56 THE BACTERIOLOGICAL EXAMINATION OF MILK 



samples it is best however to dilute the milk, or secure a deposit by 

 the gravity flask method or centrifuge. 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. 



In the course of our investigations we have tried a large number 

 of staining methods, and we have obtained the best results from 

 one or other of the following : — 



(a) Staining after clearing with 5 per cent, acetic acid. — The 

 slides are thoroughly cleaned in the ordinary way, and immedi- 

 ately before use are again washed with equal parts of alcohol and 

 ether. Several loopfuls of the milk to be examined are now 

 placed on the slide and allowed to dry at the temperature of the 

 room, being protected from the air by means of a small glass 

 cover. When the film is dry it is._fi2cedj preferably with alcohol and 

 ether, in the manner above described. It is then washed alter- 

 nately with a 5 per cent, solution of acetic acid and distilled water 

 until there is but little apparent film left upon the slide, which is 

 then dried between layers of " papier de soie " or fine filter paper. 

 The specimen may now be stained by means of any of the ordinary 

 aniline dyes, washed in distilled water, again dried, and examined 

 under the microscope. We have tried ether, chloroform, various 

 strengths of alcohol, and many other clearing agents, but we have 

 obtained the best results from acetic acid. 



{b^ Saponification. — If it be desired to retain the background of 

 casein and fat, it will be found best to saponify the milk in the 

 following manner : — 



Prepare the film of milk as before, but before drying it add an 

 equal number of loopfuls of a sodium carbonate or sodium hydrate 

 solution (5 per cent, to 50 per cent, dilution). The loopfuls of 

 milk and soda solution should be placed in immediate proximity 

 to each other on the slide, and thoroughly mixed by means of the 

 platinum loop. By this means an even distribution of the bacteria 

 is obtained. The film is then dried by gentle heating, stained, 

 washed, and cleared with xylol. The result will be that the 

 organisms will be stained more deeply in colour than the back- 

 ground of saponified matter. 



ic) Clearing with acetic acid after saponification. — We have 

 found as a general rule that the best preparations are obtained by 

 a combination of the above two simple methods. For this purpose, 

 the films are prepared exactly as in the ordinary saponification 



