporates so readily. Directions for Use. — i. Fix tissues in the solution twelve to twenty-four hours, rarely forty-eight hours, according to thickness. 2. Wash in running water twelve to twenty-four hours. 3. Preserve in 80 per cent, alcohol until used. Tissues float at first in this solution, which will be found a most admirable general fixative. It penetrates quickly, so that pieces of tissue do not need to be so thin as with most other fixatives, but it is advisable not to let them exceed 4 to 6 mm. in thickness. Nuclear figures, red blood-globules, and protoplasm are all perfectly preserved. The greatest drawback to the fluid is the precipitation of mercury, which takes place to a varying degree in the tissues. This pre- cipitation can be removed by means of iodin, which forms a colorless, soluble compound. For this purpose it has usually been advised to add iodin to the alcohol in which the tissues are preserved. Prolonged treatment, however, is required, and iodin exerts an injurious effect on the stain- ing properties of the cells, so that in general it will be found best to embed the tissues and cut sections without removing the precipitate, and to treat the sections, just before staining, with Lugol's solution or a i per cent, alcoholic solution of iodin for ten to twenty minutes, followed by alcohol to remove the iodin. In old Zenker preparations the areas in the tissues occupied by the mercuric precipitate often .stain with alum-hemato.xylin, although the precipitate itself has been removed. Zenker preparations stain slowly but beautifully in alum- hematoxylin. Excellent results can also be obtained with eosin, followed by Unna's alkaline methylene-blue solution. Fuchsin and safranin stains are sometimes useful. Corrosive Sublimate. — Use a saturated solution (made HISTOLOGICAL METHODS. 261 by heat) in normal salt solution. The addition of 5 per cent, of glacial acetic acid is usually advisable, i. Harden thin pieces of tissue (2 to 5 mm.) for six to twenty-four hours ; 2. Wash in running water twenty-four hours ; 3. Preserve in 80 per cent, alcohol. Tissues hardened in corrosive stain quickly and brilliantly in nearly all staining solutions. It is the only fixative after which the Heidenhain-Biondi triple stain gives good results. Orth's Fluid. — Recently Qrth has highly recommended as a general fixative a solution consisting of the well-known Miiller's fluid plus 4 per cent, of formaldehyde : Bichromate of potassium, 2 to 2.5 ; Sulphate of sodium, I ; Water, 100; Formaldehyde (40 per cent, solution), 10. The formaldehyde should be added only at the time of using, for in two days the solution becomes darker, and by the fourth day a crystalline deposit begins to take place. As fixation is ordinarily complete in three to four days, this deposit does not matter. The tissue should not be over i cm. in thickness. Small pieces ^ to ^ cm. in tfiickness can be readily hardened in the incubator in three hours. The specimens should be washed thoroughly in running water six to twenty-four hours before placing in 80 per cent, alcohol. The method is particularly recommended for mitosis, red blood-globules, bone, and colloid material (in cystomata, etc.), as it gives a very good consistency to the tissues, but the histological detail is not so good as after Zenker's fluid. The addition of 5 per cent, of acetic acid would unquestionably improve it. Fletnming's Solution. — Osmic acid, 2 per cent, aqueous solution, 4; Chromic acid, i per cent, aqueous solution, 15 ; Glacial acetic acid, i. I. Fix in the solution one to three days. 2. Wash in running water six to twenty-four hours. 3. Alcohol, 80 per cent. -^♦._ 262 PATHOLOGICAL TECHNIQUE. , It is best to keep the osmic acid in a 2 per cent, solution and the chromic acid in a i per cent, solution. The mixture can then be quickly made up fresh at the time it is needed. The best stains after hardening in Flemming are Babes' saf- ranin, aniline-gentian-violet, and carbol-fuchsin. Pieces of tissue for hardening in Flemming's solution should not be over 2 mm. in thickness, because it has very slight penetrating properties. Hermann's Solution. — Osmic acid, 2 per cent, aqueous solution, 4 ; Platinic chlorid, i per cent, aqueous solution, 15 ; Glacial acetic acid, i. This modification of Flemming's solution is perhaps an even better fixative than the model on which it is based, but is more expensive. It should be employed in the same manner. Pianese's Solutibn. — Chlorid of platinum and sodium, i per cent. aqueous solution (platinic), 1 5 c.c. ; Chromic acid, \ per cent, aqueous solution, 5 " Osmic acid, 2 per cent, aqueous solution, 5 " Formic acid, C. P., I drop. Fix small pieces of tissue, not over 2 mm. thick, in the solu- tion for thirty-six hours. Wash in running water for twelve hours, then 80 per cent, alcohol. Stain paraffin sections by Pianese's special methods (see p. 293). ^ This fixative and the special staining methods are particu- larly recommended for the study of karyomitosis and of the so-called cancer bodies. Formaldehyde. — The gas formaldehyde (HCOH) is soluble in water to the extent of 40 per cent. Solutions of this strength are manufactured by different commercial houses under the names of formaline, formol, and formalose. The best strength of formaldehyde to use for fixing tissues is a 4 per cent, solution ; that is, 10 parts of the aqueous 40 per HISTOLOGICAL METHODS. 263 cent, solution, no matter what name is given to it, to 90 parts of water. The addition of 5 per cent, by volume of glacial acetic acid to this solution is said to improve its fixing properties. This fixing reagent penetrates very quickly. Its harden- ing action is not understood. It does not precipitate albu- minous bodies, but makes them quite firm. It also hardens nerve-sheaths, acting toward them and red globules like the chrome salts. Formaldehyde is very useful for preserving gross specimens, because it gives them a rather tough elastic consistence and preserves the normal color better than other hardening fluids and also the transparency of many parts, such as the cornea. Tissues, such as muscles or the con- tents of a multilocular cystoma, which have been made very hard by the fixing reagent used, can be softened even when mounted in celloidin, so that they can be cut perfectly by placing in a 4 per cent, solution of formaldehyde. In histological work it is chiefly useful in the preservation of nervous tissue and as a fixing reagent for tissue from which frozen sections are to be cut, because it improves the cutting qualities of the tissue. As a fixative for specimens that are to be imbedded in paraffin or celloidin it is not rec- ommended unless combined with other reagents, such as bichromate of potassium in Orth's fluid, because it does not appear to harden the tissue elements sufficiently to enable them to resist the shrinking effects of prolonged exposure to alcohol and other fluids in the process of imbedding. In frozen sections, however, prepared by the method described below, this shrinkage of the tissue elements is not apparent, probably because prolonged exposure to dehydrating, clear- ing, and imbedding agents is avoided. "Wright's Method for Frozen Sections. — This method has given such satisfactory results as a histological pro- cedure, and has proved, after an exhaustive testing in prac- tical work, to be such a saver of time, labor, skill, and ex- pense in obtaining satisfactory sections adequate for most routine purposes, that it seems destined to replace extensively the celloidin and paraffin methods. The essential feature of 264 PATHOLOGICAL TECHNIQUE. the method is the fastening of the section to the slide imme- diately after cutting, thus keeping even the most fragile or irregularly shaped sections intact and smooth during the processes of staining, dehydrating, and clearing which are carried out on the slide. The fixation of nuclei and cells is superior to that effected by alcohol, and there is much less shrinkage than in sections prepared by the usual methods. Inasmuch as the success of the method depends to a con- siderable extent upon the frozen sections being as thin as good celloidin sections, special attention is called to the points given on p. 241 in regard to the technique of frozen- section cutting. The method is as follows : 1. Fix pieces of the tissue not more than i cm. thick in 4 per cent, aqueous solution of formaldehyde for fifteen to twenty-four hours. Thicker masses of tissue should be soaked longer. The tissue may remain indefinitely in the formaldehyde solution. If the tissue is in small fragments or is too soft and friable to yield coherent sections, it may be imbedded in gelatin, as described on p. 279. 2. Trim the piece of tissue with a knife in such a manner that it will present a thickness to be frozen of not more than 5 mm. The other dimensions of the piece of tissue may be as large as the freezing box of the microtome will accom- modate. 3. Rinse the piece of tissue for a few seconds in water, and place it on the freezing box of the microtome with a few drops of water beneath it ; then freeze and cut frozen sections, giving careful attention to the directions for cutting such sec- tions set down on p. 241. The sections should not be over 10 or 15 microns in thickness. 4. Float the sections off the knife into water, and select a good one. Spread this section smoothly on a slide. This is easily done by passing the slide into the water and under the section, while at the same time the section is manipulated by means of a small glass rod drawn out to a small probe- pointed extremity. The slide should be free from grease. 5. Drain off superfluous water. 6. Press the section upon the slide with a piece of smooth HISTOLOGICAL METHODS. 265 blotting-paper, exerting an even but not great pressure with the ball of the thumb. The section will adhere to the slide.' 7. Without allowing the section to become too dry, pour onto it immediately a small quantity of absolute alcohol sufficient to cover the section and adjacent parts of the slide. After a few seconds drain off the alcohol. 8. Flow from a bottle quickly and evenly over the section and adjacent surface of the slide a thin solution of celloidin in equal parts of absolute alcohol and ether, drain off imme- diately, blow the breath briskly once or twice on the surface of the section, and immerse the slide immediately in water for a few seconds. Thus a thin film of celloidin is formed, which fastens the section to the slide. The solution of celloidin should be almost watery in consistence, and so thin that it will form drops readily without stringing. If it is too thin, however, it will not hold the specimen on the slide, and if it is too thick, the layer on the slide will become white when the slide is immersed in water. The film of celloidin on the slide should be so thin as to be almost invisible. 9. Stain the section by any of the usual methods applied to sections affixed to the slide. The thin layer of celloidin offers no obstruction to the staining of the section. The staining fluid may be heated on the slide, as in the staining of tubercle bacilli with fuchsin, without loosening the section. The most .satisfactory staining of these sections for general purposes is with Delafield's hematoxylin, followed by eosin. The various methods for staining bacteria in tissues are applicable to these sections. 10. Dehydrate by flooding the section and adjacent por- tions of the slide with absolute alcohol. This will remove most of the celloidin. It will not, however, loosen the sec- tion unless its action is unusually prolonged. 11. Clear by flooding the preparation and adjacent parts ' If the tissue contains much mucoid material or if it has not been thor- oughly fixed, the section may stick to the blotter and not to the slide. This difficulty will be overcome by covering the section with absolute alcohol for a few seconds just before blotting it, draining off, and then proceeding as indi- cated in this step. 266 PATHOLOGICAL TECHNIQUE. of the slide with oil of origanum. If parts of the section refuse to clear, apply alcohol again and then more oil of origanum. When clear, drain off the oil, blot with a piece of dry blotting-paper, and mount in balsam. When mounted, none of the celloidin will be visible if the proper technique has been employed. The staining fluids, alcohol, and oil of origanum are con- veniently applied from drop bottles. Boiling. — Boiling precipitates the soluble albumin in tissues as a granular material which can be readily recog- nized. The method is used particularly for the demonstra- tion of albumin in renal diseases and m edema of the lungs. By means of boihng the quickest -permanent mounts of tissues can be obtained. The method is not advocated on account of the shrinkage caused by the heat, but will some- times be found useful. Small pieces of tissue not over 1.5 cm. in diameter should be dropped into the boiling water for one-half to two min- utes ; cool quickly in cold water, and make frozen sections, or put into 'io per cent, alcohol. Any stain can be used ; methylene-blue will be found excellent. Mtiller's Fluid. — Bichromate of pota.ssium, 2 to 2.5 grams; Sulphate of sodium, i gram ; Water, lOO c.c. Harden tissues six to eight weeks. Change the fluid daily during the first week ; once a week thereafter. Ordinary tissues are then washed in running water overnight before being placed in alcohol. Nervous tissue is transferred di- rectly from the fluid to the alcohol. This famous hardening solution seems destined before long to give way entirely to better fixatives. It hardens tissues slowly, evenly, and with little or no shrinkage, but it is a poor nuclear fixative, and does not encourage any great variety of stains. The sulphate of sodium seems to serve absolutely no function. For ordinary tissues it will un- doubtedly be replaced by Zenker's or Orth's fluid, both of HISTOLOGICAL METHODS. 267 which fix very quickly, besides having all its good qualities. For nervous tissues formaldehyde followed by other solu- tions of the chrome salts is a great deal quicker and better. Tellyesniczky has recently recommended the following mixture, which has met with considerable favor, and which may be regarded as an improved Miiller's fluid : Bichromate of potassium, 3 parts ; Water, 100 " Glacial acetic acid, 5 " Fix thin sections for one to two days ; thicker sections longer. Wash out thoroughly in running water. Dehy- drate in graded alcohols. Marchi's Fluid. — Miiller's fluid, 2 parts ; Osmic acid, i per cent, aqueous solution, i part. Place small pieces of tissue in the mixture for five to eight days, wash thoroughly in running water, and harden in alcohol. For its application to degenerated nerve-fibers see page 354. DECALCIFICATION. Tissues which are to be decalcified should be sawn with a fine hair-saw into thin slices, so that they will, decalcify quickly. It is usually desirable to saw the tissue into pieces of proper size for imbedding in celloidin. Very dense bone ought not to be over 2 or 3 mm. thick ; softer tissues do not need to be thinner than 4 to 6 mm. In cutting sections after decalcifying and imbedding it is necessary to throw away the first half-dozen sections or so, because the tissue is so lacer- ated to a slight depth by the movement of small fragments of bone in the process of sawing as to be useless for micro- scopic purposes. The extent of the decalcification can be tested at any time by thrusting a needle into the tissue. The following steps in the decalcification of tissues must be carefully borne in mind. I. The tissues must first be thoroughly hardened. The 268 PATHOLOGICAL TECHNIQUE. three most useful reagents for this purpose are alcohol and Zenker's and Orth's fluids. After the two latter reagents the tissues must liave been washed thoroughly in water and placed in alcohol for at least twenty-four hours. They will then be ready for decalcification. 2. The decalcifying fluid must be used in large amounts, and, if necessary, be frequently changed. 3. After decalcification the tissues must be thoroughly washed in running water for twenty-four hours to get rid of every trace of the acid. 4. The tissues finally must be hardened again in alcohol. Of the various agents used for decalcifying bone, nitric, hydrochloric, chromic, picric, trichloracetic acids, etc., the most important is nitric acid. It acts quickly, without swell- ing the tissues or attacking injuriously the tissue-elements, and does not interfere to any marked degree with any sub- sequent staining process. Red blood-globules will be found uninjured in tissues hardened in Zenker's fluid even after re- maining four days in 5 per cent, nitric acid. This acid is used in dilute solution alone or in combination with phloro- glucin. Directions for Using Nitric Acid. — i. Decalcify in large quantities of a 5 per cent, aqueous solution of nitric acid, changing the solution every day for one to four days. 2. Wash twenty-four hours in running water to remove every trace of acid. 3. Harden in 80 per cent., and then 95 per cent., alcohol. Imbed in celloidin. According to Schaffer, it is best to transfer the tissue directly from the nitric acid to a 5 per cent, solution of alum for twenty-four hours before placing in running water, so as to avoid any possibility of the tissue swelling. PUoroglucin and Nitric Acid. — Phloroglucin is not a decalcifying agent, but is added to nitric acid to protect the tissues while allowing a stronger solution of the acid to be used than would otherwise be possible. The solution is prepared by dissolving i gram of phloroglucin in 10 c.c. of nitric acid. Solution takes place quickly, with generation of considerable heat. The fluid is reddish brown at first, but HISTOLOGICAL METHODS. 269 becomes light yellow in the course of twenty-four hours. Dilute with ico c.c. of a 10 per cent, solution of nitric acid. This gives nearly a 20 per cent, solution of nitric acid. The process of decalcification in this fluid is extremely rapid ; a few hours only, as a rule, are required. It is not advisable to dilute the solution by the simple addition of water, but by the use of less acid, because the phloroglucin must be present to the amount of i per cent, or it will not protect the tissues so well. The following slower-acting solution may be found useful : Phloroglucin, i ; Nitric acid, 5 ; Alcohol, 70 ; Water, 30. A rather deep single stain with alum-hematoxylin (either aqueous solution or Delafield's) will usually be found to give the best results with tissues decalcified with nitric acid. It is very important to leave the sections after staining in a large dish of water overnight, otherwise the stain will not be so sharp and clear. Sulphurous Acid. — A saturated solution — about 5 per cent. — is used. It works very quickly and causes little swelling. The tissues should be carefully washed out in running water as after nitric acid. The stock solution rapidly grows weak through evaporation if the bottle in which it comes is not kept tightly corked. Trichloracetic Acid. — A 5 per cent, solution of this acid has lately been recommended for the decalcification of bone and teeth. It acts more slowly than nitric acid, and seems to possess no advantages over it. Tissues must be washed out in running water, as after nitric acid. IMBEDDING PROCESSES. Sections of hardened tissues can be cut with a razor by hand, or with a microtome knife after fastening the specimen in the microtome clamp either directly or between pieces of 2/0 PATHOLOGICAL TECHNIQUE. amyloid liver. Fair sections of firm tissues can often be ob- tained in this way. Thinner sections can be got by means of the freezing microtome, but these methods are all open to the objection that unless the tissue is very cohesive, por- tions of it are likely to fall out of the sections. The best results would, therefore, naturally be expected from some imbedding process, employing a substance to in- filtrate the tissues thoroughly and to hold the different parts in proper relative position even in the thinnest sections. The two substances in common use for this purpose are celloidin and paraffin. Each has its advantages and disad- vantages. Neither can be employed in pathological his- tology to the exclusion of the other. Paraffin affords the thinner sections, but they must be small if the best results are desired, and cannot be properly handled except when fastened to the slide. Hard tissues, like muscle, and tissues of varying consistency, Hke skin, are cut with considerable difficulty by the paraffin method. Staining is rather simpler than after imbedding in celloidin. On the other hand, tissues of almost any consistency or .size can be cut by the celloidin method, which is also capa- ble of furnishing very thin sections. Both methods of imbedding should be learned and used. Celloidin sections are especially good for general work, for studying the extent and relations of pathological processes, and for much of the finer histological work. Paraffin sec- tions are better for the finest details of processes — for special work on special tissues. Celloidin. — Schering's celloidin is the best preparation of gun-cotton (pyroxylin) to use. It is sold now in a con- venient granular form in small bottles. It keeps well, dis- solves somewhat slowly, and gives a fairly transparent im- bedding mass, which is firm and tough, so that very thin sections can be cut. Other forms of gun-cotton are not so reliable ; they often contain impurities and do not yield so firm an imbedding mass. Imbedding in Celloidin.— The process consists in soaking the tissues for twenty-four hours to a number of HISTOLOGICAL METHODS. 