STAINING METHODS 15 



NEGATIVE STAINING OF BACTERIA—RECOMMENDED PROCEDURES 



Dorner's Nigrosin Solution 



Nigrosin, water soluble (nigrosin B Griibler recommended by Dorner; Amer- 

 ican nigrosins certified by Commission on Standardization of Biological 

 Stains ordinarily satisfactory) 10 g 



Distilled water 100 ml 



Immerse in boiling water bath for 30 min, 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 bac- 

 teria, in place of the Burri india ink. For its use in Dorner's spore stain, 

 see page 20. 



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 microscope. 

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



the preparation is well made. 



Benians* Congo Red 



Congo red (80 % dye content) 2 g 



Distilled water 100 ml 



Staining schedule: 



1. Place a drop of the above staining fluid on a slide. . 



2. Mix culture with the drop and spread out into a rather thick film. 



3. After film has dried, wash with 1 per cent HCl. 



4. Dry, either in the air or by blotting. 



Results : Cells unstained in a blue background. Good results are not 

 to be expected from broth cultures or from cultures in salt solutions 

 unless the cells are first removed by centrifuging. 



THE GRAM STAIN— RECOMMENDED PROCEDURES 



There are numerous modifications of the gram stain, many of which 

 have been listed by Hucker and Conn (1923, 1927). The two modifica- 

 tions given below have proved especially useful to the committee. The 

 Hucker modification is valuable for staining smears of pure cultures; that 

 of Kopeloff and Beerman (1922) for preparations of body discharges such 

 as gonorrhoeal pus, also for pure cultures of stronglj^ acid-forming organ- 

 isms. The latter is itself a variation of the modification by Burke (1921). 



