178 VICTOR BtTRKE 



These experiments were discontinued because they did not 

 give promise of demonstrating a method by which we could 

 stain the colon-typhoid group of organisms differently from the 

 gonococcus like organisms. 



A MODIFIED GRAM STAIN 



The following method of making a Gram stain with aqueous 

 solutions of dyes and without the addition of mordants to the 

 stock solution of the primary stain gives better results than any 

 other method known to us. With this method satisfactory 

 differentiation between Gram positive and Gram negative organ- 

 isms can be obtained with many of the domestic gentian violet, 

 methyl violet and crystal violet dyes. With some of the better 

 dye products certain of the steps in the process can be omitted 

 and others modified but with the poorer dyes strict attention to 

 all of the details given is advisable. 



1. Air dry thinly spread film and fix with least amount of 

 heat necessary to kill the organisms and fix them to the shde (A) . 



2. Flood smear with a 1 per cent aqueous solution of the dye 

 to be used. Mix with the dye on the slide 3 to 8 drops of a 5 

 per cent solution of sodium bicarbonate, allow to stand two to 

 3 minutes (B). 



3. Flush off the excess stain with the iodine solution ' and cover 

 with fresh iodine solution and let stand one minute or longer (C). 



4. Wash in water as long as described and blot off all free 

 water until surface of film is practically free of water, but do 

 not allow the fihn to become dry (D). 



5. Decolorize with acetone or acetone and ether (1 part ether 

 to 1 to 3 parts acetone) until decolorizer flows from slide prac- 

 tically uncolored. This usually requires less than ten seconds 

 (E). 



6. Blot dry. The slide quickly dries without blotting (F). 



7. Counter stain for five to ten seconds or longer if desired 

 with a 2 per cent aqueous solution of Safranin O (G). 



8. Wash off excess stain bv short exposure to water, blot and 

 dry (H). 



'One gram iodine, 2 gnims potassium iodide, 100 cc. distilled water. 



