APPENDIX 475 



thoroughly cleaned in the ordinary wayj and immediately 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 fixed, preferably with 

 alcohol and ether, as described below. It is then washed alternately 

 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 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. Ether, chloroform, 

 various strengths of alcohol, and other clearing agents may be used if 

 preferred. 



(6) 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 background of saponified matter. 



(c) Clearing tvith Acetic Acid after Saponification. — The best prepara- 

 tions are obtained by a combination of the above methods. For this 

 purpose the films are prepared exactly as in the ordinary saponification 

 method above described, but as soon as the films have become saponified, 

 instead of at once proceeding to stain with the desired dye, the film is 

 thoroughly cleared by several alternate washings with the 5 per cent, 

 solution of acetic acid and distilled water. The subsequent procedure 

 is as in (a). 



Methods of Fixation. — The object of fixing is to coagulate the 

 albuminous material, and cause perfect adhesion of the prepared film to 

 the slide. The following alternative methods are recommended : — 



(a) Heat. — Holding the glass slide by one extremity between the 

 thumb and index finger of the right hand, pass it, film side upwards, 

 gently through the flame three times, allowing the under surface to rest 

 on the back of the left hand between each passage. 



(b) Alcohol-ether. — Place one or two drops of a mixture of equal parts 

 of absolute alcohol and ether upon the dried film, and allow it to 

 evaporate. 



(c) Formal-alcohol. — -Formalin 1 part, absolute alcohol 9 parts. Leave 

 in contact for from three to four minutes, wash well in water, blot off 

 excess of moisture, and stain. 



(d) Perchloride of Mercury. — Saturated aqueous solution. Leave in 

 contact with the film for four or five minutes. Wash off with a stream 

 of water, and apply Gram's iodine solution in order to dissolve out any 