2/1 days in two different solutions of celloidin. The two solu- tions are spoken of as thin and thick celloidin. To make thick celloidin 30 grams of the dry celloidin are dissolved in 200 to 250 c.c. of a mixture of equal parts of ether and absolute alcohol. Diluted with an equal amount of the ether-and-alcohol mixture, it forms thin celloidin. ^ The steps of the imbedding process are as follows : Pieces of tissue which have been properly fixed and finally pre- served in 80 per cent, alcohol are first to be cut up with in- telligence. They should rarely be over 4 to 8 mm. thick ; for most purposes 2 mm. will be found sufficient. Pieces of this thickness will furnish several hundred sections, will im- bed more quickly than larger masses, and will be more rigid when mounted on a block. They should never be broader or longer than is necessary to show the whole of the process under study. Very thin celloidin sections cannot usually be obtained with tissues over i-|^ to 2 cm. square, and smaller dimensions are preferable. Beginners usually imbed larger • pieces than are necessary. The trimmed pieces of tissue are first hardened and de- hydrated for twenty-four hours in 95 per cent, alcohol, fol- lowed by twenty-four hours in absolute alcohol ; then soaked in equal parts of absolute alcohol and ether for the same length of time to prepare them for the thin celloidin. In the latter they remain at least twenty-four hours, preferably for a number of days, if at all thick, for in this solution occurs most of the infiltration with celloidin. Finally, the pieces are soaked twenty-four hours or more in the thick celloidin. They are then mounted on blocks of vulcanized fiber, ex- posed to the air for two or three minutes till the surface hardens a little, and placed in 80 per cent, alcohol for six to twenty-four hours to allow the celloidin to harden. Briefly summed up, the steps of imbedding in celloidin are as follows : 1. 95 per cent., followed by absolute alcohol, twenty-four hours each. 2. Ether and absolute alcohol, aa, twenty-four hours. 3. Thin celloidin, twenty-four hours to one or more weeks. 272 PATHOLOGICAL fECHNIQUE. 4. Thick celloidin, twenty-four hours to one or more weeks. 5. Mount on.blocks of vulcanized fiber: dry a minute or two in the air. 6. Harden celloidin in 80 per cent, alcohol, six to twenty- four hours. The second step may be omitted if time is pressing. Instead of mounting directly from the thick celloidin, it is often advisable to allow the celloidin to evaporate until a firm mass is obtained. This is particularly true when very loose tissues are to be imbedded. The simplest method is to place the pieces of tissue, which have been soaking in thick celloidin, in proper position in a glass .dish and pour thick celloidin over them. The dish is then covered, but not too tightly, and the ether is allowed to evaporate for one or more days until the proper consistency of celloidin is reached, so that it can be cut out in blocks enclosing the specimens. If the ether evaporates too rapidly, place a large dish or a bell-jar over the covered dish. Mount the blocks, after they have been cut out and trimmed, by dipping the bases in thick celloidin and then pressing them on to blocks of vulcanized fiber. In two or three minutes they can be placed in 80 per cent, alcohol. After the celloidin mounts have been in 80 per cent, alcohol for six to twenty-four hours, the celloidin is of the proper consistency for cutting. It is best to take a sharp knife or an old razor and trim the top of the celloidin down to where the first good section of the specimen can be cut ; this will save considerable wear on the microtome knife. Stepanow' has recently recomrnended the following method of imbedding in celloidin for three reasons : it is quicker, the infiltration is more perfect, and thinner sections are possible. 1. Dehydrate in alcohol, 95 per cent, or absolute. 2. Oil of cloves, three to six hours or longer. 3. Place in the following solution for three to six hours or longer : ' Zeitschrift f. wissensch. Mikroskopie, 1900, xvii., 1S5. HISTOLOGICAL ME7HODS. 2/3 Celloidin (dry and in fine granules), 1.5 gm. ; Oil of cloves, 5.0 c.c. ; Ether, 20.0 c.c. ; Absolute alcohol, i.oc.c. ; 4. Pour the block of tissue and enough of the solution to cover it into a filter of very fine paper, and allow the solu- tion to thicken, preferably in a warm place. As the solution thickens it becomes clear. 5. Cut out the block of tissue, and mount in the usual way. 6. Place in 80 per cent, alcohol (or in chloroform for one to two hours, followed by 80 per cent, alcohol). In cutting, the microtome knife should be fastened very obliquely, so that as much of the edge of the knife as pos- sible shall be used in making each section. The surface of the knife should be kept well wet with 80 per cent, alcohol, preferably from an overhanging drop-bottle. If the sections curl, as often happens when they are thin, they are best flattened by unrolling them on to the surface of the knife with a camel's-hair brush just before the last edge of celloidin is cut through, as this serves to keep them fixed in place during the process. This method can be used when the .simple transferring of sections from alcohol to water is not sufficient to uncurl them. Celloidin sections can be stained by nearly all methods, without the necessity of removing the celloidin. When necessary, however, the celloidin is readily removed by placing the sections from absolute alcohol in oil of cloves or in the alcohol-and-ether mixture for five or ten minutes, and then passing them back through absolute into ordinary alcohol. • To Attach Celloidin Sections to the Slide. — A celloidin sec- tion can be fairly well attached to a slide by transferring it from water to a shde freshly washed in alcohol and dried with a cloth. The section is then to be firmly blotted with filter-paper so as to apply it closely to the slide and to re- move all wrinkles. It should not be allowed to dry. A 18 274 PATHOLOGICAL TECHNIQUE. section treated in this way will ordinarily stand considerable manipulation without becoming loose. Celloidin sections can be more securely attached by trans- ferring them from 95 per cent, alcohol to clean slides and pouring over them ether-vapor from a bottle half full of ether. With a little practice sections can be fastened in a few seconds. Follow slowly along the edge of the celloidin, and the frills in it will soften down. Then wash the specimen with 80 per cent, alcohol to harden the celloidin. Imbedding in Paraffin. — Paraffin imbedding is particu- larly useful when very thin sections are desired. To obtain the best results the pieces of tissue should be small soft, and of uniform consistency. In pathological work it is much better to cut the sections and to stain them after they are fastened to the slide than to stain in the mass beforehand, because then a variety of stains may be used. A complete or perfect series is not so important as in embryology, but with a little care can be obtained. The first step in the preparation of hardened tissues for the paraffin bath is to cut them into small, thin, square or rectangular pieces, not over i cm. square, perhaps, for the best results, and not over 2 to 3 mm. thick. It should be stated, however, that with proper skill, a heavy, sharp knife, and a rigid microtome very thin paraffin sections can be ob- tained with tissues measuring 4X3 cm. The pieces of tissue are then thoroughly dehydrated by soaking first in 95 per cent, and then in absolute alcohol. From alcohol they are put in some substance, such as chloroform or oil of cedar, which has the property of mixing with alcohol and of dissolving paraffin. From the chloroform they are trans- ferred to a saturated solution of paraffin in chloroform, and then passed through two separate baths of the melted par- affin to get rid of every trace of the chloroform. If oil of cedar is used, the specimens are transferred directly from it into the melted paraffin, or they may be placed first for half an hour or so in chloroform to get rid of the oil of cedar. This procedure enables one to make use, for certain dense tissues, such as the skin, of the better penetrating HISTOLOGICAL METHODS. 2/5 powers of the oil, and yet avoid carrying it into the paraffin bath. One advantage of the chloroform method is that the dura- tion in the hot paraffin, the objectionable feature of the par- affin method, is shortened, because the tissues are already somewhat infiltrated with paraffin. Another advantage is that the paraffin bath purifies itself, because the chloroform rapidly evaporates. When oil of cedar is used, the paraffin must be renewed frequently. The methods of imbedding in paraffin are briefly stated as follows : Method No. i. 1. 95 per cent, alcohol, 2. Absolute alcohol, 3. Chloroform, 4. Chloroform saturated with paraffin, 5. Paraffin bath, two changes, 6. Imbed and cool quickly in cold water. 6-24 hours. 6-24 ii 6-24 If 6-24 u 2- 4 6-24 ] hours. 6-24 (( 6-24 ** 2- 8 a Method No. 2. 1. 95 per cent, alcohol, 2. Absolute alcohol, 3. Oil of cedar, two changes, 4. Paraffin, three changes, until no odor of oil of cedar. 5. Imbed and cool quickly in cold water. In the second method other substances than oil of cedar can be used, such as xylol, equal parts of oil of cloves and turpentine, or oil of cloves and xylol. For imbedding paraffin specimens metallic boxes can be used, or forms made round or square from strips of sheet lead or tin. Many prefer paper boxes, which can be made easily of any size desired from stiff writing-paper. Melted paraffin is poured into the paper box to the depth of about I cm. The pieces of tissue are then placed in the box with that side down from which sections are preferred. When all the pieces are arranged in order with about half a centimeter or more between them, the box is placed on tbe 2/6 PATHOLOGICAL TECHNIQUE. surface of a large dish of cold water, on which it floats, so that the paraffin may cool quickly without crystallizing. Sometimes it is advisable to set the paper box with the specimens in it in the paraffin oven for a short while, so as to get rid of any bubbles carried in by the specimens. After the paraffin has hardened, the paper is removed and the par- affin is divided up according to the pieces in it. One of the blocks is then fastened to the object-holder by heating the latter in a flame until it will just melt the paraffin when the block is held in proper position against it. The holder is then quickly cooled in cold water. The upper surface of the paraffin should now be shaved down to the specimen. The four sides are to be carefully trimmed; the upper and lower surfaces should be parallel and not cut too close to the specimen, otherwise the sections will not adhere to each other ; the lateral surfaces should, as a rule, be cut close to the tissue, especially if very thin sections are desired, because if a rim of paraffin is left it is likely to cause wrinkling of the sections. The holder is finally carefully adjusted in the paraffin microtome. To get good sections which will adhere to each other and form a ribbon the temperature of the room must be regulated to suit the degree of hardness of the paraffin used. An open window will often make all the difference needed to obtain good results. The harder the paraffin the warmer the room must be. The temperature can be raised by burning a Bun- sen flame near the microtome or lowered by the presence of a lump of ice. It will often be found advantageous to dip the holder and paraffin block into ice water just before cut- ting sections. The ribbons of sections as cut, usually a slideful, are laid on the surface of a large dish of warm water at about 44'^ C, and if necessary gently stretched so as to remove all wrinkles. Paint the surface of a slide with a thin layer of Mayer's glycerin-albumin mixture, wipe off" all excess with a towel so that only a faint layer is left, dip the slide under the sections, arrange them in order, lift the slide, and drain off the water. The slide is then placed in a slanting position HISTOLOGICAL METHODS. 2'jy until dry, when it is put in the incubator for two to twelve hours at a temperature of 54° to 60° C. This process attaches the sections firmly to the slide. To get rid of the paraffin in the sections they are treated with two or three changes of xylol, and then with absolute followed by 95 per cent, alcohol. If for any reason the celloidin-and-oil-of-cloves mixture is used for attaching the sections to the slide, the paraffin is removed by means of xylol, followed by origanum or berga- mot oil, and finally by 95 per cent, alcohol, because absolute alcohol will dissolve the celloidin. Serial Sections by the Celloidin Method. — i. For Tissues in General. — With a little care perfect serial sections can be made by the following method, and each slide of sec- tions can be stained in whatever way seems best. The specimen is imbedded, mounted on vulcanized fiber, and hardened in 80 per cent, alcohol in the usual way. In cut- ting moisten the microtome knife with 95 per cent, alcohol. As the sections are cut they are drawn up on the surface of the knife and arranged in regular order by means of a camel's- hair brush until a slideful is ready. They are then drawn on a clean and numbered slide held against the back of the knife. After being carefully arranged the sections are fast- ened to the slide by means of ether-vapor (see p. 274) poured over them from a half-full bottle. Care must be taken that every edge of the celloidin is fully softened down. The slides are then placed in a jar of 80 per cent, alcohol to be stained at leisure. 2. Another method, often convenient where the stain is of little importance, is as follows : The tissue is stained, in bulk, in alum-cochineal or some other staining fluid that will pene- trate, and then imbedded in celloidin in the usual way. After being mounted on vulcanized fiber the specimen is hardened in chloroform instead of in 80 per cent, alcohol. From the chloroform the specimen is transferred to oil of thyme. After it is thoroughly penetrated by the latter it is ready to be cut. The knife is to be moistened with oil of thyme. The sections as cut are arranged on the knife, and then trans- 2/8 PATHOLOGICAL TECHNIQUE. ferred to slides placed against the back of the knife. The slides covered with sections can be placed under a bell-jar as fast as they are ready until all are cut, because the oil of thyme evaporates slowly. Balsam and cover-slips can be added after the cutting is finished. 3. Darkschewitsch has recently proposed a comparatively simple method for preparing a series of celloidin sections. A glass cylinder without a neck, of about the diameter of the specimen to be cut, is filled with alcohol. Then a series of circles of filter-paper are cut of a size just to fit the bottle, numbered in order, and wet with alcohol. Each section is removed from the microtome knife by pressing one of the paper circles upon it and drawing it off The paper is then inverted so that the section is uppermost, and deposited in proper order in the bottle, where the series forms a column, each section resting upon a numbered paper. The sections can be kept indefinitely. When ready to stain the alcohol is poured off, the sections washed with water if necessary, and then the staining solution poured into the bottle. Other reagents are used in the same manner, or sections can be treated with the reagents in flat plates, as they do not readily slip off the papers. 4. Weigert's method for a series of celloidin sections was designed especially for the nervous system and is rather complicated. The process depends on transferring the sec- tions as cut to narrow strips of tissue-paper. To do this each section as cut is arranged in proper position close to the edge of the knife. Then a strip of tissue-paper twice as wide as the section is gently placed upon it, and the sections withdrawn from the knife. The success of the process depends on having but little alcohol on the knife, other- wise the specimen will not stick. Each specimen is placed on the paper to the right of the last one. The strips of paper when full are kept moist by being placed with the specimens uppermost on a moist surface composed of a layer of blotting-paper wet with alcohol, covered with a sheet of tissue-paper, and lying in a shallow dish. When all the sections have been cut, each strip of them HISTOLOGICAL METHODS. 279 is taken in turn and coated on botli sides with a thin film of celloidin in the following way : A strip of sections with the specimens below is first pressed gently down upon the sur- face of a slide covered with a thin layer of celloidin. This fastens the sections and the paper can be removed. Then a thin coat of celloidin is poured over the sections and the slide is placed on its edge to drain. When the surface of the celloidin is dry, the strips can be marked by a fine brush dipped in methylene-blue. As soon as the slides are placed in the staining solution the celloidin peels off, taking the specimens with it. Later, the strips of specimens can be divided as desired. On account of their thickness they should be cleared, after dehydrating in 95 per cent, alco- hol, in a mixture of xylol 3, carbolic-acid crystals i. To obtain serial sections by the paraffin method it is only necessary to avoid losing any of the sections from the ribbon as ordinarily cut. Perhaps the easiest and safest way is to cut long ribbons, a yard or more in length, and to place them on sheets of paper in proper order. They can then readily be divided by means of needles into short series of any desired number of sections, and fastened to numbered slides by means of albumin fixative. Wright's Imbedding Method for Frozen Sections. — This method has been devised for the purpose of holding together, in one sheet, frozen sections of small fragments of tissue, such as curettings from the uterus, so that they may be manipulated and mounted as one section by the method for frozen sections described on page 263. It depends on the fact that gelatin exposed to formaldehyde becomes in- soluble in water. The method consists in soaking the frag- ments of tissue for a few minutes or longer in a warm 20 per cent, solution of gelatin in water, then placing the fragments close together in a watch-glass with enough of the melted gelatin to form a layer about 5 mm. thick, and allowing the gelatin to solidify, after which the block of gelatin inclosing the fragments is cut out of the watch-glass and placed in a 4 per cent, aqueous solution of formalde- hyde for from eighteen to twenty-four hours. Sections are 28o PATHOLOGICAL TECHNIQUE. then cut from the mass on the freezing microtome, floated out in water, and manipulated and stained as described on page 241. The sections appear as sheets of gelatin inclos- ing sections of the fragments of tissue. The gelatin may be constantly kept on hand ready for use in corked test- tubes, each containing 8 or 10 c.c. To prevent the growth of molds in the gelatin, a little thymol or carbolic acid may be added to it. Fragments of tissue already fixed in for- maldehyde may also be imbedded in the gelatin as above described. The solidification of the gelatin in the watch- glass may be hastened by placing the watch-glass in ice- water. The action of the formaldehyde on the gelatin may be hastened by placing the vessel containing the solution of formaldehyde and the gelatin block in the paraffin oven at about 55° C. for some hours. If it is desired to do this, it is necessary that the gelatin block first be kept at room-tem- perature in the formaldehyde solution for at least an hour, in order to prevent the melting of the gelatin. STAINING SOLUTIONS. Hematoxylin and Hematein Stains. — The active coloring <. agent in most hematoxylin stains is hematein,% which is gradually formed in the ordinary solutions from the hematoxylin by oxidation, a process occupying a number of days" or weeks and spoken of as "ripening." The selec- tive stalining power of alum-hematoxylin solutions is due to the combination of this hematein with alumina. The result- ing blue-colored solution is precipitated in the tissues (chiefly in the nuclei) by certain organic and inorganic salts there present, as, for instance, phosphates. Mayer and Unna have shown that it is possible to oxidize and to ripen in an instant a solution of alum and hema- toxylin by adding to it a little peroxid of hydrogen neutral- ized by a crystal of soda. % By employing hematein or its ammonium salt, instead of hematoxylin, Mayer has been able to obtain immediately ripened solutions which compare fairly favorably with old HISTOLOGICAL METHODS. 