(_a_) True lactic acid forming bacteria, _Streptococcus acidi lactici_ (Grotenfeldt).
(_b_) Bacteria of the group _Bacterium acidi lactici_ (Hueppe) and _Bacterium lactis aërogenes_.
(_d_) Two species of butyric acid bacteria, _Bacillus esterificans_ and _Bacillus keffir_ (Kuntze).
His conclusions are: 1. In any case the presence of a yeast capable of directly fermenting milk sugar is not essential. 2. The significance of the presence of yeast lies in the fact that stimulation of the lactic bacteria occurs; further, the yeast exerts a regulating influence upon the rapidity of the fermentation proper. The variety is of minor importance, provided always that the yeast does not produce an unpleasant flavour. By the use of mixed cultures of _Bacillus esterificans_, _Bacillus keffir_, and _Streptococcus acidi lactici_, and a keffir yeast, Kuntze obtained a product that possessed to the fullest degree all the characteristic properties of a normal keffir. In such cultures he was successful in obtaining the formation of keffir-like grains. Keffir fermentation is, according to Kuntze, the result of the action of various organisms. During the initial stage butyric acid fermentation takes place, but is prevented from becoming predominant by the action of the keffir yeast. Simultaneously a true lactic acid fermentation proceeds and eventually gives place to a subsequent secondary production of butyric acid. Finally, then, we have a certain amount of unison in the results obtained by Freudenreich, Essaulow, Nikolaiewa, and Kuntze. These show that, for the production of a characteristic keffir, specific organisms are not essential, provided always that those used possess, either individually or collectively, the essential capacity of acidifying, coagulating, and fermenting the milk. For the growth of normal grains the presence of a matrix-forming organism, such as _Bacillus keffir_, is indispensable.
[Illustration: FIG. 12—_Streptococcus lacticus_ (Grotenfeldt) growing on lactose-agar, stained by Gram's method. ( ✕ 900 diams.)]
_Diseases of Keffir Grains._—According to the age and the previous treatment to which keffir grains have been subjected, the vitality of one or more of the organisms constituting the grain may have been impaired. The results of Freudenreich have shown that _Saccharomyces keffir_ and _Streptococcus a_ are unable to withstand desiccation for more than a few days, and this is sufficient to account for the frequent failures to obtain normal keffir from the grain. Further, grains succumb to a mucilaginous disease; the cavities become filled with a slimy fluid, and the grains are covered with mucilaginous matter. They lose their elasticity and become brittle or mealy, but large grains appear to be more subject to this fault than do the small ones. Such grains should be disinfected by immersion for a short time in two per cent. salicylic acid solution, followed by drying in the sun, whereby they are completely regenerated.