186 MICROSCOPIC AND CULTURAL STUDY OF BACTERIA 



use, 10 c.c. of distilled water are slightly alkalinized by the addition 

 of two drops of a 10 per cent, solution of sodium carbonate, and 

 exactly ten drops of the stain are then added. 



Staining with Giemsa Solutions. (a) Films are fixed by immersion 

 in neutral absolute methyl alcohol for one minute, air-dried, and 

 covered with the diluted stain, which is allowed to act for fifteen to 

 twenty minutes when ordinary exudates and bacteria are used; for 

 one to three hours if Treponemata or Negri bodies are sought for. 



(b) Wash in water, dry and mount. 



5. W. H. Smith's Solution Stain. (a) Stain the fixed smear with 

 anilin oil gentian violet for one minute. 



(b) Wash with water. 



(c) Flood with Gram-iodin solution for thirty seconds. 



(d) Decolorize with 95 per cent, alcohol. 



(e) Wash with ether for a few seconds. 



(/) Flood with absolute alcohol for five seconds. 



(g) Stain with saturated aqueous solution eosin for one to two 

 minutes. 



(k) Wash with absolute alcohol for a few seconds. 



(i) Clear with xylol. 



( j) Mount in balsam. 



III. Staining Bacteria in Tissues. Paraffin sections are preferable, 

 partly because very thin sections may be cut; chiefly because celloidin 

 stains somewhat with the stains ordinarily used. 



The Gram-Weigert Stain for Bacteria in Tissues. 1 (a) Stain paraffin 

 sections with anilin oil methyl violet for five to twenty minutes. 



(6) Wash in water to remove excess of stain. 



(c) Gram-iodin solution for one minute. 



(d) Wash in water to remove excess of iodin. 



(e) Decolorize with several changes of absolute alcohol until no 

 more color comes out. 



(/) Clear section in xylol. 

 (g) Mount in neutral xylol balsam. 



Mallory and Wright Modification for Celloidin Sections. 2 (a) Stain 

 sections with lithium carmine for two to five minutes. 



(b) Remove excess of stain with acid alcohol. 



(c) Wash in water. 



(d) Dehydrate in 95 per cent, alcohol. 



1 Mallory and Wright, Pathological Technic, 6th ed., 1915, p. 432. 

 * Ibid. 



