TUBERCULOSIS CULTURES ON SODIUM CHLORIDE AND PHOSPHATE AGAR AFTER

HAVING GROWN FOR SEVERAL GENERATIONS ON POTASSIUM PHOSPHATE BOUILLON.

CERTAIN VARIATIONS IN THE

MORPHOLOGY OF

TUBERCLE BACILLI OF BOVINE ORIGIN.

By C. N. McBRYDE, M. D.,

Assistant in Biochemic Division, Bureau of Animal Industry.

a

The wide variation in morphology of the tubercle bacillus is strikingly shown in the recent work of Wolbach and Ernst, which consists in a careful study of the variations in form of the human and bovine bacillus at different ages and on different media. Perhaps the most striking variation noted by these authors occurred in the case of the cultures grown on brain medium, from which long, thread-like, and branched forms were obtained, the human bacilli ranging from 7 to 14 μ and the bovine from 6 to 8 μ in length. These long, thread-like forms are described as staining irregularly and as being made up alternate faint and deeply stained areas, and some excellent photomicrographs are shown in which the curiously segmented appearance of the threads is well brought out. As the foregoing is the first instance in which long, thread-like forms have been reported for the bovine bacillus, the following observations may be of interest.

of

In examining some old cultures of Bacillus tuberculosis, grown on Dorset's egg medium, it was noticed in several instances that the paraffin used in sealing the tubes had run down and across the surface of the egg, and in each case there appeared along the edges of the streak of paraffin an increased growth or a piling up of the tubercle bacillus, which seemed to be actually encroaching on or growing over the surface of the paraffin. There were three tubes in which this had occurred, namely, cultures of bovine, swine, and dog tuberculosis, which had been cultivated for a number of generations on bouillon before being transferred to egg. The age of these cultures was twentyone weeks, but the length of time the paraffin had been in contact with the growth was not known, as the cultures had been set aside and neglected for some time. To the naked eye the growths bordering the paraffin were white in color and they separated readily from the paraffin in large masses. Cover-slip preparations from these growths, stained with carbol-fuchsin and decolorized with 25 per cent II,SO,, revealed in each case long thread-like, beaded forms, measuring from 6 to 8 μ in length, the bovine and swine reaching 8 μ in length (Pl. IV, figs. 1 and 2). It will be noticed that the threads show alternate stained

These numbers refer to the bibliography at the end of the article.

and faintly stained areas, presenting a beaded appearance very similar to the forms described by Wolbach and Ernst for the brain medium. Culture tubes of egg, prepared by flowing sterile paraffin (melting point 43° C.) over the surface so as to form a streak, as in the tubes noted above, were inoculated with some of the normal growth-that is, growth away from the paraffin-from the original bovine and. swine cultures from which the long, thread-like forms were first obtained, and another from a freshly isolated bovine culture marked Heifer No. 213, the surface of the egg near the paraffin streak being inoculated in each case. These cultures showed the same piling up of the growth along the margin of the paraffin as in the original tubes, and at twenty weeks the growth in all these tubes had coalesced at the center, completely bridging over the streak of paraffin, which averaged about 5 mm. in width. Inoculations on plain egg without the addition of paraffin were made at the same time for the sake of comparison, and preparations from these and the foregoing cultures were measured with the following results, the cover-slip preparations being made always from the inner edge of the growth covering the paraffin in the case of those tubes to which the paraffin was added:

Tubercle bacilli grown on egg in contact with paraffin and on plain egg.

The measurements were made with an ocular micrometer and represent an average of the longest forms in each preparation measured. It will be noticed in this case that of the cultures to which paraffin had been added, Swine I was the only one that showed marked lengthening, though both of the other cultures showed a tendency to lengthen out, whereas no such tendency was apparent in the case of the cultures grown on plain egg. The growths in contact with the paraffin all showed beading at seventeen weeks.

A culture tube of egg, prepared by flowing sterile paraffin across the surface, was also inoculated from the inner or encroaching edge of the growth bordering the paraffin in the original bovine culture" from which the long, filamentous forms were first obtained. This culture, labeled B1, showed the same piling up of the growth along the edge of

a This culture had been cultivated for thirty-seven generations on bouillon prior to its transfer to egg, and the individual organisms at the time of its transference from the bouillon averaged 2.5μ in length.