28 1 and well-known solutions prepared from hematoxylin by the slow process of ripening. They do not stain any better, however, and it is doubtful if, for the present at least, they become generally accepted. Most solutions of alum and hematoxylin are not stable. A continuous chemical change is the formation from hema- toxyhn, by oxidation, of hematein, which, uniting with the alum, ™res a bluish or purplish solution. The degree of blueness depends largely on the freshness of the alum. As the solution becomes older free sulphuric acid is gradually formed from the alum, causing the solution to lose its bluish or purplish tint arid to become reddish. A fhird chemical change is the continuous formation of a precipitate due to the further oxidation of the hematein, in consequence of which it is always necessary to filter alum-hemato3^1in solu- tions just before they are used. More alum than is needed to combine chemically with the hematoxylin is always added to t]je solution, for the reason that it acts as a differential decolorizer, limiting the stain largely to the nuclei of the cells. As alum-hematoxylin solutions be- come older they stain more quickly, but also more diffusely. This diffuseness of staining can be counteracted by adding enough alum-water to make the stain precise again'. A good alum-hematoxylin solution ought not to stain the celloidir\ in which the section is imbedded. If the celloidin stains more or less deeply, it shows that the solution requires more alum. _ c Aqueous Alum-hematoxylin Solution. — Hematoxylin crystals, i ; Saturated a^eous solution of ammonia alum," 100; Water, «» 300; Thymol, • a crystal. The hematoxylin crystals are dissolved in a little water by the aid of heat. The combined solution is exposed to the light in a bottle lightly stoppered with a plug of cotton. The solution will be njjpned sufficiently for use in about- 282 PATHOLOGICAL TECHNIQUE. ten days, after which time it should be kept in a tightly- stoppered bottle. The solution is very easily prepared, gives beautiful results, and will keep at its best for two to three months. The proportions of alum and of hematox- ylin are the same as in Delafield's solution. For Zenker preparations, which stain very slowly, it will be found more convenient to omit the 300 c.c. of water in the above formula. Delafield's Hematoxylin. — Hematoxylin crystals, 4 grams ; Alcohol, 95 per cent, 25 c.c. ; Saturated aqueous solution of ammonia alum, 400 " Add the hematoxylin dissolved in the alcohol to the alum solution, and expose the mixture in an unstoppered bottle to the light and air for three to four days. Filter, and add — Glycerin, 100 c.c; Alcohol, 95 per cent., 100 " Allow the solution to stand in the light until the color is sufficiently dark, then filter and keep in a tightly-stoppered bottle. The solution keeps well and is extremely powerful. So long as it is good the solution has a purpHsh tinge. It would seem advisable, both in this solution and in Ehr- lich's, to combine the alum, hematoxylin, and the water, and to ripen the solution for two or three weeks before adding the other ingredients which have a tendency to prevent oxi- dation. A fully-ripened solution would then be obtained more quickly and surely. Ehrlich's Acid Hematoxylin. — Hematoxylin crystals, 2 grams; Absolute alcohol, 60 c.c. ; Glycerin, 60 c.c. ^ Water, 60 " I saturated with Glacial acetic acid, x " j ^™monia alum. HISTOLOGICAL METHODS. 283 The solution is to be exposed to the light for a long time until it acquires a deep-red color. If it then be kept carefully stoppered, its staining powers will remain constant for years. The acetic acid is added to prevent the formation of insoluble compounds of hematein and as a decolorizer to limit the stain to nuclei. ayer's Hemalum. — Hematein, or its ammonia salt, I gram ; 90 per cent, alcohol, 50 c.c. ; Alum, 50 grams ; Water, 1000 c.c; Thymol, a crystal. Dissolve the hematein or its ammonia salt in the alcohol by the aid of heat, and add it to the alum dissolved in the water. The solution can be diluted with 20 parts of water or of weak alum solution. Mayer's acid hemalum is prepared by adding 2 per cent, of glacial acetic acid to the above solution. The acid stain is more precise than the alkaline. Mayer's Glycerm-alum-hematein Solution. — According to Mayer's latest investigations, glycerin is the only reliable preservative of hematein solutions. Unfortunately, it slows the staining power to a considerable extent and makes the stain less precise. He recommends the following solution for its keeping properties : Hematein, 0.4 grams (dissolve by rubbing up in a few drops of glycerin) ; Alum, 5 ^ims ; Glycerin, 30 c.c. ; Water, 70 " Mayer's Muchematein. — 1^^ ^ Hematein, 0.2 gratis ; Chlorid of aluminum, o.i " Glycerin, 40 c.c. ; Water, 60 " 284 PATHOLOGICAL TECHNIQUE. Rub up the hematein with a few drops of glycerin, add the chlorid of aluminum, and dissolve the mixture in the glycerin and water. Weigert's Alcohol Hematoxylin. — Hematoxylin crystals, 10 grams; Alcohol (absolute or 95 per cent), 90 c.c. The solution ripens in a week or two to a brown color, and keeps perfectly for a long time. It is used only in the Weigert stain for myelin sheaths, for which purpose it is diluted at the time of using with water and combined with carbonate of lithium (see page 344). Phosphomolybdic Acid Hematoxylin (Mallory). — Hematoxylin crystals, 1.75 grams; \ per cent, aqueous solution of phos- phomolybdic acid, 200 c.c. The hematoxylin will dissolve almost immediately if powdered, or it may be dissolved in water by the aid of heat. The solution must be exposed to the light in a bottle plugged with cotton for five to six weeks before it is fully ripened. It will keep for several months, and can be used over and over. It is employed for staining the nervous system and connective tissue. Phosphotungstlc Acid Hematoxylin {Mallory)} — ^When this formula was first published the phosphotungstic acid manufactured by Merck was not pure. It contained a trace of phosphomolybdic acid, and also some oxidizing agent which ripen hematoxylin at once. The acid obtained since then sometimes oxidizes hematoxylin and sometimes does not. It is a simple matter,' however, to ripen the solution at once. Hematoxylin, o. i gram ; Water, 80. c.c. 10 per cent, aqueous solution of phos- photungstic acid (Merck), 20. " ^Mallory: The Journal of Experimental Medicine, igoo, v., 19. HISTOLOGICAL METHODS. 285 Dissolve the hematoxylin ift a little water by the aid of heat, and add it after it is cool to the rest of the solution. If the solution does not stain, it may be ripened by the addition of any one of the following oxidizing reagents: peroxid of hydrogen (U. S. P.), 0.2 c.c. ; \ per cent, aqueous solution of permanganate of potassium, 10 c.c. ; or 10 per cent, nitric acid, 1.5 c.c. The best results are obtained by using hematein ammo- nium (hematein purissimum does not seem to work quite so well) instead of hematoxylin, and allowing the solution to ripen spontaneously for several months. Solutions a year or more old stain with great intensity. If hematein ammonium is used instead of hematoxylin and it is desired to ripen the solution at once, use half the quantity of any one of the oxidizing reagents mentioned above. This staining solution will be found particularly useful for the demonstration of fibrin and of neuroglia and fibroglia fibrils. Carmine Stains. — The active staining principle in car- mine solutions is carminic acid. In cochineal carminic acid is combined with an alkaline base. Carmine itself is a com- mercial compound containing carminic acid combined with aluminum and calcium. Carminic acid itself does not stain, but it forms compounds with certain metals, mainly with the aluminum contained in alum, which have selective staining properties. All of the alkaline and acid solutions made with carmine owe their staining properties to carminic acid combined with the aluminum, and perhaps also to the calcium contained in the carmine. Alum Carmine. — Carmine, 2 grams ; Alum, 5 " Water, 100 c.c. Boil twenty minutes, adding enough water to make up for that lost by evaporation. When cool, filter and add a crystal of thymol to prevent the growth of mould. 286 PATHOLOGICAL TECHNIQUE. Alum Cochineal. — Powdered cochineal, 6 grams ; Ammonia alum, 6 " Water, lOO c.c. Boil for half an hour ; add water to make up for that lost by evaporation. Filter and add a crystal of thymol. Mayer's Alcoholic Carmine (Paracarmine). — Carminic acid, i.ogram; Chlorid of aluminium, 0.5 " Chlorid of calcium, 4.0 grams ; 70 per cent, alcohol, lOO.O c.c. Dissolve cold or warm ; allow to settle, then filter. After staining, wash out in 70 per cent, alcohol to which is added 2.5 per cent, glacial acetic acid if a more purely nuclear stain is desired. Orth's Lithium Carmine. — Carmine, 2.5 to 5 grams; Saturated aqueous solution of car- bonate of lithium, 100 c.c. ; Thymol, a crystal. The carmine dissolves at once in the cold solution. When used as a counter-stain for bacteria in the Gram-Weigert method this solution should be carefully filtered, because organisms occasionally grow in it and may give rise to con- fusion in the stained preparations. Neutral Carmine. — Dissolve, without heating, i gram of best carmine in 50 c.c. of distilled water plus 5 c.c. of strong aqua ammoniae. Expose the fluid in an open dish until it no longer smells ammoniacal (about three days) ; then filter and put away in a bottle for future use. The odor of the solution will soon become bad, but the staining properties will remain unaffected. Aniline Dyes. — It is extremely important that all aniline dyes used in histological work should be obtained, with possibly a few exceptions, from Griibler, either directly or HISTOLOGICAL METHODS. 287 from his authorized agents. In no other way is it possi- ble to obtain with certainty the results expected. In this country Eimer & Amend, of New York City, are the chief agents for Grubler. Aniline dyes come in the form of a powder or as crystals, and most of them keep well in that condition. Methylene- blue for one, however, seems to be an exception. After the original package has been opened for a short while the dye is said to lose in intensity of staining power. It is well to keep on hand saturated alcoholic solutions of certain of the dyes, because they keep well in that form, and are ready for use when a saturated alcoholic solution is wanted. This is par- ticularly true of methylene-blue, fuchsin, and gentian-violet. Aniline dyes are derived from either aniline or toluidin, or from both together. They may be regarded as salts having basic or acid properties. The basic colors stain cell-nuclei, including bacteria, for which they show a marked affinity. The acid colors stain diffusely. The basic dyes most com- monly employed in pathological histology are methylene- blue, fuchsin, gentian-violet, and safranin. Of the acid colors, eosin, picric acid, and acid fuchsin are most in use. As a rule, every aniline dye has one or more standard solutions which are used largely to the exclusion of others, for the reason that, being required for certain purposes, they are kept in stock. As they are thus always at hand, they are used where simple solutions might be used. For in- stance, Loffler's methylene-blue solution is often used, be- cause ready and convenient, when a simple aqueous solution would do as well. In the following pages we have arranged under each dye the solutions of it most in use : Methylene-blue. 1. Saturated solution in 95 per cent, or absolute alcohol. A stock solution to be used in making other solutions. It can be used as a stain by adding i part to 9 parts of water. 2. Aqueous solutions of various strengths are often used, and can be made up as needed. 288 PATHOLOGICAL TECHNIQUE. 3. Loffler's Methylene-blue Solution. — Saturated alcoholic solution of methylene-blue, 30 c.c. ;' Solution of caustic potash in water, i : 10,000, 100 " This is one of the most useful of the aniline staining solu- tions, and will keep for a long time without losing much in staining power. 4. Kuhne's Methylene-blue Solution. — Saturated alcoholic solution of methylene-blue, 10; 5 per cent, carbolic-acid water, 90. This is a stronger staining solution than Loffler's, but the resulting stain does not seem so sharp and clear. 5. Gabbet's Methylene-blue Solution. — Methylene-blue, 2 ; Sulphuric acid, 25 ; Water, 75. It is used as a decolorizer and contrast-stain for tubercle bacilli. 6. Unna's Alkaline Methylene-blue Solution. — The strongly alkaline solution of methylene-blue recommended by Unna for staining plasma-cells has been found extremely valuable as a general stain in connection with eosin, which should be used first. The solution should be diluted i : 10, or 1:1; for staining : Methylene-blue, i ; Carbonate of potassium, i ; Water, 100. (For method of using see page 321.) 7. Unna's Polychrome Methylene-blue Solution. — The poly- chrome methylene-blue solution, much used by Unna in various staining methods, is an old alkaline solution of methylene-blue, of which the above is the original formula, in which, in consequence of oxidation, methyl-violet and methylene-red have formed. Months are required for the process of oxidation to take place. The ripened solution may be obtained from Griibler. HISTOLOGICAL METHODS. 289 8. Sahli's Borax Methylene-blue Solution. — Saturated aqueous solution of methylene-blue, 24 ; 5 per cent, solution of borax, 16; Water, 40. Mix, let stand a day, and filter. Puchsin. 1. Saturated alcoholic solution to be kept in stock. 2. Ziehl-Neelson' s Carbol-fuchsin. — Saturated alcoholic solution of fuchsin, 10 c.c. ; 5 per cent, carbolic-acid water, 90 " This solution is very powerful, stains quickly, keeps well, and can be employed for a variety of purposes. 3. Aniline-fuchsin. — Saturated alcoholic solution of fuchsin, 16 c.c; Aniline-water, 84 Gentian- violet. This dye is not a definite chemical substance, but a mix- ture of crystal-violet, methyl-violet, and dextrin. 1. Saturated alcoholic solution to be kept in stock, r, 2. EhrlicKs Aniline-gentian-violet. — *'' Saturated alcoholic solution of gentian-violet, 16 c.c; Aniline-water, 84 " During the first few hours after the solution is made con- siderable precipitation takes place, so that it is best not to use it for twenty -four hours. After about ten days it begins to lose its staining power. (See under methyl-violet, page 290.) Zenker recommends a solution without alcohol : Dissolve the gentian-violet directly in the aniline-water. The color is said to be _ less easily removed from tissues when this solu- tion is used. 3. Stirling's Solution of Gentian-violet. — Gentian-violet, 5 grams; Alcohol, 10 c.c. ; J Aniline, 2 " Water, 88 " 19 > 290 PATHOLOGICAL TECHNIQUE. This solution is said to keep remarkably well. 4. Carbol-gentian Violet. — Saturated alcoholic solution of gentian-violet, 10 c.c. ; 5 per cent, carbolic-acid water, 90 -X Safranin. — Two of the many preparations by this name have been found especially useful : 1. Safranin O soluble in water. 2. Safranin soluble in alcohol. The three following solutions of safranin can be thoroughly recommended : 1. Saturated aqueous solution of " safranin O soluble in water " (to be made with the aid of heat). 2. A mixture of equal parts of — A saturated aqueous solution of " safranin O soluble in water." A saturated alcoholic solution of " safranin soluble in alcohol." 3. Rubes' Aniline Safranin. — 2 per cent, aniline-water, 100 ; " Safranin O soluble in water," in excess. Saturate the solution by heating it in a flask set in hot water to 60-80° C. ; filter. This solution is extremely powerful, stains almost in- stantly, and will keep about two months. Methyl- violet. — i. Aqueous solutions of various strengths, ^ to 2 per cent., keep well and are used for staining nuclei, bacteria, and amyloid. 2. Methyl-violet can be used instead of gentian-violet in Ehrlich's solution. Weigert recommends two permanent stock solutions by means of which the aniline oil — methyl- violet solution — can be made up easily when wanted. Solution I. — Absolute alcohol, 33 ; Anilin oil, 9; Methyl-violet in excess. Solution 2. — Saturated aqueous solution of methyl-violet. HISTOLOGICAL METHODS. 29I Thestaining solution consists of — Solution I, I ; Solution 2, 9. This mixture will keep at the most for fourteen days. 3. For staining neuroglia-fibers Weigert employs a satu- rated solution made with the aid of heat in 70-80 per cent, alcohol. Bismarck Brown. — The most common solutions are the following : 1. A I per cent, aqueous solution. 2. A saturated aqueous solution made by boiling (3-4 per cent.). 3. A saturated solution in 40 per cent, alcohol (2-2J per cent.). Unlike other aniline colors, Bismarck brown will keep in glycerin mounts and can be fixed in nuclei ,by acid alco^hol. The stain is not used so much as formerly, except -as a con- trast stain in Gram's method and for photographic purposes. Other basic stains less frequently used, an'd then generally in aqueous solutions, are dahlia, methyl-^reen, iodin-green, and thionin. , Diffuse Stains. — i . Eosin is sold in t\\fo forms — as " eosin soluble in water," and as " eosiri solublef in alcohol." The first is to be preferred, because a greater degree of differen- tiation in stain can be obtained with it. Keep on hand a Saturated aqueous solution and dilute with water as needed. The strength of solution to be used varies somewhat with the tissue and the reagent in which it has been fixed, Hit generally lies between -^ and \ per cent, when the llosirt"is used after a hematoxylin stain. These dilute solutions should contain 25 per cent, of alcohol, otherwise they will not keep well. When eosin is employed before an aniline dye such as methylene-blue, a 5 per cent, or even a saturated solution should be taken. 2. Picric Acid. — Saturated alcoholic and aqueous solu- tions should be kept in stock, to be diluted as needed. 3. Van Gieson's Picro-fuchsin Solution. — This valuable 292 PATHOLOGICAL TECHNIQUE. solution was originally made by adding to a saturated aqueous solution of picric acid enough of a saturated aqueous solution of acid fuchsin to give to the iluid a deep garnet-red color, and for certain purposes, as in staining after Zenker's fluid, this strong solution is to be preferred. Freeborn has recently given more precise directions for making up the solution according to the purpose for which it is to be used. For Connective Tissue. — (See page 322). I per cent, aqueous solution of acid fuchsin, 5 c.c. ; Saturated aqueous solution of picric acid, 100 " For the Nervous System. — (See page 329). I per cent aqueous solution of acid fuchsin, 1 5 c.c. ; Saturated aqueous solution of picric acid, 50 " Water, 50 " Picro-nigrosin {Martinotti). — Dissolve picric acid and nigrosin to >saturation in 70 per cent, alcohol. Combination Stains. — Biondi-Heidenhain Staining Solution. — Saturated aqueous solution of orange G, 100; Saturated aqueous solution of acid fuchsin or rubin S, 20; Saturated aqueous solution of methyl-green, 50. (About 20 gm. rubin S., 8 gm. orange G., and 8 gm. methyl-green; dissolve in 100 c.c. of water.) Make up the separate solutions and let them stand for seve- ral days with excess of coloring matter (shaking the bottles occasionally) until they are saturated. Then mix the solu- tions. For staining, dilute the combined solution with water I : 60 to I : 100. The following tests are used for finding out if the proper combination has been obtained: The addition of acetic acid should make the solution redder ; a drop of the solution on filter-paper should make a blue spot with green in the center and orange at the periphery. If a red zone appears outside of the orange, then too much acid fuchsin is present. HISTOLOGICAL METHODS. 293 Pianese's Staining Solutions and Staining Meth- ods. — The following stains, devised by Pianese, are recom- mended by him particularly for the study of cancer, but will be found useful in many lines of histological investigation. The first two were used by him for tissues hardened in cor- rosive sublimate or in Zenker's fluid ; the others, only after his special fixative (given on page 262). The methods are intended for paraffin sections : 1. Carmine and Picro-nigrosin. — i. Stain in neutral or lithium carmine. 2. Decolorize in acid alcohol. 3. Wash in water. 4. Absolute alcohol. 5. Aniline-gentian-violet, ten minutes. 6. lodin solution, two to three minutes. 7. Absolute alcohol, so long as any color is discharged. 8. Saturated aqueous solution of' picric acid and of nigro- sin, five minutes. 9. Decolorize in a I per cent, alcoholic solution of oxalic acid. 10. Water, several minutes. 1 1 . Absolute alc-ohol. 12. Oil of bergamot. 13. Balsam. Nuclei, red ; cell-protoplasm, light olive-green ; connec- tive tissue, dark olive-green ; elastic fibers, bluish ; bacteria and blastomycetes, violet. II. Methylene-blue and Eosin in Borax Solution. — Keep three solutions on hand : («) Saturated solution of methylene-blue in a saturated aqueous solution of borax. ip) \ per cent, solution of " bluish eosin " in 70 per cent, alcohol. (c) Saturated aqueous solution of borax. For use mix together 2 parts of the filtered solution a, i of b, and 2 of c. The different steps of the staining pro- cess are as follows : 294 PATHOLOGICAL TECHNIQUE. 1. Absolute alcohol. 2. Staining solution, ten to twenty minutes. 3. Decolorize in a i per cent, solution of acetic acid. 4. Wash in water. 5. Absolute alcohol. 6. Xylol. 7. Xylol balsam. Nuclei, blue ; red blood-globules, cell-protoplasm, granules of eosinophiles, connective tissue, etc., rose-red. "" III. a. Malachite-green, Acid. Fuohsin, and Nigrosin. — Malachite-green, i. gram; Acid fuchsin, ' .4 " Nigrosin, .1 Water, 50 c.c. ; Alcohol saturated with acetate of copper, 50 1. Absolute alcohol. 2. Stain in 20 drops of above solution diluted with 10 c.c. of distilled water for twenty-four hours. 3. Decolorize in a J per cent, aqueous solution of oxaUc acid. 4. Wash in water. 5. Absolute alcohol. 6. Xylol balsam. Resting nuclei, light red; protoplasm, reddish yellow. In the karyokinetic figures, nuclein green ; fibrillae of the achro- matic spindle and of the mitoma, bright red; centrosome and polar bodies, red ; the rest of the cell-body, a reddish- yellow color. III. b. Malachite-green, Acid Fuchsin, and Martius Yellow.— Malachite-green, .5 gram; Acid fuchsin, .1 " Martius yellow, .01 " Distilled water, 150 c.c; Alcohol, 96 per cent, 50 " 1. Stain in the solution without diluting, half an hour. 2. Absolute alcohol. HISTOLOGICAL METHODS. 29S 3. Xylol. 4. Xylol balsam. Nuclei of resting and dividing cells, green ; cell-protoplasm, connective tissue, etc., rose-colored ; " cancer-bodies," mainly red, but in masses of them some are red and some green. IV. Acid Fuchsin and Picro-nigrosin. — Saturated alcoholic solution of acid fuchsin, 6 drops ; Martinotti's picro-nigrosin, 8 " Distilled water, 10 c.c. 1. 