342 PATHOGENIC BACTERIA IN MILK 



cultures instituted in the following manner : — A stout platinum 

 loop, previously sterilised, was dipped into the centre of the milk, 

 and three parallel streaks made upon the oblique surface of the 

 medium in one tube. Then without redippingthe loop in the milk, 

 similar streaks were made consecutively on the oblique surface of 

 the media in four other tubes. The tubes were incubated at 

 37° C. The following day a growth was observed in Tube 2, 

 resembling a typical diphtheria colony. A subculture was 

 prepared in Loffler serum, and cover glass specimens made. Sub- 

 cultures were also made, and a pure culture of the organism sus- 

 pended in broth was injected beneath the skin of a guinea-pig (563 

 grammes). The biological characters and staining properties, as 

 well as the post-mortem signs, all indicated a true diphtheria 

 bacillus. Bowhill also centrifugalised samples of the milk and made 

 cultures from the sediment. But the result was negative. 



Eyre also, by shaking the sample bottles, uniformly distributed 

 the suspended particulate matter of the milk. He then plated 

 out and enumerated 10,960 micro-organisms per c.c. The 

 milk was also very thoroughly centrifugalised (roughly 20,000 

 revolutions). On removing the tubes from the centrifuge, the 

 milk in each was seen to have separated into three distinct layers. 

 The uppermost, consisting of cream, formed practically one quarter 

 of the column of fluid ; below this came the separated milk : whilst 

 the rounded portion of the lower extremity of the tube was filled with 

 sediment. The contents of one tube were used for microscopical 

 examination. The cream in other tubes was used for culturing on 

 blood-serum, after the manner adopted by Bowhill. The centri- 

 fugalised sediment in the remaining tubes was similarly planted on 

 blood - serum. All the cultures were incubated at 37° C, and 

 showed a growth next day. In fourteen out of the twenty cultures 

 made, organisms morphologically resembling the B. diphthericB 

 were present. Every " cream " culture possessed them, but only 

 three out of five " sediment " cultures appeared to do so. These 

 organisms, on being isolated and tested in the usual way, were 

 found to be bacilli of diphtheria. 



Klein relied upon animal inoculation. The milk to be tested 

 was injected subcutaneously into the groin of a guinea-pig. By 

 the fifth day, the inguinal glands were found to be swollen to about 

 the size of a filbert, and surrounded by soft oedematous tissue. 

 Film specimens were made from the juice of the incised gland and 

 stained. They showed numerous bacilli resembling the diphtheria 

 bacillus in size and shape. Cultures made on agar and ascitic agar 



