474 APPENDIX 



under examination, and place three of them in the incubator at room 

 temperature and three of them at 37 C. Place four of them in a water 

 bath heated to 80 C. for fifteen minutes, and then enclose each of the 

 four tubes in a Buchner's tube. These primary cultures may be tested 

 in forty-eight hours for B. coli, the presence of indol, and B. cnteritidis 

 sporogenes. 



4. Secondary or Sub-cultures. From the primary cultivations, make 

 sub-cultures on selected media for the isolation of organisms making 

 their appearance on the plates, or what is often preferable, make a set 

 of plates for qualitative examination only. 



5. Examination for Special Micro-organisms. The milk must be centri- 

 fugalised or the particulate matter allowed to gravitate by sedimentation. 

 It is, as a rule, useless to attempt examination microscopically or other- 

 wise without first using the centrifuge or sedimentation flask. The 

 deposit is then to be stained for the particular organism for which 

 search is being made (see p. 476). 



For centrifugalisation, take two or three samples of the milk under 

 examination to the amount of about 40 c.c. each, and place it in the 

 sterilised tubes of the centrifuge. In these tubes the milk may be 

 centrifugalised for ten or fifteen minutes at 3000 revolutions a minute. 

 At the end of such a period the milk in each tube has separated into 

 three layers at the top there is a dense layer of cream, at the bottom 

 there is the sediment or "slime" containing all the particulate matter, 

 between these two is the separated milk. Aspirate off the cream by 

 means of a sterile glass tube connected with an aspirator or vacuum 

 pump, and examine separately ; aspirate all the separated milk except 

 2 c.c. The remaining sediment is so compact and dense that the tube 

 may now be inclined and the sediment fully exposed without displace- 

 ment. By means of a sterilised platinum loop a small portion may be 

 taken up and spread on the surface of half a dozen slides, and stained. 

 The remainder of the sediment is well mixed with the 2 c.c. of milk and 

 used for inoculation of guinea-pigs. 



For sedimentation, take two conical sedimentation glasses and fill 

 them with the milk under examination, allowing them to stand in the 

 refrigerator for twelve to fourteen hours. It is customary to add a few 

 small carbolic crystals to each flask. On the completion of sedimenta- 

 tion the milk has separated into three main strata : the cream at the top, 

 the sediment at the apex of the flask, and the separated milk in the 

 middle. The cream and milk may then be carefully decanted, and the 

 sediment will be available for examination. 



STAINING METHODS IN MILK EXAMINATION 



The only difficulty which presents itself in the preparation of milk for 

 the microscope is the simultaneous staining of the casein and fat as well 

 as the organisms which may seriously confuse the issue. Hence the 

 removal of the two former substances is recommended, as follows : 



(a) Staining after Clearing with 5 per cent. Acetic Acid. The slides are 



