STAINING METHODS 17 



Kopeloff and Beerman^s Counter stain 



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 of 

 solution A to 0.4 ml of solution B, and allow to stay on slide 5 min or 

 more. 



3. Rinse with either of the above iodine solutions. (The committee 

 indicates no preference between the two; some workers 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, without 

 drying. (Kopeloff and Beerman omit the washing.) The amount 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 of ether to 1-3 vol of 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 counterstains. 

 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. 



9. Wash in tap water. 

 10. Dry, and examine. 



Results: Gram-positive organisms, blue; gram-negative organisms, red 

 This technic is claimed to have the advantage of not giving false positives 

 due to vacuolar bodies that resist decolorization by other gram-staining 

 procedures. 



Interpretation of the Gram Stain 



A word of caution is necessary as to the interpretation of the gram 

 stain. The test is often regarded with unjustified finahty because organ- 

 isms are generally described as being either gram-positive or gram-nega- 

 tive. Many organisms, however, actually are gram-variable. Hence, 