70 per cent, alcohol. 2. Stain in the solution six hours. 3. Decolorize in dilute acetic acid. 4. Absolute alcohol. 5. Xylol. 6. Xylol balsam. Resting nuclei, red ; nuclein of karyokinetic figures, yellow : cell-protoplasm, dark olive-green ; " cancer-bodies," mainly olive-gray, but some or portions of them may be ruby-red. V. Light Green (Liohtgriin) and Hematoxylin. — Ehrlich's acid hematoxylin, 15 c.c. Saturated solution of Lichtgriin in 70 per cent. alcohol, 5 " Distilled water, 15 " 1. Distilled water. 2. Stain in above mixture half an hour. 3. Wash thoroughly in several waters. 4. Alcohol. 5. Oil of bergamot. 6. Balsam. Nuclei, green ; " cancer-bodies " take the hematoxylin stain. VI. Acid Fuchsin and Hematoxylin. — Ehrlich's acid hematoxylin, 15 c.c. I per cent, solution of acid fuchsin in 70 per cent, alcohol, 15 " Distilled water, 15 " Stain as in V. 296 PATHOLOGICAL TECHNIQUE. Nuclei, red ; protoplasm, brick-red ; " cancer-bodies " take the hematoxylin stain. Orcein, a vegetable dye obtained from certain tinctorial lichens, is used mainly for staining elastic fibers. It is soluble in alcohol, and is employed either in a neutral or acid (HCl) alcoholic solution. loditt is the oldest of the histological stains, but is now but little used for that purpose. The tincture of iodin, a saturated solution in alcohol, is used for getting rid of the precipitate of mercury formed in tissues fixed in corrosive sublimate or in Zenker's fluid. I/Ugol'S solution, a solution of iodin in water containing iodid of potash, is of varying strength. Iodin in this form is much used as a test for starch, amyloid, glycogen, and corpora amylacea. In Gram's stain and its modifications iodin produces some chemical change in the coloring material em- ployed, in consequence of which, when appropriate decolor- izers are used, the stain remains fast in certain structures, while from others it is easily entirely extracted. The strength originally employed by Gram for his stain- ing method was — Iodin, I gram; Iodid of potash, 2 grams; Water, 300 c.c. Weigert in his modification of this method employed a stronger solution : Iodin, I gram; Iodid of potash, 2 grams ; Water, lOp c.c. Recently he has recommended the following strength both for fibrin and for neuroglia-fibers : Iodid of potash, 5 grams 1 ^ ^ , . , . ,. -■,r , > saturated with lodm. Water, 100 c.c. J The only difference in the action of the various solutions HISTOLOGICAL METHODS. 297 probably is that the strong solution acts practically instan- taneously, while the weaker solutions require some little time. Acid Alcohol [Orth's Discharging Fluid). — Hydrochloric acid, i c.c. ; 70 per cent, alcohol, 99 " Aniline "Water {Aniline-oil Water). — Shake together 5 parts of aniline with 95 parts of water, and filter the re- sulting milky fluid. It should come through perfectly clear. Carbolic-acid water is made in like manner by shaking together 5 c.c. of melted carbolic-acid crystals and 95 c.c. of water. The solution should be filtered. Mayer's glycerin-albumin mixture for attaching paraffin sections to slides is composed of equal parts of the white of egg and of glycerin. The mixture should be thor- oughly beaten and then filtered, or after standing for some time can be decanted. Add a little camphor or carbolic acid to prevent decomposition. Egg-albumin is dissolved by acids and alkalies, so that when such reagents are to be used the sections are best attached to the slide by some other substance. For this purpose Sckallibaum' s solution, of celloidin i part in 3 or 4 parts of oil of cloves, is often useful. Cover the slide with a thin layer of the solution. Arrange the sections in order on the slide and place it in the thermostat at 54° to 60° C. for several hours, or heat for a few seconds to half a minute over the flame until the oil of cloves runs together in drops. After cooling, remove the paraffin with xylol, pass through origanum oil to 95 per cent, alcohol, and proceed as with other paraffin sections. Clearing Reagents. — The object of clearing reagents is to render certain tissue-elements more prominent than others. This result may be brought about by dilute acetic acid (2-5 : 100), which swells up the ground substance, so that nuclei, elastic fibers, fat, myelin, and micro-organisms are more distinct, or by alkalies, which destroy the cells and ground substance and leave only elastic fibers and bacteria but little changed. This method is used almost wholly for fresh tissues. 298 PATHOLOGICAL TECHNIQUE. The same result is more commonly obtained by soaking the tissues in substances which by reason of their high index of refraction render the tissues more or less transparent. Any structure which it is desirable to study is usually pre- viously stained and thus easily rendered prominent. This second method is most applicable to hardened tissues. For soaking and clearing the tissues a variety of reagents of different chemical properties are used. Glycerin and acetate of potash are not so much employed as forjnerly, because balsam mounts are more generally preferred. Of the other reagents (ethereal oils and coal-tar products), the the choice depends mainly on two factors — the kind of stain which has been employed, and the substance in which the sections have been imbedded. Many of the clearing re- agents either dissolve celloidin or will not clear it from 95 per cent, alcohol, and nearly all of them will extract aniline colors more or less rapidly. Most of the clearing reagents can be used after hema- toxylin and carmine stains. For celloidin or paraffin sections stained by either of them oleum origani cretici, oil of ber- gamot, or the mixture of the oils of cloves and thyme is recommended in the order given. For aniline stains the best clearing reagent is xylol, which, however, clears directly only from absolute alcohol. It can be used, however, for celloidin or other sections dehydrated in 95 per cent, alcohol by a simple method original with Welch, and lately brought into notice by Weigert. Blot the section on the slide with smooth soft filter-paper, and then pour on a few drops of xylol ; repeat the blotting, fol- lowed by xylol two or three times, and the section will be found to be perfectly clear. Oleum Origani Cretici. — Colorless to light brown in color ; clears readily from 95 per cent, alcohol without dis- solving celloidin ; affects aniline colors slowly. Ordinary origanum oil is impure oil of thyme, and should not be used. Oil of Bergamot. — Light green in color ; clears quickly from 95 per cent, alcohol ; does not dissolve celloidin, but HISTOLOGICAL METHODS. 299 after repeated use of the same lot of oil it will sometimes soften it a little. Affects aniline colors slowly, with the ex- ception of eosin, which it extracts very quickly. Oil of Cloves. — Straw-colored ; clears quickly from 95 per cent, alcohol ; dissolves celloidin ; extracts aniline colors, especially methylene-blue. Oil of Thyme. — Colorless ; clears readily from 95 per cent, alcohol ; makes sections brittle ; does not dissolve cel- loidin ; • affects aniline colors. Oil of Lavender. — Clears celloidin sections readily from 95 per cent, alcohol. ' Oil of Cedar-wood. — Pale straw-color ; clears from 95 per cent, alcohol, but, unfortunately, clears celloidin sections very slowly ; does not affect aniline colors. Aniline {Aniline Oil). — Colorless when perfectly pure and fresh, but soon oxidizes and turns brown ; does not dissolve celloidin ; clears readily from 70 per cent, alcohol ; will clear from water by Weigert's method; extracts aniline colors slowly. Xylol. — Colorless ; does not dissolve celloidin ; does not affect aniline colors ; clears directly only from absolute alco- hol ; but will clear even celloidin sections from 95 per cent, alcohol if they be blotted on the slide, and the xylol be then poured over them ; the process of blotting followed by xylol must be repeated two or three times. Dunham's Mixture of the Oils of Cloves and Thyme. — Excellent for sections stained in hematoxylin or carmine. Not nearly so expensive as pure origanum or bergamot oil. Oil of cloves, I part; Oil of thyme, 4 parts. Filter if cloudy ; clears celloidin sections readily from 95 per cent, alcohol without dissolving the celloidin. Weigert's Mixture of Carbolic Acid and Xylol. — Carbolic-acid crystals, i part ; Xylol, 3 parts. 300 PATHOLOGICAL TECHNIQUE. Recommended for clearing thick sections of the central nervous system after carmine and hematoxyHn stains only. The next mixture is more used now-a-days. Weigert's Mixture of Aniline and Xylol. — Aniline, 2 parts ; Xylol, I part. Mounting Reagents. — The most generally used reagent for permanent mounts is Canada balsam. Damar and colo- phonium are only exceptionally preferred. Canada balsam occurs in commerce as a very thick, tena- cious, pale, straw-colored fluid. It should be evaporated over a water-bath to drive off all volatile substances, which might affect aniline colors, until it becomes solid and brittle on cooling. Dissolve it then in xylol, which does not affect aniline colors, to a rather thin, syrupy consistency. Two pounds of Canada balsam will evaporate to about one pound ; add xylol enough to make thfe mixture up to two pounds. In this condition it is often called xylol balsam. Canada balsam has a high index of refraction, so that tis- sues mounted in it become very transparent, and only those parts are visible which are stained. Other solvents of Canada balsam, such as chloroform and benzine, may be used, but cannot be Recommended for sections stained with aniline colors. For tissue stained with osmic acid, chloroform, bal- sam, prepared in the same way as xylol balsam, should always be used, btherwise the osmic acid stain will fade rapidly. Damar has a lower index of refraction than Canada bal- sam ; is soluble in xylol, chloroform, etc. ; dries slowly, and is generally recommended for Golgi preparations. Colophonium dissolved in benzine is employed by Nissl for mounting stained preparations of ganglion-cells. HISTOLOGICAL METHODS. 3OI METALLIC STAINS OR IMPREGNATIONS. Experimental investigation has shown that certain metals can be used for staining certain tissue-elements, either be- cause they are directly reduced from solutions of appropriate salts or because they are taken up and retained by certain tissue-elements, which are rendered prominent when the metallic salt is reduced later. The most valuable metals for this purpose are silver, gold, and osmium. Silver is used, generally in the form of silver nitrate, to stain of a brown or dark -brown color the cement substance between epithelial and endothelial cells and the ground sub- stance of connective tissue. The method finds its chief use in pathology in demonstrating the endothelial covering of a doubtful surface, in outlining the endothelial cells of patho- logically altered blood- and lymph-vessels, and in staining the ground substance of the connective tissue of the cornea when that organ is used experimentally for the study of in- flammation. In combination with certain other salts, espe- cially bichromate of potassium, nitrate of silver is much em- ployed in the Golgi methods to stain ganglion-cells and their processes in the central nervous system. The difficulty of the silver method lies in the fact that the salt forms with albuminous fluids granular and thread-like coagula which can easily give rise to false pictures. For this reason the method is limited almost entirely to natural surfaces, which should be washed off" with water or a 2 per cent, solution of nitrate of sodium before the silver solu- tion is applied. It is generally advisable to use the nitrate of silver in a very dilute solution, i : 250 or 500. The solu- tion is allowed to act on the surface for about a minute, and is then washed off with water. The tissue is next exposed in water to the action either of sunlight or of diffuse light. The outlines of the cells soon appear as dark lines, brown to black in color. The tissue to be stained should be kept stretched, because a precipitation of the silver occurs wherever there is a fold in the surface. Although nitrate 302 PATHOLOGICAL TECHNIQUE. of silver penetrates but a slight distance, it is possible to stain the outlines of the endothelial cells of the lymphatics and blood-vessels as well as the ground substance of the connective tissue — in a rabbit's diaphragm, for instance — by treating the upper or lower surface with the silver solution. The thoracic organs should be removed, and then the upper, surface of the tendinous portion of the diaphragm left in situ is exposed to the action of the silver salt in the manner already described. The outlines of the endothelial cells of blood-vessels are usually stained by injections of the silver salt through an artery. In the same way the limits of the epithelial cells of the alveoli of the lung can be stained by injections through a bronchus. Although generally employed in solution, nitrate of silver is sometimes used in the solid form, and for the cornea this method is preferable. Chloroform the animal, preferably a rabbit, deeply; rub the cornea with a stick of nitrate of silver hard enough to remove the surface epithelium. Allow the salt to act about ten minutes, then kill the animal, re- move the eye, cut out the cornea, wash it, and expose to diffuse daylight for half an hour. It is then placed in a mixture of glycerin and water, 30 parts to 70, very slightly acidulated with acetic acid (about -jJg- per cent.) for twenty- four hours, so as slightly to swell and to soften the tissues. Sections of the cornea are best made with the freezing microtome. Incise the periphery a little at four points equally distant from each other, so that the cornea will lie flat. A direct stain with alum-hematoxylin gives by all odds the best results. The sections may be mounted in glycerin or balsam. The latter method is perhaps the better. De- hydrate the sections in 50 per cent., then in 70 per cent., alcohol, clear in aniline oil, wash with xylol, and imbed in balsam. This method avoids the shrinkage which is caused by using strong alcohol. Gold, in the form of the simple or double chlorid, is em- ployed to stain the protoplasm of cells of connective tissue, and more particularly the axis-cylinders of nerve-fibers and HISTOLOGICAL METHODS. 3O3 their terminal processes. Like nitrate of silver, it acts as a fix- ing and hardening reagent as well as a stain. Unfortunately, it penetrates tissues but a very slight distance, and, so far as staining is concerned, is inconstant in action. Its chief use in pathology is in connection with experimental work on the cornea and in regeneration. The conditions under which the reduction of the gold salt takes place are not exactly understood, but both penetration and reduction are aided by the action of organic acids, such as formic, citric, and tartaric acids, on the tissues both before and after the treat- ment with the gold salt. Of the many methods proposed, the following are recommended: Lbwit's Pormic-acid Method. — i. Place very small bits of fresh tissue in a mixture of formic acid i part, and water I to 2 parts, until they become transparent (a few seconds to several minutes). 2. Transfer to chlorid of gold, i to 1.5 parts to 100 of water, for fifteen minutes. 3. Formic acid, i part to water 3 parts, for twenty-four hours. 4. Concentrated formic acid twenty-four hours. Preserve in glycerin or balsam. All the steps except the first should be performed in the dark. Ranvier's Formic-acid Method. — i. Boil together 8 c.c. of a I per cent, solution of chlorid of gold and 2 c.c. of formic acid. When the solution is cold place very small bits of tissue in it for one hour, in the dark. 2. Wash quickly in water. 3. Expose to diffuse light in a mixture of formic acid 10 c.c. and water 40 c.c. Reduction takes place slowly (twenty- four to forty-eight hours). 4. Harden in 70 per cent, then 90 per cent, alcohol in the dark. Osmic Acid (perosmic acid, osmium tetroxid) is used as a fixing reagent and for staining fat and myelin, by which it is reduced. As osmic acid is quickly reduced by organic substances, care must be taken in making up the solution. 304 PATHOLOGICAL TECHNIQUE. Remove the label from the sealed tube in which the acid comes, and place the tube, after cracking off one end, in a glass-stoppered bottle containing enough water to make a 2 per cent, solution. If desired, the tube can be broken after it is in the bottle by violent shaking. It should be borne in mind that osmic acid is very-irritating to the bron- chial mucous membrane. In a I or 2 per cent, solution osmic acid is used to stain fat in teased preparations or frozen sections of fresh tissues. In Marchi's method it is used to stain fat in tissues which have been hardened for some time in Miiller's fluid. As a fixing reagent it is usually combined with other reagents, as in Flem- ming's solution, both for its property as a fixative and for the purpose of staining any fat present. Preparations stained in osmic acid may be kept indefinitely in alcohol. When sections are mounted they should be cleared in chloroform, and preserved in chloroform balsam prepared in the manner described elsewhere. Xylol and other clearing reagents cause the stain to fade. STAINING riETHODS. The purpose of staining is to render prominent the differ- ent tissue-elements, so that they may be readily recognized and studied. The constant tendency now-a-days is toward selective or differential sl^ining methods, by which but one tissue-element will be colored to the exclusion of all others, or at least of any element that might be confused with it morphologically. These selective stains, which really are micro-chemical color reactions, enable us to differentiate from each other with ease and accuracy cellular and inter- cellular elements, or pathological products which otherwise look alike. The list given on page 305 does not pretend to be either complete or perfect in arrangement, but will give some idea of the various elements which we wish to stain. Those for HISTOLOGICAL METHODS. 305 Cell. Nucleus. Cuticle. Protoplasm. Bacteria, ' Nucleolus. Resting nucleus. Linin. Do not stain by Gram. Stain by Grai?i. 3. Stain by tubercle-bacillus method. Nucleus of ameba coli. Centrosome and polar bodies. Mastzellen. Plasma-cell of Unna. {Five kinds of gran- ules described by Ehrlich. NissPs granules in ganglion-cells. Dendritic processes of ganglion-cells. Axis-cylinder and terminal processes. Contractile elements of striated muscle-fiber. Red blood-globules. - Cilia of bacteria. Certain dots or lines in ependymal cells. So-called cilia in certain renal cells. Bile-capillaries. Granules. Intercellular substances. Cement substance of epithelial and endothelial cells. Ground substance of connective tissue. Connective-tissue fibrillce and reticulum. Fibroglia fibrilla. Myoglia fibrillin. Myxomatous tissue ; mucin. Elastic fibers. Intercellular substances of cartilage. Ground substance of bone. Myelin. ^ Neuroglia-fibers. Clubs of actinomyces. Capsules of bacteria. ' Fibrin. Mucin. Pathological products. Hyaline substances. ■ Fat. Hemosiderin. Hematoidin. . Hemoglobin. Amyloid. Glycogen. Hyalin. Colloid. Keratohyalin ^ Eleadin. 20 306 PATHOLOGICAL TECHNIQUE. which we now possess more or less perfect differential stains are printed in italics. The simplest selective stain is, of course, that for nuclei, and it can be obtained with a great variety of staining re- agents. The most difficult element to stain differentially, although it can be done under certain conditions with a fair amount of success, is probably the axis-cylinder and its ter- minal processes. Tissue-elements and pathological products differ from each other, not only in form and consistency, but also in chemical properties. While perfect preservation of form is sufficient to distinguish certain cells or elements from each other — as, for instance, polynuclear leucocytes from lymph- oid cells — differentiation based on micro-chemical tests is always to be preferred when possible. A few of the tests employed are colorless, like the precipitation of mucin by acetic acid. Certain tests, like the methylene-blue or gold stain for axis-cylinders, can be applied to fresh tissues only. Others, like the various amyloid reactions, can be obtained with fresh or hardened tissues. Most of the micro-chemical reactions, however, can be employed only with tissues which have been properly preserved. It is exceedingly important, therefore, that a tissue-element be so fixed and hardened that its peculiar chemical properties be preserved intact, otherwise a differential stain for it is impossible. Each tissue-element is a law unto itself For example, the peculiar chemical properties of red blood-globules depend on the presence in them of hemoglobin. As a differential stain of the red blood-globules depends on fixing this substance in them, it is necessary to find out the chemical properties of hemo- globin, such as the fact that it is soluble in water or dilute alcohol, but not in salt solution, and that it is fixed in the red blood-globules by heat, absolute alcohol and ether equal parts, corrosive sublimate, formaldehyde, bichromate of po- tassium, etc. While differential stains depend in part on the chemical properties of the tissue-elements, they also depend to a cer- HISTOLOGICAL METHODS. ^oy tain extent on the chemical properties of the staining re- agents and the decolorizers used. Some of the tissue-elements can be stained differentially in a number of ways, sometimes after one fixing agent, some- times after another. The simplest differential stains are those where certain tissue-elements stain directly in a given solu- tion after they have been properly fixed. Good examples are — Ehrlich's triple stain for certain protoplasmic granules in leucocytes, and the direct stain for elastic fibers with an acid alcohohc solution of orcein. Other differential stains depend on the property of certain elements to hold colors they have once taken up when treated with decolorizers. The best example of this is the tubercle bacillus, which holds certain stains through various acids, or aniline hydrochlorate, followed by alcohol, and, if necessary, by a contrast-stain. Still another varied group of elements (certain bacteria, fibrin, neuroglia-fibers, etc.) depends for a differential stain in part on changes produced in gentian- or methyl-violet by iodin, in part on the decolorizer employed for extracting the coloring reagent. Although the steps of the various staining methods differ considerably, they may be roughly arranged in the following order : 1. Staining. 