NEW MODIFICATION OF THE GRAM STAIN 397 



to allow the Gram positive organisms to retain the stain while 

 the color was removed from the Gram negative bacteria and the 

 background of milk. Hastings, Evans and Hart (1912), in their 

 cheese work used a decolorizing solution of aniUn oil one part 

 and xylol two parts. This solution although removing the stain 

 from the Gram negative organisms and the milk, was slow 

 in action and caused the organisms to appear distended and less 

 brilhant in the final preparation. Consistent results could not 

 be obtained using alcohol as a decolorizer as the stain was re- 

 moved from the Gram positive bacteria before the milk was 

 sufficiently destained. The results obtained with acetone, as 

 a decolorizer, were similar to those where alcohol was used. 



With exception of Bismarck brown,^no counter stain exhibited 

 sufficient range of affinity between the nucleo-proteins of the 

 cells and the casein of the milk to allow for different intensities 

 of color even if destained. A few successful smears were made 

 where an aqueous solution of safranin was used as a counter- 

 stain, provided the slides were well washed before the application 

 of the safranin. A heavy precipitate will be deposited on the 

 smear if the Bismarck brown is not frequently dissolved and 

 filtered. 



The above method has been used for the routine examination 

 of milk samples for an entire season at a cheese factory where all 

 grades of milk were being received, and it proved helpful in 

 eliminating milk which would develop gassy curds. The smears 

 were checked with duplicate samples stained with methylene 

 blue and no appreciable difference in the count could be observed. 



REFERENCES 



Breed, R. S., and Brew, J. D. 1916 Counting bacteria by means of the 



microscope. N. Y. Agr. Exp. Sta. Tech. Bui. 49: (Condensed form) 



N. Y. Agr. Exp. Sta., Circ. 58. 

 Conn, H. J., et al. 1919 Methods of pure culture study. Jour. Bact., 4, 



107-132. 

 Hastings, E. G., Evans, Alice E., and Hart, E. B. 1912 The bacteriology 



of cheddar cheese. U. S. Dept. Agr. Bur. An. Ind. Bui. 150. 



'If Bismarck brown stains the background of milk too deeply, slide may be 

 immersed for a few seconds in a weak aqueous solution of acid fuchsin, after 

 counter staining. 



