cH.xiii PATHOGENIC BACTERIA IN MILK 215 



10 to 15 minutes of this treatment the cover-glasses are 

 removed from the hot mixture and washed with absolute 

 alcohol. If treatment with ether is not adopted a better 

 preparation is obtained, if, after pouring off all the super- 

 natant fluid, the tube is filled with distilled water, again 

 rotated for a few minutes, the fluid poured off, and the films 

 made. At least four films should be made and examined. 



No reliance can be placed on a negative examination as 

 evidence of freedom of the milk sample from tubercle bacilli, 

 since if the bacilli are present in only small numbers they may 

 be missed. A positive result cannot be accepted as furnishing 

 conclusive evidence of tubercle bacilli, since that organism 

 may be confounded with other acid-fast bacilli, which some- 

 times occur in milk. Their presence furnishes very strong 

 presumptive proof sufficient for practical but not for research 

 purposes. Del^pine states that by adopting the treatment 

 with ether and alcohol described above, no difiiculty is caused 

 by acid-fast bacilli. 



The procedure the writer prefers for the Ziehl-Neelsen 

 method is as follows : 



Spread the sediment uniformly and not too thinly over cover- 

 slips, making at least 4 preparations. Dry and fix in the ordinary 

 way. 



Filter a little carbol fuchsine solution into a clean test tube. 

 Heat to boiling, and then pour on sufficient to completely cover 

 the cover-slip, preferably held in Cornet forceps. Leave on for five 

 minutes without further heating. 



Wash in water and decolorise thoroughly but carefully in a 

 watch-glass containing 25 per cent sulphuric acid. 



Decolorise further for 30 seconds in absolute alcohol. 



Wash in water. Counterstain with methylene blue staining 

 for one minute. 



Wash in water. Dry and mount in xylol balsam. 



The tubercle bacilli are stained red, the tissue elements and 

 other bacteria are blue. 



Antiformin has recently been introduced as a means of 

 more reliably detecting the presence of tubercle bacilli in 

 microscopic preparations. This substance is the name given 

 to a mixture of an alkaline hypochlorite and an alkaline 

 hydrate. When sputum, for example, is treated with 15 to 

 20 per cent antiformin the tenacious lumps are broken down 