2. Differentiating. 3. Decolorizing. 4. Dehydrating. 5. Clearing. 6. Mounting. Very often two or more of the steps are combined in one, as when aniline oil is used for decolorizing, dehydrating, and clearing sections stained for certain bacteria. Sometimes the staining process occupies more than one step, as in Weigert's myelin-sheath stain. In alum-hematoxylin the differentiating reagent, the excess of alum, is combined with the stain ; in Gram's method the differentiating reagent, iodin, forrns a step by itself. 308 PATHOLOGICAL TECHNIQUE. NUCLEAR STAINS. For general histological work few stains are more valua- ble or can be more highly recommended than alum-hema- toxylin, either alone or in contrast with eosin. Prop- erly made and used, the solution stains the nuclei sharply and of varying degrees of intensity, depending on the cha- racter of the cells. Besides the nuclei, however, it stains other tissue-elements in delicate shades of blue, so that they are readily visible, and thus more or less differentiated from those structures which fail to stain. Of the carmine stains, lithium carmine, followed by picric acid, will be found the most brilliant, generally useful, and permanent. Safranin gives, perhaps, the most permanent stain of any of the basic aniline dyes, and confines itself very sharply to the nuclei. It is much used after certain fixing reagents, such as Flemming's and Hermann's solutions. Fosin, followed by methylene-blue, gives beautiful results, especially when Unna's alkaline solution of methylene-blue is used. The advantages of this solution are that it stains readily tissues hardened in Zenker's fluid and brings out nuclei and nuclear figures with great sharpness, while at the same time it stains the protoplasm of certain cells so that they are easily dis- tinguished from other cells. The Heidenhain-Biondi triple stain is useful after fixation in corrosive sublimate, but can- not be employed with celloidin sections, so that its field is limited. The other aniline dyes are used on occasion or for some definite purpose, but not so generally as those men- tioned above. Alum-hematoxylin Stains. — Most alum-hematoxylin solutions will over-stain if the sections are left too long in them. The proper time required depends on the fixing re- agent used and on the degree of ripeness of the staining solution. It is therefore advisable to wash a section in water occasionally and decide from the color it has acquired if it be sufficiently stained, or to mount it in water on a slide and examine with the low power of the microscope. HISTOLOGICAL METHODS. 309 The best results are obtained with alum-hematoxylin solu- tions by staining sections just deeply enough, washing them thoroughly in several changes of water, and leaving them in a large dish of water over night. This thorough washing is done to rid the tissues of every trace of alum or of acid, so that the color will become a clear blue and will keep in- definitely. Many microscopists prefer to stain deeply and diffusely in an old, quickly-staining alum-hematoxylin solution, and then to employ a decolorizer. The agents most used for the pur- pose are alum (i per cent, aqueous solution for one to two hours), hydrochloric acid (jlj to \ per cent, aqueous solution, or even the ordinary acid alcohol), and acetic acid (i to 3 per cent, solution) for a few seconds only. After being suf- ficiently decolorized the sections must be thoroughly washed in water, preferably for a number of hours, otherwise the stain will fade. The objection to this method is that a pure nuclear stain only is obtained, because the acid removes the color completely from all the rest of the tissue. Under cer- tain circumstances, as when hematoxylin is used as a con- trast-stain to fuchsin in staining for tubercle bacilli, such a sharp limitation to the nuclei is desirable. Alum-hematoxylin stains well and quickly tissues hard- ened in alcohol, in corrosive sublimate, and in picric acid. It stains much more slowly tissues hardened in solutions containing chrome salts, such as Zenker's and Miiller's fluids. For counter-staining eosin will usually be found to give the most beautiful contrast, although, picric acid. Van Gieson's mixture, and neutral carmine are often of service. A good alum-hematoxylin solution should have a bluish or purplish color, and should stain celloidin very faintly or not at all. Aqueous Alum-hematoxylin; Delafield's Hema- toxylin; Bhrlich's Acid Hematoxylin (see pages 281 and 282). 4 I . Stain in one of the above solutions two, five, or thirty minutes, or sometimes even longer. 3IO PATHOLOGICAL TECHNIQUE. 2. Wash in several changes of water, and then leave sec- tions, if possible, for several hours or over night in a large dish of water; or better still, wash in running tap water for ten to thirty minutes. 3. Contrast-stain, usually an aqueous solution of eosin, ^ to J per cent., for one to five minutes. 4. Alcohol, 95 per cent., two or three changes to dehydrate and to remove excess of contrast-stain. 5. Clear in oleum origani cretici or in Dunham's oils-of- cloves-and-thyme mixture. 6. Canada balsam. The staining of the nuclei by Ehlrich's alum-hematoxylin solution is not so sharp as that obtained by the simple aque- ous solution. The more customary method of using Delafield's alum- hematoxylin solution is to filter a few drops of it into a dish of water and to stain sections for a long time, even over night, with the very dilute solution thus obtained. It is sometimes advisable to use the aqueous solution in the same way. Mayer's Hemalum (see page 283). — i. Stain three to five minutes or longer. 2. Wash out in i per cent, alum solution until the stain is precise. 3. Wash thoroughly in several changes of water. 4. Alcohol, 95 per cent. 5. Oleum origani cretici. 6. Canada balsam. The staining is rather diffuse, so that it has to be washed out to some extent with alum-water. Mayer's acid hemalum is more precise, and usually does not need to be decolorized, so that the second step can be omitted. Hemalum is used for staining tissues in bulk. Twenty- four hours are required for large pieces. Heidenhain's Hematoxylin Stain. — i. Stain twenty- four to forty-eight hours in a simple \ per cent, aqueous solution of hematoxylin dissolved by the aid of heat. 2. Transfer the sections directly to a |^ per cent, aqueous HISTOLOGICAL METHODS. 31 1 solution of simple ciiromate of potassium for twenty-four to forty-eight hours, changing the solution frequently until no more color is given off by the sections. 3. Wash thoroughly in water. 4. Alcohol. 5. Oil. 6. Canada balsam. Heidenhain's Iron Hematoxylin. — This staining method is particularly useful for the demonstration of the centrosome, but also stains nuclei and a variety of other structures, according to the degree of differentiation. 1. Fix in corrosive sublimate, Zenker's fluid, or alcohol. 2. Stain very thin paraffin sections (not over 5 to 6// thick) in a 2.5 per cent, solution of the violet iron alum (sulphate of iron and ammonium) for three to twelve hours. The sections should be placed vertical in the solution, so that no precipitate may fall on them. 3. Wash off quickly in water. 4. Stain in a 0.5 per cent, ripened alcoholic solution of hematoxylin for twelve to thirty-six hours. 5. Wash off in water. 6. Differentiate in the iron-alum solution, controlling the results under the microscope. The section should be washed off before each examination in a large dish of tap water, which immediately stops the decolorization. 7. Wash in running water for a quarter of an hour. 8. Alcohol, xylol, xylol balsam. A counterstain with Bordeaux R. before, or with rubin S. after, the iron stain is sometimes useful. Mallory's Chlorid of Iron Hematoxylin.* — The results which can be obtained by this method are equally quick and satisfactory after all of the usual fixing reagents except, perhaps, formaldehyde. Celloidin or paraffin can be employed for embedding. 1. Stain sections on the slide for three to five minutes in a ID per cent, aqueous solution of ferric chlorid. 2. Drain and blot the sections ; then pour over them a ' Mallory: The Journal of Experimental Medicine, 1900, v., 18. 312 PATHOLOGICAL TECHNIQUE. few drops of a freshly prepared i per cent, aqueous solution of hematoxylin. If all of the hematoxylin is precipitated by the excess of ferric chlorid, pour off the solution and add a fresh supply. In three to five minutes the sections will be colored a dark bluish-black. 3. Wash in water. 4. Decolorize and differentiate in a J4^ per cent, aqueous solution of ferric chlorid. The sections should be kept con- stantly moving in the solution. The differentiation will be complete in a few seconds to one or more minutes. 5. Wash in water. 6. Dehydrate in alcohol. 7. Clear in oleum origani cretici. 8. Xylol balsam. In the above directions definite strengths have been assigned to the solutions, but they may vary greatly with- out affecting the result. The important point is to get the sections stained deeply, and then to decolorize slowly. The differentiation can be stopped at any moment by transferring the sections to water. Sometimes it is advisable to examine the sections under the microscope to see if enough color has been extracted. The strength of the hematoxylin solution is unimportant ; it is simply necessary to have enough hematoxylin to com- bine with all of the iron in and on the section. The sim- plest way is to dissolve by the aid of heat a pinch of the crystals in a few cubic centimeters of water. A little ex- perience will determine about how much is needed. If a solution of hematoxylin more than one or two days old is used, the color obtained is grayish-blue, and not so bright. This method gives a sharp, permanent, dark-blue stain to nuclei ; it also stains fibrin of a grayish to dark-blue color ; if the decolorization is not carried too far, the contractile ele- ments of striated muscle are brought out very sharply. In Zenker preparations the red globules appear of a greenish- gra^ cglor. Coiinective tissue is tinted a pale yellow. The nucleifs^of the amoeba coli stains sharply by this method. Carmine Stains. — The ordinary carmine solutions give HISTOLOGICAL METHODS. 313 good nuclear stains, but of the finer details in a specimen they bring out much less than a direct alum-hematoxylin stain. They are much less used now than formerly, except as contrast-stains to bacteria and to fibrin in the methods of Gram and Weigert, for which purpose lithium carmine will usually give the best results. Alum Carmine ; Alum Cochineal (see pages 285 and 286). — I. Water. 2. Stain in either of the above solutions for five to twenty minutes. 3. Wash thoroughly in water. 4. Alcohol, 95 per cent. 5. Oleum origani cretici. 6. Canada balsam. Over-staining does not occur. The solutions cannot be recommended for tissues which stain with difficulty. When used for staining in bulk, twenty-four to forty-eight hours are required. Lithium Carmine (see page 286). — i. Water. 2. Stain two to five minutes. 3. Transfer directly to acid alcohol, one or more changes for several minutes or more, until the sections are well differ- entiated. 4. Wash in water. 5. Alcohol, 95 per cent. 6. Oleum origani cretici. 7. Canada balsam. This method gives an intense and permanent bright-red nuclear stain. Over-staining is impossible. A trace of picric acid added to the alcohol used^ for dehydration affords a beautiful contrast-stain. Aniline Dyes as Nuclear Stains.— Any of the basic anilme dyes may be used as nuclear stains after the following general method : 1. Stain paraffin sections in a strong solution of the dye preferred in water or in dilute alcohol for five to thirty minutes. 2. Wash in water. 314 PATHOLOGICAL TECHNIQUE. 3. Dehydrate in absolute alcohol. 4. Clear in xylol. 5. Xylol balsam. With celloidin sections use 95 per cent, alcohol, blot with filter paper, and clear in xylol. As a matter of fact, however, certain dyes and certain solutions are generally used in preference to the others. Most of the colors are more or less affected by all clearing reagents except xylol. With paraffin sections and those from which the celloidin has been removed it is very easy to dehydrate in absolute alcohol and to clear in xylol. With celloidin sections, however, this is impossible, because the absolute alcohol will dissolve out the celloi- din, and this is usually not desirable. For celloidin sec- tions, therefore, blot with filter paper, and then pour on xylo^ffepeat the blotting, fpllowed by xylol, two or three tim^jBntil the specimen is perfectly clear. Mount in xylol ^^^washing okt the excess of color it is sometimes found advantageous to acidulate very slightly either the water or the first alcohol with acetic or hydrochloric acid. This pro- Rp, if not carried too far, tends to make the nuclear stain arper. « Safranin is one of the very best nuRar-staining aniline dyes. Tissues may be hardened in alcohol, corrosive subli- mate, Flemming's, Hermann's, or Zenker's fluids. Any one of the solutions of safranin given on page 290 may be used. I. Stain paraffin sections two to five minutes to twenty- four hours according to the staining solution and fixing re- agent used. .fl*'. . 2. Wash iifB'ater. 3. AbsoluCe'^alGohol, several changes, until the section appears properly differentiated. 4. Xylol. .;'■ 5. Xylol balsam. For celloidin sections dehydrate in 95 per cent, alcohol, clear in bergamot or origanum oil, and wash out in xylol. HISTOLOGICAL METHODS. 31 5 To render the stain more precise a few drops of acid alcohol are sometimes added to the first alcohol. The Eosin and Methylene-blue Stain. — This stain, used on paraffin sections of tissues fixed in Zenker's fluid, can be recommended as the very best general stain yet devised. It is a sharp nuclear stain, and, at the same time, brings out with a great deal of differentiation all the various other structures in the different tissues. It has been in constant use for many years as the routine stain for all tissues in the pathological laboratories of the Harvard Medical School and Boston City Hospital. Fix in Zenker's fluid or in corrosive sublimate. Alcohol and formaldehyde are not nearly so good. 1. Stain paraffin sections in a 5 to 10 per cent, aqueous solution of eosin for twenty minutes or longer. Sometimes it is advisable to get a deeper eosin stain by placing the sec- tions in the paraffin oven for fifteen to twenty minutes. 2. Wash in water to get rid of excess of eosin. 3. Stain in Unna's alkaline methylene-blue solution (see page 288), diluted 1-4 or 5 with water, for ten to fifteen minutes. 4. Wash in water. 5. Differentiate ^d dehydrate in a dish of 95 per cent, alcohol, keeping the section in constant motion, so that the decolorization shall be uniform. Control the result under the microscope. When the pink color has returned to the section and the nuclei are still a deep blue, finish the dehy- dration quickly with absolute alcohol. 6. Xylol. ^, ■ ^ ' ^ 7. Xylol balsam. .-*" ''^^^: ' For celloidin sections use 95 per cent. alcoaEf)lo|^ and pour on xylol ; repeat the last two steps until tne specimen is clear. It is important to get a deep stain with eosin, because the methylene-blue washes it out to a considerable extent. The eosin must be used first, because methylene-blue is readily soluble in an aqueous solution of eosin, and therefore is quickly extracted if the eosin is used after it, while on the "I 3I_6 PATHOLOGICAL TECHNIQUE. other hand eosin is very slightly soluble in an aqueous solu- tion of methylene-blue which is precipitated by any excess of eosin. DiflEiise or contrast-stains are useful to make promi- nent certain of the tissue-elements left uncolored by the nuclear stain. A greater richness of detail is obtained with diffuse stains if, after rather deep staining, the sections be washed out for some time in alcohol, because certain struc- tures possess a greater affinity than others for certain diffuse stains, and by holding them are brought out sharply. Of the diffuse stains, eosin, picric acid, and acid-fuchsin in Van Gieson's mixture are the ones most frequently em- ployed. Eosin is most frequently used as a contrast to alum-hema- toxylin stains, but is often serviceable with alum-cochineal, methylene-blue, gentian-violet, etc. It brings out particu- larly well red blood-globules and smooth and striated muscle- fibers. The strength of the solutions used after hematoxy- lin varies from -^ to \ per cent, according to the tissue and the fixative used. Zenker's preparations stain intensely in eosin, so that for them a very dilute solution is advisable. When desired as a contrast-stain to basic aniline dyes, eosin should be used first in a 5 or even 10 per cent, solution, because otherwise it is likely to be washed out by the nuclear stain. Picric acid is used for contrast with the carmine stains, more rarely with alum-hematoxylin. Striated muscle-fibers and cornified epithelium are rendered especially prominent by it. To stain with picric acid it is only necessary to add a few drops of a saturated aqueous solution to a dish of water, or of a saturated alcoholic solution to a little alcohol, and allow sectidnsj^d remain in the solution for a few seconds. Van GieseSSi^ stain (see p. 291), a mixture of picric acid and acid fuchsin, is excellent as a contrast-stain to alum- hematoxylin, especially when it is desirable to render promi- nent connective-tissue fibrillae or certain pathological prod- ucts. The nuclear stain with alum-hematoxylin must be HISTOLOGICAL METHODS. 317 rather deep, because the picric acid to some extent extracts or overpowers it. 1. Stain deeply in alum-hematoxylin. 2. Wash in water. 3. Stain in Van Gieson's solution three to five minutes. 4. Wash in water and dehydrate directly in 5. Alcohol, 95 per cent. 6. Oleum origani cretici. 7. Canada balsam. Neutral Carmine (see page 286). — Neutral carmine is a diffuse stain, and is employed more especially for the central nervous system and for bone. Filter one or two drops of the solution into 20 c.c. of dis- tilled water, and leave the sections in the dilute solution over night. It is advisable to place a piece of filter-paper on the bottom of the dish for the sections to rest on, otherwise they may be stained on the upper side only. In double stains with hematoxylin and carmine the sections should be stained first in the hematoxylin and then thoroughly washed in water for six to twelve hours before they are stained in the carmine. After the carmine they are again to be thoroughly washed in water. Combination Stains. — Biondi-Heidenhain Stain (see p. 292). — Tissues must be hardened in corrosive sublimate. 1. Stain paraffin sections six to twenty-four hours with the dilute solution. 2. Wash out a little in 90 per cent, alcohol. 3. Dehydrate in absolute alcohol. 4. Xylol. 5. Canada balsam. It is important to place the sections directly from the staining fluid into the alcohol, because water washes out the methyl-green almost instantly. ■* Staining; in Mass. — The staining of tissues in mass is a procedure much less employed in pathological than in nor- mal histology, but still occasionally useful. For patholog- ical tissues a variety of stains is generally necessary. It is therefore much better to make a series after one of the 3l8 PATHOLOGICAL TECHNIQUE. methods described, and then to stain the sections in what- ever way seems best. For staining in bulk only a hmited number of solutions are available — either those, like alum-carmine and alum- cochineal, which do not stain beyond a certain point, or those, like lithium and borax-carmine and Heidenhain's hematoxylin, which may be decolorized so as to leave only the nuclei stained, The process of staining differs from that for sections only in the length of time required for each step. Tissues \ cm. thick will need from one to two days in the staining solution. MITOSIS. For the study of karyomitosis it is important that the tissue be perfectly fresh — that is, just removed from a living animal or from one just dead — and that it be fixed in a suitable reagent as quickly as possible. The best results cannot be obtained with tissues put into a hardening fluid over half an hour after removal from a living animal. On the other hand, mitotic figures can be demonstrated in tissues which have been dead for some time (twenty-four hours or more) before being put into a fixing reagent, but the details of the figures are not so perfect as those in perfectly fresh tissues, and the figures are not so numerous, because some of them have completed their changes and can no longer be recognized. It is therefore evident that mitosis can be studied much better in tissues from the lower animals, or in tissues removed by operation from the human body, than in the organs and tissues removed at post-mortem examinations. The choice of fixing reagents for the study of mitotic figures is important. The figures can often be demonstrated after hardening in alcohol or even in Miiller's fluid, but for their careful study quicker and more perfect fixing reagents must be used. Nearly all of the reagents employed pene- trate slowly, so that it is absolutely necessary for the best results that the tissue to be hardened be cut into very thin slices, rarely over 4 mm. in thickness and preferably not over HISTOLOGICAL METHODS. 