276 CLEAN MILK 



Stain with aqueous fuchsin, aqueous methylene blue, Loffler'sr 

 methylene blue or other simple stains (i. e., not combined with 3l 

 mordant), thus: flood the smear with the dye, wash off in water 

 thoroughly, and dry. Simple (" aqueous ") stains consist of the dry- 

 stain or dye, 1 part, alcohol 10 parts, water 100 parts. 



Note : Young cultures stain well with methylene blue, and pe- 

 culiarities of staining are best shown with this stain, such as polar 

 granules, vacuoles, metachromatic granules, etc. The intensity with 

 which this stain colors the bacteria is important, certain species stain- 

 ing faintly, others deeply. 



In old cultures, methylene blue stains only the more vigorous 

 survivors: those which have died (as a result of auto-intoxication, 

 etc., not those killed artificially by heat) losing the power to stain 

 well with this dye; with aqueous fuchsin, the tendency with young' 

 cultures is to overstain; with old cultures, the degenerate forms 

 stain, as well as those not degenerate. 



TECHNIQUE OF ZIEHL-NIELSEN STAIN FOR TUBERCLE BACILLI AND 



SPORES. 



For tubercle bacilli: Prepare a smear of the suspected material; 

 cover the smear with an excess of the Ziehl-Nielsen stain; heat 

 five minutes, without boiling, however. Wash in water ; decol- 

 orize until a faint pink only remains with acid alcohol (HC1 

 5 parts, alcohol 100 parts.) Wash and counter-stain with 

 methylene blue. Wash and mount. The tubercle bacilli are 

 seen red in a blue field. Since bacilli not unlike the tubercle 

 bacillus in appearance, and staining by the same method, are 

 found at times on certain grasses, hay, etc., and in butter, etc., 

 conclusive proof that tubercle bacilli are present in a given 

 farm product cannot be had from the staining reaction alone, 

 but requires confirmation from careful study of the effects upon 

 a guinea-pig of inoculation with the bacterium in question. 



