480 APPENDIX 



pound of butter and place it in a sterilised flask with 150 c.c. of sterile 

 salt solution. Place the flask in the water bath at about 35° C, and 

 shake gently until the butter has melted. The contents of the flask now 

 appear as a milk-like emulsion. A small quantity of this mixture may 

 be used for plate cultivation on gelatine and agar, as in milk. The 

 remainder should be placed in a sedimentation flask in the refrigerator 

 for twenty-four hours. By this means the particulate matter of the 

 butter, including the contained organisms, are deposited. After remov- 

 ing the superficial solidified fat by means of a sterile spatula, the turbid 

 fluid may be decanted, and the sediment collected for microscopical 

 examination or the injection of guinea-pigs. 



Examination of Cheese. — With a knife previously sterilised by 

 p issing through the flame, cut off from the piece of cheese under 

 examination a thin slice parallel to the surface. Remove this, and 

 with a second sterile knife cut perpendicularly downward from the 

 bared surface. Pass down into the latter cut a coarse .sterile platinum 

 needle, of which a small portion near the extremity has been slightly 

 roughened with a file. 



Inoculate with this needle a sufficient number of tubes of bouillon 

 from which plate cultivations can subsequently be made for isolation 

 purposes, and placed under both aerobic and anaerobic conditions. 



Examination of Milk for Pus Cells. — Place 10 cc. of the milk to be 



examined in each tube of the centrifuge (Plate 5, p. 74) and centrifugalise 

 for two minutes. Pour off the supernatant fluid, and with a sterilised 

 needle or pipette take up a small quantity of the sediment remaining in 

 the tube. Spread the sediment evenly over the surface of an ordinary 

 glass slide, and dry over the flame of a Bunsen burner or on the drying 

 stage. Wash the fixed film with ether (or alternately with absolute 

 alcohol and ether) until all the superfluous fat is removed, and stain. 

 The preparation may be stained (a) by one of the ordinary solutions 

 such as Loffler's blue, etc. ; or (b) by Gram's method. Examine under 

 the microscope with a ^th oil immersion lens. 



Inoculation of Guinea-pig's in Milk Examination. — It will 



be sufficient to remark that the simplest forms of inoculation are all that 

 are usually required in milk investigation, namely, the intra-peritoneal and 

 subculaneous. In some cases it may be sufficient to inoculate a few c.c. of 

 the original milk ; but, as a rule, it is advisable to centrifugalise, or use 

 the sedimentation flask containing about 250 c.c. From the deposit or 

 sediment two guinea-pigs may be inoculated, the one subcutaneously in 

 the groin, the other intra-peritoneally. Particularly is this necessary in 

 making a reliable and exhaustive search for the B. tuberculosis. Micro- 

 scopic examination alone for this organism is not reliable (see p. 478). 

 The details of the process as carried out in practice are as follows : — 



After centrifugalisation the deposit is mixed with the 2 c.c. of milk 

 remaining in the tube after aspiration of that which is superfluous. Two 

 guinea-pigs (of say 250 grammes weight each) are taken and inoculated 

 with the deposit from about 40 c.c. of milk. The fluid is inoculated 

 subcutaneously on the inner side of the leg under strict aseptic precau- 