319 2 mm. The amount of fixing reagent used should always be at least ten to fifteen times as great as the volume of the tissue, and should be changed if it becomes cloudy. The most important fixing reagents are — 1. Flemming's solution. 2. Hermann's solution. 3. Pianese's solution. 4. Zenker's fluid. 5. Corrosive sublimate. 6. Orth's fluid. The first three solutions penetrate with much difficulty, so that tissues placed in them should be especially thin. The most generally useful stain for mitosis is probably safranin. The time of staining varies with the solution used. Babes' is the quickest. The mitotic figures should be stained deeply : then, when treated with alcohol slightly acidulated with hydrochloric acid, they will retain the color, while the resting nuclei will yield up most of theirs and become very pale or even colorless. In consequence of this intense stain mitotic figures can then be very readily found. Directions for Staining Karyomitotic Figures with Safranin. — i. Stain paraffin sections five minutes to twenty- four hours, according to solution used. 2. Wash in water. 3. Wash in 95 per cent, alcohol to which are added a few drops of acid alcohol. 4. Wash in pure 95 per cent, alcohol, followed by absolute alcohol. 5. Xylol. 6. Xylol balsam. For celloidin sections dehydrate in 95 per cent, alcohol, blot, and pour on xylol ; repeat the last two steps until the specimen is clear. Safranin can be used after any of the above fixing reagents. Other useful stains are carbol-fuchsin and aniline-gentian- violet, used in the same way as the safranin. The Gram- Weigert method gives good results after Flemming's solu- tion. 320 PATHOLOGICAL TECHNIQUE. After fixing in corrosive sublimate mitotic figures can be demonstrated by the Biondi-Heidenhain solution, which stains resting nuclei blue-violet and mitotic figures green. After Pianese's solution his special staining mixtures should be used (see page 293). His methods are said to give beautiful, results. SPECIAL STAINS FOR CERTAIN TISSUE=ELEMENTS OTHER THAN NUCLEI. MASTZELLEN. Mastzellen are found in the connective tissue, more especially in chronic inflammatory processes. Their proto- plasmic granules stain intensely like bacteria with the basic aniline dyes. Several methods of staining the granules are given. With Unna's stains for plasma-cells a differential color-stain is obtained for the granules of the mastzellen. Bhrlich's Method. — A. General Stain. — Harden in alcohol. 1. Stain with a saturated aqueous solution of dahlia. 2. Wash out with acidified water. 3. Dehydrate in alcohol; clear and mount in Canada balsam. B. Specific Stain. — Only the protoplasmic granules are stained. Harden in alcohol. 1. Stain twelve hours in — Absolute alcohol, 50 c.c. Water, lOO " Glacial acetic acid, 1 2.5 " Dahlia, q. s., so that the solution is almost saturated. 2. Wash out in alcohol, clear, and mount. C. Ehrlich- Westphal Method. — Nuclei red ; granules blue. Harden at least a week in alcohol. HISTOLOGICAL METHODS. 32 1 1. Stain in the following solution twenty-four hours : Alum-carmine solution, 200 ; Saturated solution of dahlia in absolute alcohol, 200 ; Glycerin, 100; Glacial acetic acid, 20. (Stir repeatedly, then allow the mixture to stand for some time.) 2. Decolorize for twenty-four hours in absolute alcohol. 3. Oil, Canada balsam. Unna's Isolated Stains for Mastzellen. — Harden in alco- hol. Nuclei blue ; protoplasmic granules of " Mastzellen " red. A. — I. Stain in polychrome methylene-blue solution, plus a little alum, for three hours to over-night. 2. Wash in water. 3. Absolute alcohol, oil of bergamot, balsam. B. — I. Stain in polychrome methylene-blue solution one- quarter of an hour. 2. Wash in water. 3. Decolorize in glycerin-ether mixture for five to ten minutes. 4 Wash a long time in water. 5. Absolute alcohol, oil, balsam. PLASMA-CELLS. These are certain cells, much studied by Unna, which are very abundant in subacute and chronic pathological pro- cesses, and which are characterized by protoplasm which stains quite deeply in alkaline methylene-blue solutions. The two methods best suited for their demonstration furnish at the same time a differential color-stain for mastzellen. The granules of the latter are stained red, the plasma-cells are stained blue. Unna's Diflferential Stains for Plasma-cells and Mast- zellen. — Harden tissues in absolute alcohol. A. — I. Stain paraffin sections in polychrome methylene- blue one-quarter of an hour to over-night. 2. Decolorize in a small dish of water, to which are added a few drops of glycerin-ether mixture. 21 322 PATHOLOGICAL TECHNIQUE. 3. Wash thoroughly in water. 4. Absolute alcohol, xylol, balsam. B. — I. Stain in polychrome methylene-blue solution five to fifteen minutes. 2. Wash in water. 3. Decolorize and dehydrate in a J per cent, alcoholic solution of neutral orcein (about fifteen minutes). 4. Absolute alcohol, xylol, balsam. CONNECTIVE-TISSUE FIBRILL/E AND RETICULUM. Several methods are available for the demonstration of connective fibrillse and reticulum. The simplest is by means of Van Gieson's picric acid and acid-fuchsin solution, but it is applicable to the coarser fibers only. With Ribbert's method contrast stains are incompatible. The stain with aniline blue is believed to be better than any yet proposed, but is limited to tissues hardened in corrosive sublimate solution or in Zenker's fluid. '' A. Mallory'e Aniline Blue Stain.^ — The following method is not absolutely differential because, besides connective-tis- sue fibrillae and reticulum, it also stains certain hyaline sub- stances, but these latter usually are so different morpho- logically tliat confusion cannot arise. The method is also useful for the study of fibrin, fibroglia fibrils, smooth and striated muscle-fibers, and amyloid. 1. Fix in Zenker's fluid. 2. Imbed in celloidin or paraffin. 3. Stain sections in a ^V P^r cent, aqueous solution of acid fuchsin for five minutes or longer. 4. Tpnsfer directly to the following solution and stain for twenty minutes or longer : Aniline blue soluble .in water (Griibler), 0.5 ; Orange G (Griibler), 2.0- One per cent, aqueous solution of phos- phomolybdic acid, loo.o; 'Mallory: The Journal of ExpeHmental Medicine, 1900, v., 15. HISTOLOGICAL METHODS. 323 5. Wash and dehydrate in several changes of 95 per cent, alcohol. 6. Clear in xyl6l or in oleum origani cretici. 7. Xylol balsam. The fibrillse and reticulum of connective tissue, amyloid, mucus, and certain other hyaline substances stain blue ; nuclei, protoplasm, fibroglia fibrils, axis-cylinders, neuroglia- fibers, and fibrin red ; red blood-globules and myelin-sheaths yellow ; elastic fibers pale pink or yellow. The various structures do not stain with equal intensity, so that certain ones are brought out with great sharpness. This is par- ticularly true of the fibrillae and reticulum of connective tissue, and of fibrin and smooth and striated muscle-fibers. If it is desired to bring out the connective tissue as sharply as possible, omit the staining with acid fuchsin. Then the nuclei and protoplasm stain yellow, and the blue fibrillae and reticulum stand out more prominently. B. Van Gieson's Stain. — The proportions given are those recommended by Freeborn. Occasionally it will be found necessary to increase the proportion of the acid fuchsin. 1. Harden in chrome salts or in corrosive subHmate. The results after alcohol are not so good. 2. Stain deeply in alum-hematoxylin. 3. Wash in water. 4. Stain for three to five minutes in I per cent, aqueous solution of acid fuchsin, 5 '^•'^• Saturated aqueous solution of picric acid, lOO " 5. Dehydrate in 95 per cent, alcohol. 6. Oleum origani cretici. 7. Canada balsam. C. Unna's Orcein Stain. — i. Harden in alcohol. 2. Stain in the concentrated solution of polychrome methylene-blue five minutes. 3. Wash in water. 4. Decolorize, differentiate, and stain in a i per cent, solu- tion of orcein in absolute alcohol fifteen minutes. 5. Wash in absolute alcohol. 324 PATHOLOGICAL TECHNIQUE. 6. Oil of bergamot. 7. Balsam. Nuclei, dark blue ; protoplasm, pale blue ; elastic and con- nective-tissue fibers, deep orcein red ; smooth muscle-fibers, bluish ; mastzellen granules, red ; protoplasm of plasma- cells, deep blue. D. Mall's DiflFerential Method for Reticulum.^ — i.' Digest frozen sections of fresh tissue, 40 to 80 /i thick, for twenty- four hours in the following solution : Parke, Davis & Co.'s pancreatin, 5 grams ; Bicarbonate of sodium, 10 " Water, 100 c.c. 2. Wash carefully in clean water. 3. Place sections in a test-tube half full of water, and shake thoroughly in order to remove all the cellular debris. 4. Spread out on shde, and allow to dry. 5. Allow a few drops of the following solution to dry on surface : Picric acid, 10 grams ; Absolute alcohol, 33 c.c. ; Water, 300 " 6. Stain for about half an hour in the following solution : Acid fuchsin, 10 grams ; Absolute alcohol, 33 c.c. ; Water, 66 " 7. Wash in the picric acid solution for a moment. 8. Alcohol, xylol, balsam. FIBROQLIA F1BRILS.2 Connective-tissue cells produce, in addition to reticulum, the fibrils of white fibrous tissue, and elastic fibrils, a fourth variety of fibrils, which bear the same relation to the con- ' Mall ; Johns Hopkins Hospital Reports, 1896, i., 171. 2 Mallory : " A Hitherto Undescribed Fibrillar Substance Produced by Connective-tissue CeWs," Journal of Medical Research, 1903, vol. i. HISTOLOGICAL METHODS. 325 nective-tissue cells that neuroglia fibrils bear to neuroglia cells. Fibrils which stain by this same method are present also in the true basement membrane of the coil-glands of the skin, of the glands of the breast, and many tumors derived from them, of the tubules of the kidney, and beneath the endothelium of arteries and veins. The stroma of a more "or less scirrhous carcinoma of the breast furnishes excellent material for their study. Mallory's Differential Stain. — i. Fix in Zenker's fluid. The tissue should be as fresh as possible, and cut into thin sections (2 to 4 mm. thick) for the best results. 2. Stain celloidin or paraffin sections in a i per cent, aqueous solution of acid fuchsin overnight in the cold; or twenty to thirty minutes in the paraffin oven (56° C). 3. Wash quickly in water (not over five seconds). Water extracts acid fuchsin very rapidly. 4. Differentiate in a 0.25 per cent, aqueous solution of permanganate of potassium for twenty to forty seconds. This step must not be prolonged beyond the exact time needed or the section will be decolorized. 5. Wash quickly in water (not over five seconds). 6. Dehydrate in alcohol. 7. Clear in oleum origini crsetici or in xylol. 8. Mount in xylol balsam. While not an absolutely differential stain for these fibrils, the method, for the most part at least, is perfectly satisfac- tory. It stains intensely red, not only these fibrils and the cell nuclei, but also fibrin, the contractile elements of striated muscle-cells, the coarse, differentially staining fibrils of smooth muscle-cells, neuroglia fibers, and the cuticular surfaces of epithelial cells. Ordinary connective-tissue fibrils appear from brownish-yellow to colorless ; elastic fibrils, unless degenerated, are bright yellow. .^^ These fibrils can also be stained by several other methods. The most reliable is, perhaps, phpsphotungstic acid hema- toxylin after fixation in Zenker's fluid. I. Place sections in a |^ per cent, aqueous solution of per- manganate of potassium ten to twenty minutes. 326 PATHOLOGICAL TECHNIQUE. 2. Wash off in water. 3. 5 per cent, aqueous solution of oxalic acid one to two hours. 4. Wash thoroughly in several changes of water. 5. Stain in phosphotungstic acid hematoxylin twenty- four to forty-eight hours. 6. Wash off in water. 7. Alcohol, xylol, balsam. In sections stained by the aniline-blue connective tissue stain these fibrils appear bright red. They can also be demonstrated by either of the iron-hematoxylin methods if the differentiation is not carried too far. ELASTIC FIBERS. Elastic fibers are not affected by dilute caustic soda or potash, or by acids. These reagents are often used, there- fore, to demonstrate elastic fibers in the fresh condition, as, for example, in sputum, because they render them prominent by clearing or destroying the other tissues. The fibers show a marked affinity for osmic acid, staining with greater rapid- ity than most other tissue-elements. For bringing out elastic fibers in sections of hardened tissues there are two excellent differential stains, of which the first, Weigert's, is particularly recommended for the sim- plicity of the method and the sharpness and intensity of the picture it gives. A. Weigert's Stain for Elastic Fibers.' — Fixation in alcohol or formaldehyde preferable, but other fixing reagents give good results. Imbed in celloidin or paraffin. I. Stain sections twenty minutes to one hour in the fol- lowing solution : Fuchsin, 2 ; Resorcin, 4 ; Water, 200. Boil the solution in a porcelain dish ; when briskly boil- ing add 25 c.c. of liquor ferri sesquichlorati ; stir and boil ' Weigert : Centralblatt filr allg. Pathologic, 1898, ix., 289. HISTOLOGICAL METHODS. 327 for two to five minutes. A precipitate forms. Cool and filter. The filtrate is thrown away. The precipitate remains on the filter-paper until all the water has drained away or until the precipitate has thoroughly dried. Then return filter and precipitate to the porcelain dish, which should be dry, but which should contain whatever part of the precipi- tate remained sticking to it. Add 200 c.c. of 95 per cent, alcohol, and boil. Stir constantly, and fish out the filter-paper as the precipitate is dissolved off". Cool ; filter ; add alcohol to make up the 200 c.c. Add 4 c.c. of hydrochloric acid. 2. Wash off in alcohol. 3. Blot with filter-paper, and add xylol quickly ; repeat the blotting, followed by xylol, two or three times until the section is perfectly cleared. 4. Xylol balsam. Sections can be stained for several hours. If the rest of the tissue is overstained, differentiate in acid alcohol ; if the sections are too deeply stained, the color cannot be washed out. Diffuse staining can be avoided by diluting the stain. The elastic fibers appear dark blue, almost black, on a clear background. The nuclei can be stained red with carmine before or after the staining of the fibers. The solution keeps for months. If it be desired to keep sections for some time before mounting, wash them in alcohol and place in water. B. Unna's Orcein Method for Elastic Fibers. — Unna's latest method of using orcein is as follows, and can be highly recommended : I. Stain sections in the following solution : Orcein (Griibler), i ; Hydrochloric acid, i ; Absolute alcohol, 100. Place the sections in a dish and pour over them enough of the solution to cover them. Warm gently in an incu- bator or over a small flame for ten to fifteen minutes until the solution thickens, or leave in the solution at room- temperature overnight. 328 PATHOLOGICAL TECHNIQUE. 2. Wash off thoroughly in dilute alcohol (70 per cent.). 3. Wash in water to get rid of all the acid and to fix the color. 4. Alcohol. 5. Oil. 6. Balsam. The washing in water is not absolutely essential. Elastic fibers are stained of a deep silky-brown color, con- nective tissue a pale brown. If it is desirable to have only the elastic fibers stained, wash for a few seconds in i per cent, hydrochloric-acid alcohol before washing in water. The nuclei can be brought out by staining in Unna's poly- chrome methylene-blue solution after washing the sections in water. SMOOTH AND STRIATED MUSCLE=CELLS. Smooth and Striated Muscle-cells. — For the demon- stration of muscle-cells double stains, such as alum-hema- toxylin and eosin or eosin and methylene-blue, are sufficient. For certain purposes, however, the aniline-blue connective- tissue stain is much to be preferred. For the demonstration of the coarse, differentially staining myoglia fibrils of smooth muscle-cells three methods are available, but all of them stain also the fibroglia fibrils of connective-tissue cells. (See p. 322.) 1. Heidenhain's iron-hematoxylin stain is useful, especially for cross-sections of the fibrils. (See p. 31 1.) 2. Benda's Stain for Myoglia Fibrils.' — i. Fix fresh material in Zenker's fluid for twenty-four hours. 2. Wash for a number of hours in water. 3. Make frozen sections. 4. Place sections in a 0.5 per cent, solution of chromic acid for twenty-four hours. 5. Wash off in water. 6. Place in a 0.25 per cent solution of permanganate of potassium for about three minutes. ^ Erganzungsheft zum XXI. Band, 1 902, des Anatoniischen Anzeigers, p. 214. HISTOLOGICAL METHODS. 329 7. Wash off in water. 8. Place in Pal's mixture of sulphite of sodium and oxalic acid for five minutes. 9. Wash off in water; take up section on slide. 10. Cover with the following solution : Crystal-violet, saturated solution in 70 per cent, alcohol, i part; Acid alcohol, i " Aniline-water, 2 parts. 11. Blot with filter-paper. 12. Cover with dilute Lugol's solution. 1 3. Blot with filter-paper ; dry. 14. Differentiate in aniline oil and xylol, equal parts. 15. Xylol; xylol balsam. 3. Mallory's differential stain for fibroglia fibrils is much simpler than the above method of Benda's, and gives equally good results. (See p. 322.) THE CENTRAL NERVOUS SYSTEM. In the preservation of the central nervous system the special structures which require consideration are the gan- glion-cells, including both the dendritic and the axis-cylinder processes, the myelin-sheaths, and the neuroglia-fibers. No one fixing reagent is suited for the best preservation of all of them, unless possibly it be formaldehyde. The main fixing fluids for the nervous system until within a very short time have been various solutions of the chrome salts, particularly of bichromate of potassium, either alone or in combination with sulphate of sodium, as in the well- known Miiller's fluid. The chief objections to the chrome salts as fixatives are that they penetrate and harden very slowly, and do not preserve properly either the ganglion- cells or the neuroglia-fibers. On the other hand, they prob- ably preserve the axis-cylinders as well as any reagent we yet know, and are invaluable for their property of enter- ing into some chemical combination with myelin, in con- sequence of which it is possible to obtain by the method 330 PATHOLOGICAL TECHNIQUE. originated by Weigert a differential stain of the myelin- sheaths. The new fixing reagent, formaldehyde, seems likely to find its greatest use histologically as a fixative of the central nervous system. It penetrates and hardens up to a certain degree with great rapidity. It also preserves in certain struc- tures the special chemical properties on which certain differ- ential stains depend. Small pieces of nervous tissue are properly fixed in the standard solution (4 per cent, solution of formaldehyde gas) in four days. A whole brain will be so hardened in ten days to two weeks that thin serial sections can be made through it without fear of the slices altering their shape in the least. The process could undoubtedly be hastened by injecting the arteries. It must be borne in mind, however, that for most purposes formaldehyde must be followed by other reagents before the structures and their chemical properties preserved by it are properly fixed so that they will not be altered when trans- ferred to alcohol. In other words, formaldehyde may be looked upon as a very quick preliminary fixing reagent. The hardening of brains entire in it is not recommended, except in certain cases — for instance, of cysts, hemorrhages, or occasionally of tumors — where the gross lesions and the tracts or structures affected by them are of more importance than the finer histological changes. For the proper preser- vation of ganglion-cells and of neuroglia-fibers very small pieces must be taken and fixed by the special methods given ; but if the main object is to trace system-degenera- tions, much larger pieces, or even the whole brain, may be taken, because the myelin-sheaths change comparatively very slowly after death. The stains for the central nervous system may be divided into two classes — general and differential. For nearly all of them preliminary fixation in formaldehyde is advisable or possible. This renders the immediate preservation of ner- vous tissue very simple, and at the same time allows a variety of mordanting and staining methods to be used later. The staining of the various histological elements of the HISTOLOGICAL METHODS. 