32 THOMPSON YATES LABORATORIES REPORT 
The litmus milk, or plain milk tubes, were then placed in an apparatus described by me 
in another part of these Reports, where they were kept automatically at a temperature 
between 75 0 C. and 8o° C. for 20 minutes. They were afterwards placed in wide 
mouth stoppered bottles, into which the requisite amount of pyrogallic acid and caustic 
potash was poured to insure anaerobiosis ; the bottles were then placed in the incubator 
at 37° C. 
Usually all the tubes that ' went ' typically had ' gone ' after 1 8 hours' 
incubation. By ' typical ' I mean the reaction described by Klein : the milk is clotted, 
and the clot turned into clear whey with an acid reaction, a broken or scattered layer 
of cream remaining on the top broken up by the gas which may be seen evolved in 
bubbles from the whey. If litmus milk was used, the whey and cream which was 
bleached in the anaerobic bottle will be seen gradually to assume a pink colour on being 
exposed to the air, instead of its original violet colour ; this is due to the altered 
reaction. The tube generally smells more or less of butyric acid. 
With many of my samples the butyric acid smell was masked by a putrid 
smell ; sometimes both could be distinguished ; this occurred chiefly in the samples 
of grains, and was probably due to the presence of some other spore-bearing 
micro-organism causing putrefactive changes. On making one or two subcultures in 
milk, or a milk culture from the exudation of an animal killed by injection of a culture 
with a putrid smell, the smell would often be found to have disappeared and the milk 
culture to be in every respect typical. A certain proportion of tubes ' went ' in the 
ways Klein describes as atypical and intermediate. An atypical tube is one devoid of 
gas formation, and, consequently, the layer of cream remains unbroken ; in the 
intermediate type the clot is not fully peptonised into whey, the tube contains one to 
two-thirds excavated clot and the rest is whey, there is also less gas formation. 
With the following samples whenever a tube ' went ' typically, atypically, or in 
the intermediate way, a portion of the whey was inoculated into Guinea-pigs beneath 
the skin over the abdomen ; 2 c.c. of the whey were inoculated in most cases, and, as 
the Guinea-pigs were all fully grown, this dose would correspond to Klein's dose for 
standard virulence, namely, 1 c.c. per 200-300 grams of Guinea-pig. According to 
Klein, the pathogenicity to Guinea-pigs is the means of distinguishing his Bacillus 
enteritidis sporogenes from Botkin's butyric acid bacillus, which gives the same reaction 
in milk cultures, but is non-pathogenic. As a result of subcutaneous inoculation, if 
the culture is virulent, the Guinea-pig will be found dead in 18 to 36 hours. On 
examination it will be found that the skin is separated from the subjacent tissues over 
a considerable area by dissolution of the connective tissue ; the skin may have sloughed 
through to the outside, or the muscles of the abdominal wall may have sloughed into 
the abdominal cavity. The space between the skin and the abdominal and thoracic 
walls contains more or less badly-smelling sanguineous fluid, but the fluid may be 
very scanty or almost absent in the most rapidly fatal cases. 
