STAINING METHODS iv«-7 



Carbol Crystal Violet (Nicolle) 



original statement of formula emended statement 



Solution A 



Sat. ale. gentian violet 10 ml. Crystal violet (90% dye content) 0.4 g. 



1% aqu. sol. phenol 100 ml. Ethyl alcohol (95%) 10 ml. 



Solution B 



Phenol 1 g. 



Distilled water 100 ml. 



Mix solutions A and B. 

 This formula is sometimes preferred either as a general stain or in the Gram technic 

 If properly prepared it is permanent; but it has a tendency to gelatinize if the amount 

 of dye is too great. To prevent this sort of deterioration the quantity of dye in the 

 above amended formula has been reduced to 0.4 g. from the 1.0 g. recommended in 

 previous editions of this leaflet. Even when the solution is so prepared as to be 

 permanent, however, it seems to have no advantage over the ammonium oxalate 

 crystal violet given above. 



Anilin "Gentian Violet" (Ehrlich) 

 original statement of formula emended statement 



Solution A 



Sat. ale. sol. gentian violet 5-20 ml. Crystal violet (90% dye content) 1.2 g. 



Anilin water (2 ml. anilin shaken Ethyl alcohol (95%) 12 ml. 



with 98 ml. water and filtered) 100 ml. Solution B 



Anilin 2 ml. 



Distilled water 98 ml. 



Shake and allow to stand for a few min- 

 utes, then filter. 



Mix Solutions A and B. 

 This formula is given largely for its historic interest. It is a quite unstable solution, 

 and has no special value today. It was, however, one of the first important bacterial 

 staining fluids and was formerly regarded as the standard formula for the Gram stain. 

 It is not, however, certain what was the "anilin gentian violet" originally employed in 

 the Gram stain, even though ascribed to Ehrlich. As a matter of fact Ehrlich seems to 

 be properly credited only with the idea of using anilin water in the formula, as he ap- 

 parently did not recommend any one definite formula. 



Negative Staining of Bacteria — Recommended Procedures 

 dorner's nigrosin solution 



Nigrosin, water soluble (nigrosin B Gnibler recommended by Dorner; 

 American nigrosins certified by Commission on Standardization of Biologi- 

 cal Stains ordinarily satisfactory) 10 g. 



Distilled water 100 ml. 



Immerse in boiling water bath for 30 minutes; then add as preservative: 



Formalin 0.5 ml . 



Filter twice through double filter paper and store in serological test tubes, about 5 ml. 



to the tube. 



This staining solution is used for the negative demonstration of 

 bacteria, in place of the Burri India ink. For its use in Dorner's spore 

 stain, see p. IV46-II. 

 Staining schedule: 



1. Mix a loopful of the bacterial suspension on the slide with an 



equal amount of the staining solution. (If prepared from 

 growth on solid media, the suspension must not be too heavy.) 



2. Allow the mixture to dry in the air, and examine under micro- 



scope. 

 Results : Unstained cells in a background which is an even dark gray 

 if the preparation is well made. 