33 1 central nervous system and the fixing reagents best suited for each of them will be considered under the following headings : General Stains. |- Protoplasmic granules ; Stains for ganglion-cells, -j Dendritic and axis-cylinder processes ; l Axis-cylinders and their terminal processes. Stains for the myelin-sheath. Stains for the neuroglia-fibers. General Stains. — General stains include the ordinary- nuclear stains, with or without a contrast-stain, and certain diffuse single or combined stains which color the nuclei, the cell-protoplasm, including to a varying extent the dendritic processes of the ganglion-cells, the axis-cylinders, and the neuroglia-fibers. The different stains vary somewhat in regard to the structures which they bring out most prominently. The best fixation for the general stains is, on the whole, that used for mordanting the myelin-sheaths ; in other words, formaldehyde combined with or followed by a chrome salt (see page 342). Occasionally the fixatives employed for other tissues are indicated. Alum-hematoxylin, followed by eosin, is always a useful stain. The eosin, if deep enough, brings out fairly well both the dendrites and the axis- cylinders. The basic aniline dyes, especially methyl ene-blue, find their greatest use in Nissl's method of staining the protoplasmic granules of the ganglion-cells. The various carmine solutions, particularly neutral, ammonia, and picro-carmine, have long been the favorite diffuse stains for the central nervous system, but the uncer- tainty of their action and the difficulty of always getting a good staining solution has gradually led to the introduction of more reliable methods. Of these, the simplest, quickest, and in many ways the most generally useful is — A. Van Gieson's Stain. — Although this mixture of acid fuchsin and picric acid may be made up in the way originally recommended, the following exact proportions, given by Freeborn for staining nervous tissues, will be found gener- ally preferable : 332 PATHOLOGICAL TECHNIQUE. I per cent, aqueous solution of acid fuchsin, 1 5 c.c. ; Saturated aqueous solution of picric acid, 50 " Water, 50 " 1. Stain sections first rather deeply in alum-hematoxylin. 2. Wash in water. 3. Stain in above solution three to five minutes. 4. Dehydrate in alcohol. 5. Oil, Canada balsam. The nuclei appear bluish red, the ganglion-cells and pro- cesses red, the axis-cylinders brownish-red, the myelin- sheaths yellow, the neuroglia-fibers orange red, connective- tissue fibrilljE deep red. After certain reagents this solution will not give a sufficiently intense stain. In such cases a mixture of i part of a I per cent, solution of acid fuchsin to 2 parts of a saturated solution of picric acid is recommended. B. Phosphotungstic-acid. hematoxylin (see page 284) will be found of much value as a general stain for the central nervous system if employed in the manner recommended for neuroglia-fibers after fixation by the method there given, because a greater differentiation of the various tissue-ele- ments is obtained than by any other method. Preliminary staining in Van Gieson's mixture is often an advantage. C. Phosphomolybdic-acid Hematoxylin (see page 284). — I. Stain sections twenty minutes to one hour. 2. Wash out in two or three changes of 50 per cent, alcohol until the celloidin becomes completely decolorized (about five minutes). 3. Dehydrate in 95 per cent, alcohol. 4. Oil, Canada balsam. The ganglion-cells are often overstained, especially if the tissue has been hardened but recently. The method is particularly good for bringing out the axis-cylinders and the neuroglia-fibers. D. Aniline Blue Stain (see page 286). — The method recommended for connective- tissue fibers will also be found very useful for the study of the nervous .system. The best results are obtained after fixation in Zenker's fluid, but fair HISTOLOGICAL METHODS. 333 results can also be obtained after fixation by the method advised for neuroglia-fibers — namely, formaldehyde, picric acid, and bichromate of ammonium, provided the following changes are made in the method : Use a i per cent, solution of acid fuchsin ; mordant for several minutes in the phospho- molybdic acid ; do not stain more than one to three minutes in the aniline blue solution. E. Nigrosin. — i. Stain sections in a concentrated aqueous solution of nigrosin five to ten minutes. 2. Decolorize and dehydrate in weak, then in strong, alcohol. 3. Oil, Canada balsam. The stain is not very sharp, but is simple and useful, par- ticularly for low-power observation. Stains for Nissl or Tigroid Bodies. — A. Nissl's Stain. — I. Carefully harden pieces of tissue not over i to 1.2 cm. in diameter in 96 per cent, alcohol. 2. Cut sections without imbedding, as follows : Remove excess of alcohol from tissue with filter-paper ; dip base of specimen in thick celloidin ; mount on block ; harden in 96 per cent, alcohol. Moisten knife with 96 per cent, alcohol. Sections should always be under 3-5-5- mm. in thickness. In order to be able to compare the number of cells, etc., in one case with those in another, the sections should be of uni- form thickness. Preserve sections in 96 per cent, alcohol. 3. Stain the sections in the following solution heated over a flame until it bubbles noisily (6o°-70° C.) : Methylene-blue, B patent,' 3.75 ; Venetian soap, i-7S ; Distilled water, looo. 4. Wash out in — Aniline oil, 10 parts ; 96 per cent, alcohol, ^ 90 " until the color is no longer given off" in coarse clouds. 1 Nissl personally prefers and uses the make of Carl Buchner und Sohn, Munich. 334 PATHOLOGICAL TECHNIQUE. 5. Transfer section to slide; dry with filter-paper and cover with oil of cajuput. 6. Blot with filter-paper, and then wash with a few drops of benzine. 7. Add a few drops of benzine-colophonium (made by dissolving colophonium in benzine for twenty-four hours and then decanting). 8. Hold the slide above the flame until all the benzine is driven off. (Nissl no longer recommends burning off the benzine.) 9. Cover-slip. Warm the slide, so that the colophonium will spread out evenly between the cover-slip and the slide. The specimen is now mounted in a medium in which dif- fusion of color cannot take place, so that the stain is practi- cally permanent. The best results are obtained with tissues which have not been hardened in alcohol over one to four days. Contact with water, weak alcohol, and ether must be avoided. B. LenhoBsek's Stain. — The following method will be found simpler, but the specimens are not permanent. 1 . Harden sections in go per cent, alcohol, then in 96 per cent., or in formaldehyde followed by alcohol. Do not keep the tissues too long in alcohol. 2. Imbed sections in celloidin or paraffin, or cut without imbedding, as in Nissl's method. 3. Stain sections in a completely saturated solution of thionin for five minutes. 4. Wash for a few seconds in water. 5. Differentiate in aniline, i part; Absolute alcohol, 9 parts. Do not decolorize too long. 6. Clear in oleum cajuputi. 7. Xylol. 8. Canada balsam.. The granulations can be shown by other stains, such as safranin, fuchsin, dahlia, alum-hematoxylin. Ganglion-cells ; Dendritic and Axis-cylinder Pro- cesses. — Golgi's Methods. — Golgi's methods, although of BISTOLOGICAL METHODS. 335 the greatest value in the study of the normal histology of the central nervous system, are of very little use in the study of its pathology. The reason of this is the very peculiarity that makes the method of value in normal histology — namely, that it picks out here and there a cell and stains it with all its wealth of processes more or less completely, while the neighboring cells are left colorless. If all of the cells and their processes were stained, the picture presented would be a confused mass. In pathological histology, where the presence or absence of certain cells or processes is of paramount importance, it is of primary necessity that every cell within a given area shall be perfectly stained. Golgi introduced three different methods of obtaining the stain now called after his name. They are spoken of as the slow, the mixed, and the short methods. Golgi himself employed principally the first two methods, and they are still used for the study of the developed brain and cOrd. The quick method exclusively has been used by Ramon y Cajal and other recent investigators for the study of em- bryonic nervous tissue. The following points are to be borne in mind : The tissue should be as fresh as possible, and should be cut into small pieces, not over i to ij cm. thick — for the quick method even thinner. With the corrosive-sublimate method, how- ever, larger pieces can be used. Large quantities of the solutions should be used — at least ten times the volume of the specimen It is best to keep the specimens in the solution in the dark, especially in using the corrosive-sublimate method. Golgi's Slow Method. — i. Harden the tissues in a 2 per cent, solution of bichromate of potassium two to six weeks. In summer fifteen to twenty days are sufficient ; in winter, unless the temperature is kept at 25" C, one to one and a half months will be required. Keep the specimens in the dark. Large amounts of the solution should be used, and it should be frequently changed, especially during the first week. 2. Transfer either to (a) a f per cent, solution of nitrate 336 PATHOLOGICAL TECHNIQUE. of silver for twenty-four to forty-eight hours ; a longer time will do no harm ; or to {b) a \ per cent, solution of corrosive sublimate — small pieces eight to ten days, large pieces two months or more. Change the solution frequently during the first few days ; later only when the solution gets yellow. This second procedure is recommended for larger pieces of tissue than can properly be impregnated by {a). Golgi's Mixed Method. — i. Harden small pieces of tissue for three to five days or longer in a 2 per cent, solution of bichromate of potassium at 25" C, in the dark. 2. Transfix to a mixture of — 1 per cent, solution of osmic acid, 2 parts ; 2 per cent, solution of bichromate of potas- sium, 8 " for three to eight days. 3. Place in a f per cent, solution of nitrate of silver for twenty-four to forty-eight hours. Golgi's Quick Method. — i. Small pieces of fresh tissue are placed directly in the following solution : I per cent, solution of osmic acid, i part ; 3.5 per cent, solution of bichromate of potassium, 4 parts, for several days (three to eight). 2. They are then transferred to a large amount of a f per cent, solution of nitrate of silver for one, two, or six days. The length of time the tissues should remain in the osmic-acid and bichromate-of-potassium solution depends on what elements it is desired to impregnate. In the human cord the time is in general the following: 1. Neuroglia, 2-3 days; 2. Nerve-cells, 3-5 " 3. Nerve-fibers and collaterals, 5-7 " The further treatment of the tissues impregnated by these methods is as follows : Alcohol must be avoided as much as possible. The tissues are usually firm enough to cut after the impregnation ; if not, place in absolute alcohol for fifteen to thirty minutes. The sections should be rather thick, -^-^ HISTOLOGICAL METHODS. 337 to ■jJjj- mm. They may be made free hand with a razor or in the microtome. For either method the tissues can be held between pieces of elder-pith, or may be quickly imbedded in celloidin by dehydrating" for a few minutes in absolute alco- hol and then placing in a thick solution for five minutes. From the celloidin they are mounted in elder-pith or on blocks, and placed for a short time in 80 per cent, alcohol to harden. Treatment of Sections. — i. Dehydrate quickly in alcohol. 2. Clear in oil of cloves or bergamot. 3. Wash off with xylol. 4. Mount without a cover-glass in xylol damar, and dry quickly at 40° C. The mounted sections must be protected from the light and from dust as much as possible. Cajal has modified Golgi's quick method by repeating the steps (Cajal's so- called double method) so as to get a more perfect impregna- tion. The same osmic-acid and bichromate-of-potassium solution may be used over again, or a fresh solution, con- taining about one-half as much osmic acid, is made up fresh. The silver solution should be taken fresh each time. Len- hossek, Weigert, and others have obtained very good Golgi preparations with tissues first fixed in formaldehyde. Of the -various methods proposed for fixing the Golgi stains so that contrast-stains could be used with them and the specimens protected by cover-slips, the simplest and most practical seems to be that advocated by Kallius. The Method of Kallius for Fixing Golgi Stains. — The method depends on the employment of a photographic developer to reduce the bichromate of silver to metallic silver. I. Place sections for several minutes in a solution com- posed of I part of the following developer : Hydrochinon, I ; Sulphite of sodium, 8; Carbonate of potassium. 1-5 ; Water, 575. 22 338 PATHOLOGICAL TECHNIQUE. plus one-third to one-half as much absolute alcohol until the sections become gray to black in color. If too much alcohol is added, the carbonate of potassium will be pre- cipitated, but will redissolve on the addition of a little more developer. 2. 70 per cent, alcohol for ten to fifteen minutes. 3. Hyposulphite of sodium (20 per cent, aqueous solu- tion). 4. Wash thoroughly in a large amount of water for twenty- four hours. 5. Alcohol, oil, Canada balsam ; cover-glass. Cox's Modification of Golgi's Corrosive-sublimate Method. — The same black pictures are obtained by this method as by Golgi's, but with this difference, that nearly all of the cells in the section are impregnated. This is an ad- vantage when the topographical arrangement of the cell- layers is desired, but a disadvantage when it comes to the study of individual cells, because on account of the luxuri- ance of the impregnation such a study is rendered impos- sible. Small pieces of nervous tissue are placed in the fol- lowing solution : Bichromate-of-potassium 5 per cent, solution, 20 ; Corrosive-sublimate 5 per cent, solution, 20 ; Distilled water, 30-40; Simple chromate-of-potassium 5 per cent, solution, 16. The time required for impregnation is a month in summer and two to three months in winter. The after-treatment is the same as for Golgi preparations. Axis-cylinders and their Terminal Processes. — The three methods most in use for the study of central and peripheral nerve-fibers and their terminations are the gold, the Golgi, and the methylene-blue methods. All three may give beautiful results, but, as a rule, they are very unreliable. Their use is confined almost wholly to the study of normal tissues. Gold Stain for Nerve-fibers. — For the application of the gold method to fresh tissues see p. 302. HISTOLOGICAL METHODS. 339 Various attempts have been made to devise a reliable method of employing chlorid of gold for staining nerve- fibers in sections of hardened tissues. The results have not been altogether successful. The best results can probably be obtained by — A. GerlacKs Method. — i. Harden tissues in a 1-2 per cent, solution of bichromate of ammonium for one to three weeks ; cut sections without passing through alcohol, which must be avoided. 2. Place the sections in a very dilute solution (ybij per cent.) of the double chlorid of gold and potassium very slightly acidulated with hydrochloric acid, for ten to twelve hours, until they become slightly violet in color. 3. Wash in a solution of hydrochloric acid i, to water 2000-3000. 4. Place for ten minutes in a -jig- per cent, solution of hy- drochloric acid in 60 per cent, alcohol. 5. Absolute alcohol, oil of cloves, Canada balsam. Another method frequently recommended is the following : B. Freud's Gold Stain for Nerve-fibers. — i. Harden tissues in Erlicki's or Miiller's fluid, followed by alcohol. Imbed in celloidin. 2. Stain sections three to five hours in i per cent, solution of chlorid of gold, and 95 per cent, alcohol, equal parts. 3. Wash in water. 4. Reduce in — Caustic soda, I ; Distilled water, 6, for two to three minutes. 5. Wash in water. 6. Place for five to fifteen minutes in a 10 per cent, solu- tion of iodid of potassium. 7. Wash in water. 8. Alcohol, oil, Canada balsam. C. Stroebe's Aniline-blue Stain for Nervefibers in Hard- ened Sections. — Harden tissues in Miiller's fluid, i. Stain •340 PATHOLOGICAL TECHNIQUE. one-half to one hour iii a saturated aqueous solution of ani- line-blue. 2. Wash in water. 3. Transfer to a small dish of alcohol to which are added 20 to 30 drops of a I per cent, alcoholic solution of caustic potash (caustic potash I to alcohol 100 : let the mixture stand for twenty-four hours ; then filter). In one to several minutes the sections become bright brownish-red and trans- parent. 4. Transfer to distilled water for five minutes. The sec- tion becomes bright blue again. 5. Stain in a half-saturated aqueous solution of safranin one-quarter to one-half hour long. 6. Wash out and dehydrate in absolute alcohol. 7. Xylol, Canada balsam. D. Chlorid-of-iron and dinitroresorcin method for the study of degenerated peripheral nerves : 1 . Place fresh pieces of peripheral nerves for several days in a solution of — Chlorid of iron, i part; Distilled water, 4 parts. 2. Wash out thoroughly in water. 3. Transfer to a saturated solution of dinitroresorcin in 75 per cent, alcohol for several weeks. 4. Wash, dehydrate, imbed, etc. A permanent green color is formed which stains the nerves green and brings out the green axis-cylinders very sharply. The stain will succeed with preparations which have been hardened in Flemming's solution or Miiller's fluid. Golgi's methods are sometimes employed for the study of the terminal processes of nerve-fibers (for directions see P- 334). Methylene-blue Stain for Nerve-fibers. — The methylene- blue method is due to Ehrlich. Many modifications of the original procedure have been suggested with a view to mak- ing the results surer or the specimens more permanent. Tissues can be stained either by injection or by immersion. HISTOLOGICAL METHODS. 34I The methylene-blue used should be Griibler's " rectified methylene-blue for vital injection." For injection in the blood- or lymph-vessels of live, of dead animals a i to 4 per cent, solution in normal salt solu- tion is recommended. The injected organs are exposed to the air until a bluish tint is visible. As soon as the greatest intensity of stain is reached (five minutes to two hours) the color in the preparation is fixed by placing small bits of the tissue in a freshly-filtered, cold, saturated, aqueous solution of picrate of ammonium, or, better still, in the solution given below, recommended by Bethe. Very small or thin pieces of tissue intended for staining by immersion (the method employed for human tissues) are placed in a very dilute solution (t6~-iV P^*" cent.) of methyl- ene-blue in normal salt solution. Lavdowski recommends very highly a solution of methylene-blue in egg-albumin, either alone or combined with chlorid of sodium or ammo- nium. The white of egg is freed from the thicker portions or filtered. When the experiment is to last some time, add to the egg-albumin an equal part of a J per cent, solution of chlorid of sodium or of a ^ per cent, solution of chlorid of ammonium. The tissue, protected by a large dish, is exposed to the air for fifteen minutes to twelve hours, until the maxi- mum stain is obtained. The stain may then be fixed by the method already given, or, better still, in the following manner : Bethe' s Method of Fixing Methylene-blue Stains of Nerve- fibers. — I. Wash off excess of color with normal salt solu- tion. 2. Place in — Molybdate of ammonium, i gr. ; Distilled water, 10 c.c. ; Peroxid of hydrogen, i " Hydrochloric acid, i drop. A precipitate forms on making up the solution, but disap- pears on shaking. The solution will keep eight days, but is best made up fresh each time. It should be used as cold as 342 PATHOLOGICAL TECHNIQUE. possible, preferably surrounded by a mixture of ice and salt. Leave the tissue in the cold solution for from two to five hours, and then for a while longer at the room-temperature. 3. Wash one half to two hours in running water. 4. Dehydrate and harden as quickly as possible (not over twelve to twenty-four hours) in cold absolute alcohol. (The color is soluble in warm alcohol.) 5. Clear in xylol. 6. Imbed in paraffin. The sections may be mounted directly or brought into water and stained with alum-cochineal for contrast. If a little osmic acid be added to the fixing solution after the speci- mens have been in it for a while, a more permanent methyl- ene-blue stain is obtained. Stains for the Myelin-sheath. — The myelin-sheath of nerve-fibers is a form of fat, and like it possesses the property of reducing osmic acid, by means of which a selec- tive 'sheath stain can be obtained. Unfortunately, however, the oimic acid penetrates to but a very slight depth. Three methods employing osmic acid are given, but they are all expensive and not so satisfactory as those employing hema- toxylin. The differential hematoxylin stain, originated by Weigert, and ordinarily used, depends on some chemical reaction which takes place between the myelin and a chrome salt, in consequence of which the myelin is fixed so that it will not later be dissolved out by alcohol or ether, and at the same time is so mordanted that it can be deeply stained with hematoxyhn, to which it clings when treated with certain decolorizers. This reaction between the myelin and the chrome salts in general use takes place very slowly at the ordinary temperature; six weeks to several months are usually required. Weigert's latest- method depends on the interaction of two chrome salts in the same solution, in consequence of which the time needed for this reaction or mordanting is reduced to four days. The solution may be used alone, but is best combined with formaldehyde or used after it. HISTOLOGICAL METHODS. 343 A. "Weigert's Myelin-sheath Stain. — In this method five steps are involved, but the first two can be, and oftien are, combined in one. These five steps are fixation, primary mordanting, secondary mordanting, staining, and differentia- tion. These different steps will be considered separately. 1. Fixation. — Place the tissues in a 4 per cent, solution of formaldehyde (10 per cent, solution of formalin) for four days to several weeks or indefinitely, using several times the volume of the tissue. Change the solution at the end of twenty-four hours, and thereafter whenever it becomes cloudy. Large masses of nervous tissue, like the medulla and pons or the basal ganglia, should be fixed in formaldehyde for one to three weeks. 