STAINING METHODS 1V46-9 



BURKE AND KOPELOFF-BEERMAN MODIFICATIONS 



ALKALINE GENTIAN VIOLET 



Solution A Solution B 



Gentian or crystal violet- 1 g. NaHCOs 1 g. 



Distilled water 100 ml. Distilled water 20 ml. 



BURKe's IODINE SOLUTION 



Iodine, 1 g.; KI, 2 g.; distilled water, 100 ml. 



KOPELOFF AND BEERMAN's IODINE SOLUTION 



Iodine 2 g. 



Normal NaOH (40.01 g. per liter) 10 ml 



After the iodine is dissolved, make up to 100 ml. with distilled water. 



burke's counterstain 

 Safranin O (85% dye content), 2 g.; distilled water, 100 ml. 



KOPELOFF AND BEERMAN's COUNTERSTAIN 



Basic fuchsin (90% dye content), 0.1 g.; distilled water, 100 ml. 



Staining schedule: 



1. Dry thinly spread films in the air without heat. 



2. Flood with Solution A; mix on the slide with 2-3 drops (or 



more, depending on size of flooded area) of Solution B, and 

 allow to stand 2-3 min. 



Kopeloff and Beerman mix the two solutions in advance, 

 1.5 ml. Sol. A to 0.4 ml. Sol. B, and allow to stay on slide 

 5 min. or more. 



3. Kinse with either of the above iodine solutions. (The Com- 



mittee indicates no preference between the two; some work- 

 ers prefer one, some the other.) 



4. Cover with fresh iodine solution and let stand 2 min. or longer. 



5. Rinse with tap water; then blot water from surface of smear, 



ivithout drying. (Kopeloff and Beerman omit the washing.) 

 The amomit of drying is important in this step. One must 

 get rid of all free water, but not allow the cells to dry. 



6. Follow the blotting very quickly with decolorization in ether 



and acetone (1 vol. ether to 1-3 vol. acetone), adding to 

 the slide drop by drop until practically no color comes off 

 in the drippings (usually less than 10 sec.) In this step 

 the speed of decolorization can be varied by varying the 

 ratio of ether to acetone; the more acetone the more rapid 

 the process. It is sometimes desirable to slow down the 

 process by using a ratio of 1:1. 



7. Dry in the air. 



8. Counterstain 5-10 sec. in one of the above given counter- 



stains. Burke's (i.e. safranin) is preferred. The Kopeloff 

 and Beerman counterstain is too powerful to be used when 

 the shorter staining time recommended by Burke is followed. 



'The authors specify either crystal violet or methyl violet 6B. Probably any of the 

 gentian violets now sold under the Commission certification are satisfactory; i. e. either 

 crystal violet or one of the bluer grades of methyl violet (e. g., methyl violet 2B). 



