148 Methods of Observing Bacteria 



results for staining many micro-organisms. It differs from 

 the Gram method in that anilin oil instead of alcohol is 

 used for decolorizing. To secure the most brilliant results 

 it is best first to stain the tissue with alum, borax, or 

 lithium carmin, and then 



1. Stain in Ehrlich's anilin-oil-water gentian violet, 



five to twenty minutes; 



2. Wash off excess with normal salt solution; 



3. Immerse in dilute iodin solution (iodin i, iodid of 



potassium 2, water 100) for one minute; 



4. Drain off the fluid and blot the section spread out 



upon the slide, with absorbent paper; 



5. Decolorize with a mixture of equal parts of anilin 



and xylol; 



6. Wash out the anilin with pure xylol; 



7. Mount in xylol balsam. 



Eosin and Methylene-blue (Mallory) make a beautiful 

 contrast tissue stain for routine work, and also demonstrates 

 the presence of most bacteria. The success of the method 

 seems to depend largely upon the quality of the reagents 

 used and a careful study of their effects. Hardening in 

 Zenker's fluid is highly recommended as a preliminary. The 

 details as given by Mallory are as follows: 



1. Stain paraffin sections in a 5 to 10 per cent, aqueous 



solution of eosin for five to twenty minutes or 

 longer; 



2. Wash in water to get rid of the excess of eosin; 



3. Stain in Unna's alkaline methylene-blue solution 



(methylene-blue i, carbonate of potassium i, water 

 100), diluted i : 10 with water, for one-half to one 

 hour, or use a stronger solution and stain for a few 

 minutes only; 



4. Wash in water. 



5. Differentiate and dehydrate in 95 per cent, alcohol, 



followed by absolute alcohol until the pink color 

 returns in the section; 



6. Clear with xylol; 



7. Mount in xylol balsam. 



The nuclei and micro-organisms will be colored blue, 

 the cytoplasm, etc., red. 