2. Primary Mordanting. — Cut the tissues fixed in formal- dehyde into slices not over i cm. thick, and place in the following solution for four to five days at room-temperature : Bichromate of potassium, 5 ; Fluorchrom, 2 ; Water, ad 100. Steps I and 2 may be combined by adding 4 per cent, of formaldehyde to the mordanting solution and placing the fresh tissues directly in the mixture. 3. Secondary Mordanting. — Transfer the tissues to the following solution for twenty-four to forty-eight hours : Acetate of copper, 5.0; Acetic acid, 36 per cent, solution, 5.0; Fluorchrom, 2.5 ; Water, ad loo.o (For method of preparation see page 284.) Weigert always transfers blocks of tissue to the secondary mordant, of which the function is to intensify the staining reaction. Many workers, however, prefer to employ the secondary mordant on sections only. In this case they place the tissues directly from the first mordant into graded alcohols, imbed in celloidin, and cut sections before step 3. Either way gives good results, but the first is the simpler. 344 PATHOLOGICAL TECHNIQUE. Weigert also recommends the following iron solution as a secondary mordant, but if applied to blocks of tissue, they must be well washed in running water before being dehy- drated and imbedded in celloidin, because otherwise the iron will rust the knife badly. Of course, if this mordant is applied to sections only, prolonged washing is not necessary : Iron alum (ammonioferric alum), 5 ; Acetic acid, 5 j Water, ad lOO. After the secondary mordant the tissue is dehydrated in graded alcohols, imbedded in celloidin, and sections cut in the usual manner. 4. Staining. — Stain sections in the following solution for twelve to twenty-four hours : Ripened 10 per cent, solution of hematoxylin in absolute alcohol, 10 ; Saturated aqueous solution of carbonate of lithium, I ; Water, 90. Keep on hand as a stock solution a 10 per cent, solution of hematoxylin in absolute alcohol. At least ten days of exposure to light are required to ripen this solution so that it can be used for staining. Combine with the carbonate of Hthium and the water at the time of using in such propor- tions as are wanted for immediate use. Wash the sections thoroughly in water before differentiation. Another staining solution which Weigert recommends highly is the following — it consists of two parts : [a) Liquor ferri sesquichlorati (officinal), 4 c.c. ; Water, 96 " {B) Ripened 10 per cent, solution of hema- toxylin in absolute alcohol, 10 " 96 per cent, alcohol, 90 " Mix thoroughly equal parts of these two solutions just before using, and pour over the sections. Stain overnight HISTOLOGICAL METHODS. 345 or longer at room-temperature. Pour off solution and wash with water. 5. Differentiation. — The sections are differentiated in the following solution which it is sometimes advisable to dilute with water : Borax, 2.0 ; Ferricyanid of potassium, 2.5 ; Water, loo.o. After the first staining method given above the decolor- ized tissues appear yellow ; after the iron-hematoxylin solu- tion they are colorless. Moreover, this latter method stains the very finest fibers and at the same time the coarse fibers, such as occur, for example, in the nerve-roots. After differentiation the sections should be thoroughly washed in water, dehydrated in alcohol, cleared in the anilin oil-xylol or carbol-xylol mixture, and mounted in xylol balsam. The steps of the process may be summed up as follows : 1. Fix in 10 per cent, formaldehyde four days or longer. 2. Mordant in the bichromate of potassium-fluorchrom solution four to five days. 3. Mordant in the acetate of copper-fluorchrom solution twenty-four to forty-eight hours, or in the iron solution twenty-four fo forty-eight hours. 4. Dehydrate in graded alcohols. 5. Imbed in celloidin. 6. Stain sections in the alcoholic hematoxylin solution or in the iron-hematoxylin solution for twelve to twenty-four hours. 7. Wash off in water. 8. Differentiate in the borax-ferricyanid of potassium solution. 9. Wash thoroughly in water. 10. Dehydrate in alcohol. 11. Clear in the anilin oil-xylol or carbol-xylol mixture. 12. Mount in xylol balsam. B. Pal's Modification of Weigert's Myelin-sheath Stain. 346 PATHOLOGICAL TECHNIQUE. — I. Fixation and primary mordanting as for Weigert's method. 2. Place sections for several hours in a ^ per cent, aqueous solution of chromic acid, or for a longer time in a 2-3 per cent, solution of bichromate of potassium. This step is often omitted, especially when the tissues have been but recently mordanted. 3. Transfer sections to Weigert's alcoholic hematoxylin solution for twenty-four to forty-eight hours (if necessary for an hour in the incubator at 37° C). This solution is as follows : Ripened 10 per cent, solution of hematoxylin in absolute alcohol, lO. Water, 90. 4. Wash in water plus i to 3 per cent, of a saturated aqueous solution of carbonate of lithium until the sections appear of a uniform deep-blue color. 5. Differentiate for twenty seconds to five minutes in a |- per cent, aqueous solution of permanganate of potassium until the gray matter looks brownish-yellow. 6. Transfer to the following solution : Oxalic acid, I ; Sulphite of potassium, i ; Water, 200. for a few seconds until the gray matter is colorless or nearly so. 7. Wash thoroughly in water. 8. Dehydrate in 95 per cent, alcohol. 9. Oil, Canada balsam. Steps 5 and 6 sometimes have to be repeated when the differentiation has not been complete. Of all the numerous modifications of Weigert's original myelin-sheath stain, the only one that has found general acceptance is Pal's. It has the following advantages : It gives very clear pictures ; everything except the sheaths is completely decolorized, so that contrast-stains are possible ; HISTOLOGICAL METHODS. 247 it is more successful with thick sections than Weigert's method ; the separate steps are quicker. On the other hand, the danger of decolorizing the sheaths of the finer fibers is greater. C Exner's Stain. — The tissue should be obtained as soon as possible after death, although the method will succeed with tissues even over twelve hours old. 1. Place pieces of brain or cord not over ^ crfl. thick in a I per cent, aqueous solution of osmic acid, using at least ten times as much fluid as the volume of the specimen. 2. Change the osmic-acid solution on the second day. 3. After five or six days wash thoroughly in water. 4. Dehydrate, imbed, etc. 5. Examine sections in glycerin rendered slightly ammo- niacal. The myelin-sheaths appear gray to black. The prepara- tions are not permanent. This procedure has been almost entirely replaced by Wei- gert's method, which has numerous advantages. Lately, however, it has been brought forward again by Heller, who uses a photographic developer to reduce the osmic acid and to make possible permanent mounts. He has lately pub- lished the following method for sections, but it cannot be unconditionally recommended : D. Heller's Myelin-sheath Stain. — i. Harden as for the Weigert method (Heller used Miiller's fluid). 2. Imbed in celloidin. 3. Place sections in a i per cent aqueous solution of os- mic acid for ten minutes in thermostat or for half an hour at room-temperature. 4. Wash in water. 5. Reduce in the following developer: Sulphate of sodium, 125 ; Carbonate of sodium, 70; Water, 500 ; Pyrogallic acid, 15. 348 PATHOLOGICAL TECHNIQUE. 6. Wash in water. 7. Differentiate in an aqueous solution of permanganate of potassium, \ per cent, or less. 8. Remove the brown of the permanganate of potassium in a I per cent, aqueous solution of oxalic acid. 9. Wash in water. 10. Alcohol, oil, Canada balsam. E. Robertson's Modification of Heller's Myelin-sheath Stain. — I. Harden in Weigert's fluorchrom-copper solution plus 4 per cent, of formaldehyde ; in other words, use the mordant for neuroglia-fibers (page 350) eight to ten days. 2. Wash off in water. 3. Alcohol ; imbed in celloidin. 4. Stain sections in a i per cent, solution of osmic acid half an hour in the dark. 5. Place in a 5 per cent, aqueous solution of pyrogallic acid for half an hour. 6. Differentiate in a -1^ per cent, aqueous solution of per- manganate of potassium one to four minutes. 7. Remove brown color in i per cent, oxalic acid three to five minutes. 8. Alcohol, oil, balsam. It is important to wash carefully in water between each of the staining steps. Stains for Neuroglia-fibers. — It is possible to obtain a differential stain of the neuroglia-fibers in man by several different methods. The tissue must be as fresh as pos- sible. The best results are obtained with tissues placed in the fixing solution within one hour after death. After four to six hours the results are only fair ; after twenty-four hours they are practically nil. The chemical property in the neuroglia-fibers on which the differential stain depends has undergone some chemical change or has disappeared. It is retained longest where the fibers are most numerous, as about the central canal. For most of the methods formaldehyde must be used as the fixative, either alone or combined with certain other reagents mentioned below, but one method depends on fixa- HISTOLOGICAL METHODS. ^4g tion in alcohol. According to Weigert, who first fully recognized the value of formaldehyde in preserving neu- roglia-fibers, the best strength to use is a 4 per cent, solu- tion (J. e., a 10 per cent, solution of formaline, etc.). The pieces of tissue in which it is desired to stain the neuroglia- fibers must be cut very thin — never over ^ cm. thick, and preferably thinner. With thicker pieces only the surface sections are of any value. The tissues after fixation must be mordanted, so as to render the staining more intense ; without mordanting only a few of the fibers will stain. The methods of staining neuroglia-fibers are given in the order of their publication. The second method (Weigert's) gives a more intense stain than the first, but has the draw- back of staining degenerated nerve-fibers. The third method is particularly useful for class purposes, because a large number of sections can be stained all at one time. A. Mallory's Differential Stain for Neuroglia-fibers. — I. Fix in a 4 per cent, aqueous solution of formaldehyde four days or more. 2. Place in a saturated aqueous solution of picric acid four to eight days. Steps I and 2 may be combined by adding 10 parts of the 40 per cent, formaldehyde solution to 90 parts of the saturated aqueous solution of picric acid. 3. Transfer to a 5 per cent, aqueous solution of bichro- mate of ammonium for four to six days in the incubator at 37° C, or for three to four weeks at room-temperature. ,^/ Change the solution on the second day. 4. Place directly in alcohol. 5. Imbed in celloidin. 6. Fasten sections to slide by means of ether-vapor. 7. Stain in aniline-gentian-violet fifteen to twenty minutes- 8. Wash off with normal salt solution. 9. lodin solution, i : 2 : 100, for one minute, or a stronger solution for a few seconds. 10. Wash off with water. 11. Dry with filter-paper. 3 so PATHOLOGICAL TECHNIQUE. 12. Decolorize in equal parts of aniline and xylol. 13. Wash off thoroughly with xylol. 14. Xylol balsam. The neuroglia-fibers, fibrin, nuclei, and to some extent the red blood-globules, are stained blue. The other tissue- elements are colorless. By very faintly tinting the aniline- and-xylol mixture with fuchsin, which is readily soluble in aniline, the other tissue-elements are easily brought out, but the finer neuroglia-fibers are likely to lose their blue color. Weigert' s Differential Stain for Neuroglia-fibers. — A. Fix thin pieces of tissue, not over \ cm. thick, in a 4 per cent, solution of formaldehyde for at least four days. B. Mordant in the following solution for four to five days in the incubator or for eight days at room-temperature : Acetate of copper, 5 gr. ; Acetic acid, 36 per cent, solution, 5 c.c. ; Fluorchrom, 2.5 gr. ; Water, ad 100 c.c. Boil the fluorchrom and water in a covered dish, turn off the gas, add the acetic acid and then the acetate of copper ; stir briskly until the latter is dissolved, then cool. The solution remains clear. (Steps I and 2 may be combined by adding 4 per cent, of formaldehyde to the above solution ; change on the second day ; harden eight days.) C. Wash off in water ; dehydrate in alcohol ; imbed in celloidin. D. Reduction of copper salt in sections : 1. Place the sections, which must not be over .02 mm. thick, in a ^ per cent, aqueous solution of permanganate of potassium for ten minutes. 2. Wash off with water. 3. Decolorize and reduce for two to four hours in the fol- lowing solution, carefully filtered : Chromogen, 5 o-r. ; Formic acid (sp. gr. 1.20), 5 c.c; Water, ad 100 " HISTOLOGICAL METHODS. 35 1 to 90 c.c. of which are added just before using 10 c.c. of a 10 per cent, solution of sulphite of sodium. The sections lose their color in a few minutes, but are best kept in the solution as long as above directed. The sections can now be stained in the manner to be de- scribed, but the color of the fibers will be more inten-se if the following steps are added, and a slight yellowish contrast- stain is obtained for the ganglion and ependymal cells and for the larger nerve-fibers. This step has one disadvantage, however : the connective-tissue fibers stain blue after it. E. Further reduction of copper salt : 1. Wash twice in water. 2. Place sections in a carefully filtered saturated (5 per cent.) aqueous solution of chromogen overnight. 3. Wash in water. 4. The sections are now ready for staining or may be pre- served until wanted in — 80 per cent, alcohol, 90 c.c. 5 per cent, oxalic acid, 10 " F. Staining of neuroglia-fibers : 1. Lift section from large dish of water on slide freshly cleaned with alcohol ; blot with filter-paper (method recom- mended by Weigert for attaching sections to slide). 2. Stain in the following mixture : Saturated solution of methyl-violet in 70-80 per cent, alcohol, 100 c.c. ; (saturated with aid of heat ; decanted when cold). 5 per cent, aqueous solution of oxaHc acid, 5 " The oxalic acid is added to render the preparations more lasting. The staining is practically instantaneous. 3. Wash off with normal salt solution. 4. lodin solution : 5 per cent, iodid-of-potassium solution saturated with iodin. It is simply poured on and then off, as the reaction is instantaneous. 352 PATHOLOGICAL TECHNIQUE. 5. Wash off with water and blot with filter-paper. 6. Decolorize thoroughly in equal parts of xylol and aniline oil. 7. Wash repeatedly with xylol or the stain will not keep. 8. Canada balsam. The sections keep better if exposed for from two to five days to diffuse light before being put away. C. Differential Stain of Neuroglia-flbers by Means of Phosphotungstic-acid Hematoxylin (i^a//^ry). — i. Fix and mordant tissues in exactly the same way as in the first method given for neuroglia-fibers — viz. : {a) Fix in 4 per cent, aqueous solution of formaldehyde four days. (B) Saturated aqueous solution of picric acid four days. (c) 5 per cent, aqueous solution of bichromate of am- monium four to six days in incubator or three to four weeks at room-temperature. {d) Alcohol, celloidin, etc. 2. Place the sections in a |- per cent, aqueous solution of permanganate of potassium for fifteen to thirty minutes. 3. Wash in water, 4. Five per cent, aqueous solution of oxalic acid one to two hours. 5. Wash in two or three changes of water. 6. Stain in phosphotungstic-acid hematoxylin for twenty- four hours to two or three days. 7. Wash quickly in water. 8. Place in a strong (20 to 30 per cent.) alcoholic solution of ferric chlorid for five to twenty minutes, rarely longer. It is best to make up the solution as needed, as it does not keep well : the exact strength used is not important. 9. Wash thoroughly in water. 10. Alcohol, oleum origani cretici, xylol balsam. The nuclei, neuroglia-fibers, and fibrin stand out sharply of a clear blue color; everything else is decolorized or appears of a pale yellowish or grayish tint. The results obtained are practically identical with those obtained by either of the modified fibrin stains, and the method has the HISTOLOGICAL METHODS. 353 decided advantage of being applicable to any number of sections at once. Benda's Stain for Neuroglia Fibrils. — Hardening. — i. Fix fresh material for at least two days in 90 to 93 per cent, alcohol. 2. Place thin sections (not over 5 mm. thick) in 10 per cent, nitric acid for twenty-four hours. 3. Two per cent, aqueous solution of bichromate of potas- sium, twenty-four hours. 4. One per cent, aqueous solution of chromic acid, forty- eight hours. 5. Wash in running water for twenty-four hours, harden in graded alcohols, imbed in paraffin. Staining. — Iron-alizarin-toluidin-bbie Stain. — i . Mordant the sections for twenty-four hours in a 4 per cent, solution of iron-alum. 2. Wash off in running water. 3. Twenty-four hours in dilute amber-yellow aqueous solution of sodium sulphalizarate. 4. Dip in water and blot with filter-paper. 5. Stain in a I per cent, aqueous solution of toluidin-blue, warm until steam rises, then let it stain about fifteen minutes in the cooling fluid ; or stain one to twenty-four hours in cold toluidin-blue. 6. Dip in 10 per cent, acetic acid or in very dilute picric acid. 7. Dry with filter-paper and dip in absolute alcohol. 8. Differentiate in beech creasote about 10 minutes, con- trolling result with microscope. 9. Dry with filter-paper ; xylol balsam. Degenerations of the Nervous System. — The same methods apply to the study of degenerations in nervous tis- sue that apply elsewhere, except in the demonstration of fat. Both myelin and fat reduce osmic acid, so that the ordinary test for fat in the hardened tissues fails. Marchi and Algeri, however, have shown that after myelin has been mordanted for eight days or over in Muller's fluid or other solution of the bichromates, it loses the property of reduc- 23 354 PATHOLOGICAL TECHNIQUE. ing the osmic acid, while fat retains the property unimpaired. On this peculiarity is based their method for differentiating fat from myelin. Marchi and Algeri's Method for Staining Fatty De- generated Myelin-sheaths of Nerve-fibers. — i. Harden in Miiller's fluid or in formaldehyde, followed by Muller's fluid, for eight days to three months. 2. Transfer tissue for five to eight days directly into the following solution : Muller's fluid, 2 parts ; I per cent, osmic-acid solution, i part. 3. Wash out thoroughly in water. 4. Dehydrate in alcohol. 5. Imbed in celloidin. .6. Clear in chloroform and mount in properly prepared chloroform balsam (see page 300). EXAMINATION OF THE BLOOD. The specific gravity of the blood varies but slightly and averages 1055. For clinical purposes the method of Ham- merschlag is the best for estimating the specific gravity. The method depends upon the physical law that a body which re- mains suspended in a fluid must have the same specific grav- ity as that fluid. The fluid selected is a mixture of chloro- form (specific gravity 1 526) and benzol (specific gravity 0.889). A drop of blood does not mix with either fluid. A small test-tube holding about 10 c.c. is half filled with a mixture of benzol and chloroform. This mixture should have the specific gravity of from 1050 to 1060. A drop of freshly-drawn blood is allowed to fall into this mixture, care being exercised that the drop falls directly into the fluid. Chloroform or benzol is added according as the drop of blood sinks to the bottom or floats on the surface of the fluid. It is necessary in adding either of the fluids to thoroughly mix them by gentle rotating movements without breaking the HISTOLOGICAL METHODS. 355 blood-drop. If the drop floats on the surface, it is better to add sufficient benzol to make it sink to the bottom, and then add chloroform until it becomes suspended in the fluid. Too large a drop of blood is liable to be broken up in mix- ing the fluids, and this must be avoided. When the drop re- mains suspended in the thoroughly mixed fluids the latter is filtered and the specific gravity tested. Apparatus Used in the ISxamination of the Blood. — An accurate examination of the blood can be made only by the strictest observance of cleanliness and attention to de- tails. It has been deemed wise, therefore, to devote the greater part of this article to a description of the various steps of the process for the benefit of those who are unfa- miliar with the technique. With this object in view it is not necessary to describe the numerous instruments which have been devised for examin- ing blood or to refer to many of the staining fluids. The Thoma-Zeiss hemocytometer, or blood-counting instrument (Fig. 117), is the one which is generally employed to count the red and white blood-corpuscles, and consists of a glass slide on which the blood-corpuscles are counted and a pi- pette for mixing the blood and diluting fluid. The counting slide has a thin square plate of glass cemented on its surface : a circular opening in the center of this plate is nearly filled by a glass disc -^-^ mm. thinner than the square plate which surrounds it. A series of horizontal and vertical lines on the surface of the disc divides it into squares, the sides of which are -^ mm. long. Additional lines placed close together divide this surface into quadrants. Each quadrant contains sixteen of the small squares. The pipette consists of a capillary tube which expands into an ovoid chamber above. The chamber contains a glass pearl, which assists in mixing the blood and diluting fluid. The capillary tube has a capacity of i mm. and is graduated to tenths. Above the ovoid chamber is a line marked 10 1 mm. A dilution of I to 100 or i to 200 is obtained by sucking the blood up to the mark i. or 0.5, and the diluting fluid up to the mark loi. 356 PATHOLOGICAL TECHNIQUE.