STAINING METHODS iv«-ll 



Solution A Solution B 



Auramine O (90% dye content) . . 0.1 g. Ethyl alcohol (70%) 100 ml. 



Liquefied phenol 3 ml. Cone. HCl 0.5 ml. 



Distilled water 97 ml. NaCl 0.5 g. 



Staining schedule: 



1. Stain dried smears 2-3 min. in Solution A. 



2. Wash in tap water. 



3. Destain 3-5 min. in Solution B, freshly prepared. 



4. Dry, and examine under a monocular microscope, using 8 mm. dry objective and 



a 20X ocular; illumination should he a low voltage, high amperage microscope 

 lamp, supplied with a l)lue (ultraviolet transmitting) filter, a complementary 

 yellow filter having been provided for the ocular. 

 Results: Acid-fast bacteria, bright yellow, fluorescent; other organisms, not visible; 

 background, nearly black. 



Much's Method 

 Much (1907) 



Much's method No. 2, which is now quite widely used, employs carbol gentian violet 

 of essentially the formula given on page iv^t-S for carbol fuchsin except that in the 

 place of basic fuchsin the author calls for methyl violet BN. Preparations are 

 stained cold for 24 hours or by gentle application of heat until steaming. They are 

 then washed in water and treated with Lugol's iodine (see p. iv^6-8) from 1 to 5 

 minutes. After a second washing they are treated with 5% nitric acid for 1 minute 

 followed by 3% hydrochloric acid for 10 seconds. They are then decolorized 1 minute 

 in equal parts of acetone and 95% 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 counterstaining with 1% aqueous safranin (5 to 10 seconds) or with 

 Bismarck brown (1 minute). The counterstain is applied immediately after the 

 decolorization, the acetone-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% of a 10% 

 aqueous sodium chloride solution is added just before use. Smears are stained either 

 by steaming 3 to 4 minutes, then allowing them to cool until a precipitate forms, or 

 else by standing overnight in a 37° incubator and cooling in an ice box for 20 minutes 

 to allow precipitation to occur. After the precipitation, the smears are washed with 

 tap water and decolorized 1 to 10 minutes in acid alcohol (5 ml. of nitric acid, sp. gr. 

 1.42, to 95 ml. of 95% ethyl alcohol); washed again with water, and finally for 1 minute 

 with 95% ethyl alcohol. They are counterstained with 1% 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 dis- 



tilled water in a small test tube. 



2. Add an equal quantity of freshly filtered Ziehl's carbol fuchsin 



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 prepara- 



tion with one loopful of Dorner's nigrosin solution (p. 7). 



5. Smear as thinly as possible and do not dry too slowly. 



