6o THE BACTERIOLOGICAL EXAMINATION OF MILK 



1. Allow two or three drops of the stain to fall upon the slide 

 and remain in contact with the film {or five seconds. 



2. Wash off the stain with the iodine solution applied from a 

 drop bottle for five or six seconds. The film should then be black 

 or dark brown. 



3. Wash off the iodine solution with a mixture of i part 

 acetone and 2 parts alcohol absolute, but allow to remain in 

 contact for two or three seconds only. 



4. Wash off with absolute alcohol, applied until no more stain 

 comes away. 



5. Wash in water, blot off superfluous water, and set aside to 

 dry. If thought desirable the preparation may be counterstained 

 by the application of a very weak solution of Ziehl-Neelsen. 



As the above manipulation follow one another with some 

 rapidity, it is well to have the bottles containing the reagents 

 clearly labelled and arranged in their proper order, so as to come 

 readily to hand. A very easily made and convenient stand for 

 this purpose is illustrated on Plate 8. 



{h) Ziehl-Neelsen method of staining*— 



If milk is under examination it should first be centrifugalised.^ 

 The centrifuge tubes must be thoroughly cleaned with water, 

 followed by soaking for some minutes in strong sulphuric acid or 

 nitric acid, then rinsed in tap water, distilled water, and finally 

 rectified spirit. It is better not to place more than about an inch 

 depth of milk in the tube. The centrifuge should be whirled for 

 at least two minutes. 



The film is prepared in the usual manner, either from the 

 particulate matter of the milk after centrifugalisation or from the 

 lesions of an experimental animal, and fixed with alcohol and 

 ether. The procedure then is as follows : — 



1. Allow two or three drops of Ziehl's carbol-fuchsin to fall 

 upon the film, and heat either over the flame or upon a heating 

 stage as shown on Fig. 8 until the steam rises. 



2. Allow to act for three minutes, replacing with fresh stain 

 any loss by evaporation. 



* If no centrifuge is available the milk must be placed in a sterilised conical 

 flask and treated in the following manner : To 50 c.c. of milk set for sedimenta- 

 tion, 10 c.c. of liquefied carbolic acid crystals are added. The mixture is 

 thoroughly shaken and poured into a tall conical glass. After standing for 

 twenty-four hours a little of the sediment is taken by means of a sterilised 

 pipette and the film made. 



