Fig. 14.—Granule Bacillus from Yoghourt. Shredded preparation of a fresh skim-milk culture at 37° C. for six hours. Stain: aqueous methylene blue. (Enlarged 1:500.) In Figs. 15 and 17 will be noticed the chain arrangement of the bacillus, which, in spite of the supposed data of Luerssen and Kuhn, will be generally noticed in the granule bacillus. Fig. 16.—Granule Bacillus from Yoghourt. Agar Milk Sugar Culture cultivated for forty-eight hours at 37° C. Below is the true branching, above, the distorted involution form. This production of involution forms occurs chiefly in old cultures, and is an indication of degeneration. Stain: aqueous methylene blue. (Enlarged 1:700.)
Fig. 15.—Granule Bacillus from Yoghourt, cultivated after the usual Agar method, for twenty-four hours at 37° C. Stain: aqueous methylene blue. (Enlarged 1:500.) Fig. 17.—Bacteria W. from Milk, cultivated twenty-four hours at 37° C. Methylene blue. (Enlarged 1:500.) The similarity in the pictures ought to serve as a proof of the near relation of the granule form and non-granule varieties.
Fig. 18.—Agar Milk Sugar Culture. From the original Bulgarian Yoghourt. In the centre, and beneath, the characteristic hairy irregular colonies of the granule bacillus (Bacillus bulgaricus group), to the left, the smooth contoured yeast colonies. The colonies of the former organism always remain microscopic in size. (Incubated several days at 20° to 25° C. Magnified X 10.) Fig. 19.—Agar Milk Sugar Culture. Surface colony of granule bacillus from calf's stomach. The great resemblance this colony bears to those formed by the granule bacillus from Yoghourt will be apparent. This fact, as well as close agreement in other cultured features, induced Küntze to place these organisms in one group. (Incubated two days at 37° C. Magnified X 100.)
Fig. 20.—Agar Milk Sugar Culture. Deep-lying colony of granule bacillus from calf's stomach. The form of the colony is often determined by the relative presence or absence of air. (Two days at 37° C. Enlarged about 1:50.) Fig. 21.—Agar Milk Sugar. Colony of Bacterium W. from Yoghourt (non-granular variety of the granule bacteria, as far as possible identical with Luersen and Kuhn's Bacillus bulgaricus), of a cubical branching-out form. According to Küntze, the granule formation of this and related organisms is variable, while White and Avery regard it as a constant characteristic. (Incubated two days at 37° C. Magnified X 50.)
Fig. 22.—Two colonies of Bacillus acidophilus from calf's manure. Agar Milk Sugar Culture. With this organism, also, we have conformation to one type of colony, while, in other respects, temperature requirements and production, etc., we have close agreement with the granule bacillus (Bacillus bulgaricus). (Two days at 37° C. Enlarged about 1:50.) Fig. 23.—Beer-wort Gelatine. Fourteen-days-old colony of Yoghourt yeast. (Enlarged about 1:50.)]
Fig. 24.—Shredded preparation of the Bulgarian original Yoghourt. Stain: aqueous methylene blue. Granule bacillus, diplostreptococci, and yeast. (See also other photo-micrographs of Yoghourt. Enlarged 1:70.)] Fig. 25.—Granule Bacillus from Yoghourt. Cultivated in skim milk in twenty-four hours at 37° C. Stain: aqueous methylene blue. (Enlarged 1:50.) By means of this staining treatment the presence of granules (not spores) can be easily detected. Treatment with fuchsine fails to bring out these formations.
Fig. 26.—Bacteria W., Agar Milk Sugar Culture. Cultivated twenty-four hours (knobs, clubs). Stain: Gram's method coloured with aqueous fuchsine afterwards. (Enlarged 1:600.) Fig. 27.—Bacteria acidophilus from calves' manure, isolated by means of bouillon as acid as vinegar. Shredded out of the usual Agar culture. Twenty-four hours at 37° C. Stain: aqueous methylene blue. (Enlarged 1:700.)
Fig. 28.—Mucus from calf's stomach inoculated into milk after eight transferrings. Shredded preparation cultivated in milk for twenty-four hours at 37° C. Diplostreptococci and granule bacillus. Stain: aqueous methylene blue. (Enlarged 1:500.) Fig. 29.—Diplostreptococcus from Yoghourt. Pure culture in skim milk. A comparison of the illustrations will show how close a resemblance exists between bacteria found in the mucous membrane of calf's stomach and those occurring in Yoghourt. In fact, by the combined action of granule bacilli, and of diplostreptococci from calf's stomach, together with a Yoghourt yeast, it is possible to prepare normal Yoghourt.

In a review of the literature of the subject of soured milks, Makrinoff suggests the adoption of the two names, Streptobac. lebenis viscosus and Streptobac. lebenis non-viscosus, for the organisms of the so-called Bulgaricus group, and known at present as Bacillus-bulgaricus, Streptobac. lebenis, Bacillus of Massol, Granule bacillus, Bact. Mazun, Bac. lactis acidi, etc.[77]

White and Avery[78] have made a comparative study of a large number of varieties and species of lactic acid bacteria of the above type obtained from various fermented milks and milk tabloids. Their descriptions are so detailed and their conclusions are so important that we give them at length. According to this work, the whole of the thermophilic lactic acid bacilli of the so-called Bulgaricus type may be divided into two sub-types, A and B.

The Cultural Characteristics of the Bacillus Bulgaricus Group

The cultural characteristics of all the strains of Bacillus bulgaricus (granule bacillus) are as follows:

In Whey Agar.—All strains exhibit wide variation in size, 2 µ to 50 µ long and about 1 µ broad. Almost all individuals are intensely Gram-positive, and show regularity of outline. All strains show involution form, exhibiting vacuoles, and often show empty cell membranes. The latter are Gram-negative, and vary greatly in both dimensions as well as in form. All strains show tendency to chain formation, some being arranged in chains of six to twenty-five segments, which may contain both Gram-positive and Gram-negative individuals. Type B exhibits Gram-negative spherical bodies varying from 0.25 µ to 1 µ in size, adhering to the sides of some of the Gram-negative individuals.

In Whey.—In this medium there is a marked tendency toward degeneration and involution. In the early stages of incubation, at 100° to 112° F., the bacilli are uniform in size and intensely Gram-positive; in succeeding stages the irregular, vacuolated, inflated, and ruptured forms predominate. Between the eighteenth and twenty-fourth hours of incubation at 112° F. the strains of type A develop oval to kidney-shaped nodules attached to a stem extending from the cell substance. As the incubation is prolonged these nodules increase in size, often measuring 1 µ to 2 µ in length; this nodule formation occurs at the expense of the cell protoplasm, and appears to be a marked characteristic of growth in whey. Cultures of type B do not form nodules or clubs, but small spherical bodies more or less securely attached to the cell wall are seen. Again, type A assumes the form of small bacilli in chains, while type B strains develop to a greater length and exist almost exclusively as single isolated forms. True branching has been observed in strains of type B.

In Milk.—In milk there is a tendency to thread-formation consisting of four to ten segments in the case of type A, while type B shows longer and more curved forms. With increasing age of the culture there also appears to be increase in the length of the organisms. All strains are non-motile, non-sporogenous, and non-capsule-forming.

Staining Reactions.—All strains are readily stained by the usual aniline dyes.