STAINING METHODS 19 



To this add 3-4 drops of Tergitol No. 7 (a Carbide & Chemical Corp. 



product), and stir thoroughly. 



Preparation of methylene blue solution: 

 Add 30 ml of a stock 1.5 per cent alcoholic methylene blue solution to 



100 ml of 0.01 per cent aqueous KOH. 



Staining schedule: 



1. Stain 5-10 min, without heating, in the above basic fuchsin solution. 



2. Rinse in warm water. 



3. Agitate for 30-60 sec in acid alcohol (3 ml of cone HCl in 97 ml ethyl 

 alcohol). 



4. Rinse with cold water. 



5. Counterstain 3-5 min in the above methylene blue solution. 



Note : The only essential difference between this method and Darrow's 

 is that the latter author states that equally good results were obtained 

 with a weaker (0.3 per cent) fuchsin solution in phenol. In the hands of 

 one the committee's collaborators, however, Gross' 1 per cent solution 

 has proved more satisfactory. 



ACID-FAST STAINING—ALTERNATE PROCEDURES 



Fluorescence Method 

 Richards and Miller {1941) 



Although this method is not of special importance in pure culture work, special 

 mention should be made of it because of the amount of attention now given to it in 

 diagnostic work. Its real advantage is that it can be used with relatively low magnifi- 

 cation, and the large fields that can be examined assure positive diagnoses in cases 

 where the numbers of tubercle organisms are few. 



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 20 X ocular; illumination should \)Q a low-voltage, high-amperage microscope 

 lamp, supplied with a blue (ultraviolet-transmitting) filter, a complementary yel- 

 low 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 2, which is now quite widely used, employs carbol gentian violet 

 of essentially the formula given on page 13 for carbol fuchsin except that in the place 

 of basic fuchsin the author calls for methyl violet BN, Preparations are stained cold 



