When there is no special reason for haste, it is usually more convenient to adopt the method followed by the New York Board of Health, of twelve hours' exposure, the cultures are kept at body temperature over night and are ready for examination in the morning.
It is not possible to determine the pres ence or absence of diphthelia bacilli in the cultures upon the blood-serum from the gross appearances; but if it is found that the culture-medium has been lique fied during the incubation, it can safely be said that contaminating bacteria are present in such numbers as to render the culture valueless. The diphtheria ba cilli or cocci do not liquefy the medium.
The true diphtheria bacilli do not grow in fluid antitoxic serum, nor do non-virulent pseudobacilli that render bouillon acid, while virulent organisms that render bouill On alkaline grow equally well in liquid antitoxic serum and normal serum. All forms grow ex cellently upon antitoxic serum that has been coagulated at 70 degrees. De Mar tini (Centralb. f. Bakt., Parasit., u. Infr., Jan. 30, '97).
Upon the centre of a clean cover-glass is placed a drop of sterile water. With a sterile platinum loop a number of the colonies, which show themselves as fine, granular elevations upon the culture sur face, are swept off. The loop is then im mersed in the water upon the cover-glass and its contents spread evenly over the glass. The preparation after being al lowed to dry in tbe air is fixed by pass ing it three times through a moderate gas-flame. It is then stained by cover ing it with Loeffler's alkaline methylene blue solution and allowing it to stand for ten minutes. The cover-glass is then washed, dried, and mounted in Canada balsam.
The following is recommended as a dif ferential stain for the diphtheria ba cillus:— (A) One gramme of methylene-blue (Grubler's) is dissolved in 20 cubic cen timetres of 96-per-cent. alcohol, which is then mixed with 950 cubic centimetres of distilled water and 50 cubic centime tres of glacial acetic acid.
(B) Two grammes of vesuvin are dis solved in 1 litre of boiling distilled water and filtered.
The cover-glass preparations are stained in A for 1 to 3 seconds, rinsed in water, and stained in B for 3 to 5 seconds, washed in water, dried, and mounted. Stained in this manner, the bacilli are brown, and contain two, or rarely three, but never more, blue corpuscles. The
corpuscles are oval, not round, in shape, and their diameter appears greater than that of the bacilli in which they are situ ated. Neisser (Zeitschr. f. Hyg., vol. xxiv, No. 3, p. 443, '97).
The examination is made with a 1/12 oil immersion lens. In a large propor tion of the cases we see an almost-pure culture of the diphtheria bacillus; next most frequently cultures of cocci, single double, or in chains; in some cases the cocci and bacilli are about equal in num ber, and in a small number only a few diphtheria bacilli are seen scattered among great numbers of cocci. From time to time we see in the cultures ba cilli which closely resemble the diph theria bacilli, but with certain definite points of distinction, and pseudodiph theria bacilli. The diphtheria bacilli seen in such cover-glass preparations vary in length from 1.5 to 6.5 millimetres, and in diameter from 0.3 to 0.8 millime tres. They occur singly or in pairs, rarely in chains of three or four. The rods are straight or slightly curved and are not usually uniformly cylindrical through out their length, but are swelled at the ends, or pointed at the ends and swelled in the middle. The variety' in size and shape even from the same culture is char acteristic. When in pairs, the bacilli may lie with their axes in the same line or forming an acute or obtuse angle; sometimes they are crossed. The bacilli show no spores, but may contain highly refractile bodies, especially- in their swelled portions. When grown upon blood-serum and stained in the manner above desciibed, the bacilli stain in a peculiarly-characteristic way. Lack of uniformity, both in the individual ba cillus and in the numbers of groups, is marked. Thus, different parts of a ba cillus take the stain unequally; so that the ends are dark blue, while the centre shows little or no color, or vice versa. Likewise bacilli lying side by side show marked difference in coloring, one being much more deeply stained than the other. This lack of uniformity in the staining- of the bacilli seems to belong to a certain period of their growth; it is usually marked after the twelve-hour in cubation, but many disappear entirely in older cultures.