110 STAINING OF BACTERIA 



Ribbert's Method 



1. Stain for several minutes in the following solution: Prepare a hot 

 saturated watery solution of dahlia, add to 100 c.c., 50 c.c. alcohol 

 (95 per cent.) and 12.5 c.c. glacial acetic acid. 



2. Wash in water, dry between filter paper, and mount in Canada 

 balsam. 



Welch's Method.- 



1. Prepare and fix cover-glass as usual, then cover the film with 

 glacial acetic acid for a few seconds. 



2. Blow off the glacial acetic acid and replace by anilin-water 

 gentian violet. This must be poured on several times to wash off all 

 the acetic acid. 



3. Wash in a 1 to 2 per cent, solution of chloride of sodium. Mount 

 in this fluid (not in Canada balsam) and examine. 



Staining of Spores. It has previously been stated that spores, in 

 consequence of the possession of a very tough, tenacious membrane, 

 cannot be stained by the ordinary methods, but require a special 

 technique. The following are some of the methods employed : 



1. Prepare and fix cover-glass, as usual. 



2. Float film on Ziehl's carbol-fuchsin solution contained in a 

 beaker. Place beaker over a small flame or on a water bath and keep 

 the staining solution boiling for twenty to thirty minutes. 



3. Wash in water. 



4. Decolorize in the following solution: Alcohol, 2 parts; 1 per 

 cent, acetic acid in water, 1 part. Keep on washing until no more 

 red stain is given off. The bacteria are now decolorized, except the 

 spores, which are stained red. 



5. Wash in water. 



6. Mount cover-glass on a slide in water and examine with a J- 

 inch dry lens to see whether spores are really stained properly. If 

 this is the case, counter-stain for a few minutes in a weak watery 

 solution of methylene blue. 



7. Wash in water, dry between filter paper, mount in Canada 

 balsam. Result of procedure spores red, remainder of bacilli blue 



Moeller's Method. 



1. Prepare and fix cover-glass as usual, then cover it for two 

 minutes with chloroform, which removes fatty matter. 



2. Wash in water. 



3. Pour on cover-glass a 5 per cent, solution of chromic acid for 

 1J to 2 minutes. 



4. Stain with watery fuchsin solution heated over a small flame for 

 one minute. 



5. Decolorize in 5 per cent, sulphuric acid for five seconds. 



6. Wash in water. 



7. Counter-stain in dilute watery methylene-blue solution for one- 

 half minute. 



8. Wash in water, dry between filter paper, mount in Canada 

 balsam. 



