over all of the surface of the medium, and not in the form of one or two narrow streaks. Animal Inoculations.-The general methods of animal inoculation have already been described. The inoculation of animals directly with pathological material is often of important diagnostic value, and the special methods of doing so when it is necessary for diagnosis will be treated under the special headings of this section. Suppurative Processes.-The bacteria commonly found associated with these conditions are the pyogenic cocci. In general it may be said that the staphylococci will be found in the localized suppurations with abscess-formation, while the streptococcus will be found in the spreading inflammations producing little pus and accompanied by lymphangitis. The streptococcus pyogenes may often be identified by the cover-glass examination alone through its characteristic chain-formation, but this may not be apparent and the result of cultures must then be awaited. The staphylococcus pyogenes aureus cannot usually be identified with any certainty by the cover-glass examination. alone. Cultures are necessary in order to differentiate from the other staphylococci and from the streptococcus. For practical purposes the identification of the pyogenic cocci may be made by the appearances of their colonies on blood-serum and by their morphology; no secondary cultures are usually necessary. Erysipelas.-The streptococcus is most readily found in the extreme margin of the affected area where the process is newest. The skin should be cleansed with soap and water, and with alcohol. Then with a sterile knife-point or a large needle a small wound should be made, and some of the blood and exudate pressed out from the tissue beneath. From this, cultures and cover-glasses may be prepared. Peritonitis and Appendicitis.-The exudate or purulent material is best obtained at the operation by means of swabs. A great variety of bacteria may be met with in these situations, the most frequent among which is the bacillus coli communis. Pleural, pericardial, and joint exudates may contain various organisms. In purulent exudates of the pleural and pericardial cavities the pneumococcus is frequently present, as are also the pyogenic cocci. Other bacteria also occur here. The pneumococcus may be identified from the cover-glass examination alone by the demonstration of its capsule with one of the special staining methods (see p. 92), and by its peculiar morphology. In sero-purulent exudates from the joints the gonococcus may be present. This may be identified with some degree of certainty by the special staining method for the gonococcus (see p. 191). Cultures on the special media (see p. 131) should be made if it is desired to remove all doubt as to the identity of the organism. The other pyogenic cocci also occur in purulent joint-inflammations. If the exudate from these situations is not purulent, and if tuberculosis is suspected, the results of cover-glass examination for the presence of tubercle bacilli will usually be negative. The main reliance in the determination of the tuberculous nature of such an exudate is in the results of the inoculating of guinea-pigs (see p. 189). The animal is best inoculated with a sterile hypodermic syringe in the tissues of the abdominal wall, about I c.c. of the fluid being injected. Anthrax, or Malignant Pustule.-The bacilli may be found by the cover-glass examination of the contents of the small blebs and vesicles. The bacillus of anthrax may be identified by its morphology (see p. 156), its special characteristics being its large size and its square or concave extremities. The inoculation of a mouse at the root of the tail with some of the material from the pustule, and the production. of the characteristic fatal septicemia, will render the identification certain. Diphtheria.-The bacteriological diagnosis of infection with the bacillus diphtheriæ 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 resulting growth is examined by cover-glass preparations stained either with Löffler's methylene-blue solution or by one of the special methods given below. The bacillus diphtheria, if present, may then be recognized and differentiated from other bacteria present in the preparation by its characteristic morphology and peculiarity of staining, described on page 138. 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 examination. 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 comparative feebleness of the growth of the organism on these media, and because of the fact that its microscopic appearances when cultivated on such media are not sufficiently characteristic. 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 testtube 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 collected, 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. In addition to the coagulated blood-serum here recommended, another practical medium is the following: Special Culture-medium of Kanthack and Stephens.—This consists of a mixture of ascitic, pleuritic, or other similar fluid with agar-agar. The method of preparation is as follows: To every 100 c.c. of the serous fluid add 2 c.c. of a 10 per cent. solution of potassium hydrate, by which the serum-albumin of the fluid is transformed in the after-heating into alkali-albumin. To this add 1.5 to 2 per cent. agar-agar which has been softened by soaking in weakly acidulated (HCI) water, and heat the mixture in the steam sterilizer until the agar-agar is dissolved. The mixture is then filtered through filter-paper and 4 to 5 per cent. of glycerin added. It is then to be run into test-tubes and sterilized as in the case of agar-agar (see p. 88). In the diagnosis of diphtheria it is used in the form of "slants" in the same manner as the coagulated blood-serum. In order to obtain this medium clear, it is necessary that the serous fluid should be worked into the medium as soon as possible after it has been withdrawn from the body. The advantages of this medium are— 1. It can easily be prepared. 2. It is clear and transparent. 3. Its basis is a body fluid which may be readily obtained in a hospital. 4. The bacillus diphtheria and certain other pathogenic bacteria grow upon it rapidly and vigorously, while many common organisms grow upon it slowly and feebly. If a 5. It can be used in the plate method of Petri. Note.-Before adding the potassium hydrate to the serous fluid, the latter should be tested in a test-tube by heating to the boiling temperature to determine the amount of albumin present. great amount of coagulation results, add to the serous fluid twice its volume of distilled water and then proceed as above indicated. If this dilution is not made, the mass will be gelatinous and therefore useless. The serous fluid after treatment with the alkali forms also a good fluid culture-medium. Special Methods of Staining the Bacillus Diphtheriæ.— Owing to the fact that the bacillus diphtheria may be recognized by its peculiar morphology and characteristic staining in coverglass 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 importance in the identification of the organism. These special methods of staining are said to be of great advantage in cases where only a few specific bacilli may be suspected to be present among a large number of other bacteria. Hunt's Method.-1. 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 methylene-orange one minute, without heating. 6. Wash in water. 7. Dry, and mount in balsam. 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. The solution of methyl-orange should be freshly prepared, for it deteriorates in a few days. Crouch's Method.-Stain the cover-glass preparation in the following mixture for a few seconds, then wash in water and mount: 1 per cent. aqueous solution of methyl green, 5 parts; I part; 4 parts. By this method the deeply stained portions of the bacillus take on a reddish color, while the remainder of the organism is colored pale green. Method used at the Boston City Hospital.-1. Stain with Löffler's methylene-blue solution, heating the preparation for a few seconds. 2. Wash in a per cent. solution of acetic acid for a few seconds. 3. Wash in water and mount. The partial decolorization with the acetic acid brings out the deeply-blue stained granules more strongly. Another method may be mentioned here. Whether it is of any use in the routine diagnosis of diphtheria we are unable to say. We are indebted to Dr. A. C. Abbott of Philadelphia for it. The method consists in staining the preparation by Gram's method and then staining with Bismarck-brown. The granules will be found to be black or blue-black and the body of the bacillus of a brown color. Influenza (see also p. 162).-Microscopic examination of cover-glass preparations of the bronchial sputum shows very small, short, round-ended bacilli, often in very large |