160 BACTERIA IN RELATION TO PLANT DISEASES. 
Essaulof, with which conclusion von Freudenreich agrees. Essaulof believed that only Bacillus acidi 
lactici and the yeast were necessary to the formation of the symbiosis. Von Freudenreich was 
unable to obtain conclusive evidence on this point. He found other lactic acid bacteria and suggests 
that possibly the same micro-organisms do not always occur in kefir. 
“The yeast and Kern’s Bacillus are always present but the lactic acid bacteria may possibly be 
different if only they can bring about the splitting up and fermentation of the milk. If one sums up 
the results obtained hitherto we have in kefir an example of the symbiosis of several micro-organisms, 
among which is a yeast that according to most authors is not able to ferment milk-sugar, as well as 
probably a lactic acid ferment and a bacillus hitherto only cultivated by Beyerinck, which appears to 
be identical with the bacillus present in the kefir grains and described, but not cultivated by Kern. 
On the other hand, up to this time the réle of these particular micro-organisms in kefir fermentation 
has not been clearly made out.” 
His own experiments began in 1892 and were continued with interruptions up to the time of the 
publication of his paper. In his preparations he found especially yeast-cells and long, mostly bent 
bacilli, very much resembling Kerns’ pictures, but also shorter rods—younger stages of the bacillus— 
and furthermore, coccus forms, the latter, however, much more rarely. Often he found that the 
bacillus stained only at the two poles, a phenomenon which he thinks led Kern into error as to the 
presence of spores in his Dispora. He thinks that the sporogenous organisms occurring in Kern’s 
cultures were only potato bacilli and similar bacilli. ‘‘I have never observed spores in the bacillus 
of the kefir grains, Dispora caucasica, therefore, I would write Bacillus caucasicus.” 
When the kefir was clean, von Freudenreich found four different micro-organisms in it, namely, 
yeast cells, large coccus forms arranged in chains, smaller cocci and bacilli. The larger streptococcus 
and the yeast grew readily on gelatin plates, sometimes also the smaller streptococcus, but not the 
bacillus. Only once did he obtain colonies of the latter on an anaerobic gelatin plate. On the surface 
of milk serum agar plates at 35° C. one readily obtains the smaller streptococcus along with the 
larger one and also very small colonies of a bacillus believed to be identical with Bacillus caucasicus. 
Nevertheless he says that such cultures do not always succeed. Sometimes its colonies were entirely 
absent without any reason therefor being apparent. When he made streaks on slant milk serum 
agar, using kefir itself as.a substance for inoculation and keeping the tubes at 22° C. he states that 
he obtained masses of growth containing the four organisms mentioned, and he figures the microscopic 
preparations of such mixed cultures, but these figures are not very conclusive as to any symbiosis. 
He says also that Bacillus caucasicus may be isolated by stab-cultures in deep layers of agar, these 
cultures being kept at 35°C. In the stab then appear mostly only the bacillus and the small strep- 
tococcus. 
He describes the yeast as follows. Saccharomyces kefir is obtained readily in plate-cultures 
where it produces very small, coarse-grained pale colonies. On milk serum gelatin plates the colonies 
are said to be round and yellowish and better developed than on ordinary nutrient gelatin. The 
granulations on the edge of the colony are coarse. On the less thickly sown plates, the superficial 
colonies are well formed and whitish, finally yellowish. ‘The buried ones are a yellowish color. The 
center of the colony is dark brown. Stab-cultures were distinctly visible in 24 hours. Development 
on the plates also was rather prompt, the colonies being visible for the most part after 2 to 3 days. 
Beef-broth kept at 20° C. clouded in 24 hours, also milk-sugar bouillon. The growth, however, is 
not so vigorous as in beer-must. In the latter medium there was an abundant growth. Gas-pro- 
duction occurs but is less abundant than in case of beer yeasts. Maltose is fermented by the yeast. 
The yeast ferments grape-sugar with production of alcohol. It does not cause any fermentation of 
milk, but develops well in it with the formation of a peculiar taste, which is different from that due 
to beer yeast. To the eye the milk remains unchanged. On potato the yeast grew with the formation 
of a yellowish patch. The optimum temperature is about 22° C. The yeast will growat 28° C., but 
not at 35° C. This yeast consists of oval cells of variable size, on an average 3 to 5uX2 to 3u. 
Single cells are roundish, especially in potato cultures. The cells stain readily with all the ordinary 
aniline dyes, also by Gram. There is ordinarily a vacuole. In the protoplasm there are one or more 
shining granules. ‘The yeast is unlike Saccharomyces pastorianus I, II, and III, and also unlike 
Saccharomyces cerevisiae and Saccharomyces ellipsoideus, with which he compared it. There is never 
any pellicle formed by the kefir yeast, something which always goes with the other yeasts mentioned. 
He could not discover any ascospore formation. A temperature of 50° C. for 5 minutes sometimes 
sterilizes the culture, and a temperature of 55° C. always sterilizes it. He found the yeast also very 
sensitive to dry air. It.endured 2 and 3 days’ exposure, but not 4 or more days when taken from 
fluid cultures and exposed on filter paper. I omit descriptions of the two forms of streptococci 
because most observers are agreed that they only occur accidentally in the kefir. The photomicrograph 
of his larger streptococcus shows an organism with a long diameter nearly double the short diameter 
and makes one think that very likely the organism figured is not a streptococcus at all. 
