20 MANUAL OF MICROBIOLOGICAL METHODS 



for 24 hr or b}'- gentle application of heat until steaming. They are then washed in 

 water and treated with Lugol's iodine (see page 16) from 1 to 5 min. After a second 

 washing they are treated with 5 per cent nitric acid for 1 min followed by 3 per cent 

 hydrochloric acid for 10 sec. They are then decolorized 1 min in equal parts of acetone 

 and 95 per cent ethyl alcohol. Weiss (1909) has modified this procedure by staining 

 with a mixture of 3 parts of carbol fuchsin to 1 part of carbol gentian violet and coun- 

 terstaining with 1 per cent aqueous safranin (5 to 10 sec) or with Bismarck brown 

 (1 min). The counterstain is applied immediately after the decolorization, the ace- 

 tone-alcohol being removed merely by blotting. In some laboratories this method 

 of counterstaining is employed following the Much technic with carbol gentian violet 

 alone for the primary stain. 



Cooper^s Method 

 Cooper (1926) 



The Cooper method calls for staining in Ziehl's carbol fuchsin to which 3 per cent of a 

 10 per cent aqueous sodium chloride solution is added just before use. Smears are 

 stained either by steaming 3-4 min, then allowing them to cool until a precipitate 

 forms, or else by standing overnight in a 37° incubator and cooling in an icebox for 

 20 min to allow precipitation to occur. After the precipitation, the smears are washed 

 with tap water and decolorized 1-10 min in acid alcohol (5 ml of nitric acid, sp gr 1.42, 

 to 95 ml of 95 per cent ethyl alcohol) ; washed again with water and finally for 1 min 

 with 95 per cent ethyl alcohol. They are counterstained with 1 per cent brilliant 

 green or, if the smear is heavy, with a greater dilution of this same stain; washed with 

 water; dried; and examined. 



SPORE STAINING—RECOMMENDED PROCEDURES 



Dorner^s Method 

 Dorner {1922, 1926) \ 



Staining schedule: 



1. Make a heavy suspension of the organism in 2-3 drops of distilled 

 Avater in a small test tube. 



2. Add equal quantity of freshly filtered Ziehl's carbol fuchsin (page 13). 



3. Allow the mixture to stand in a boiling water bath 10 min or more. 



4. On a cover slip or slide mix one loopful of the stained preparation with 

 one loopful of Dorner's nigrosin solution (page 15). 



5. Smear as thinly as possible and do not dry too slowl3^ 



Note: If even backgrounds for exhibiting or photographing are 

 required, especially in the case of slime-producing bacteria, the following 

 procedure is recommended : 



1. Make the suspension in 0.5 ml of nutrient broth or water. 



2. Add 1 ml of 10 per cent gelatin solution. 



3. Add 1 ml of carbol fuchsin, and stain as in steps 1 and 2 above. 



4. Wash out the colloids with warm tap water, with the help of centrifuge 

 or sedimentation. 



5. Mix with nigrosin, and proceed as above. 



Results: Spores, red; vegetative cells, unstained; background, gray. 



