Gram-stained Bacterial Film 203 



excess iodine and then placed into absolute alcohol until no more color 

 comes away; unless the film is very thick, this will appear to decolorize it 

 completely. It is passed from alcohol to water, which instantly stops 

 differentiation, and then dried. Varying types of bacteria require varying 

 periods of differentiation, but it is best for the beginner to use absolute 

 alcohol until no more color comes away rather than to endeavor to control 

 the differentiation under the microscope. 



Though this is a Gram preparation by the original technique, it is cus- 

 tomary nowadays to provide a counterstain of a contrasting color to bring 

 clearly into evidence any Gram-negative organisms that may be mixed 

 with Gram positive. A 1 per cent solution of safranin is widely employed, 

 and in this case, the second red contrasting stain is allowed to act from 

 5 to 10 sec, washed off with water, and the slide then dried and examined 

 under an oil-immersion objective. 



SUMMARY 



1. Follow steps 1 through 3 of Example 7 but allow stain to act for 2 

 min. Then wash it off under tap. 



2. Treat stained smear with Gram's iodine for 1 min. Rinse under tap 

 and soak in absolute alcohol until no more color comes away. 



3. If desired, counterstain 5 to 10 sec in 1% safranin and wash it off 

 with water before drying. 



