120 STAINING OF BACTERIA 



(alcohol 70 per cent.- 100 c.c. HC1 5 drops), then in several 

 changes of pure alcohol, so that every trace of acid is removed, and 

 finally they are embedded in celloidin or paraffin. 



Mallory's Eosin-methylene Blue Stain. When staining for bac- 

 teria in tissues it is frequently necessary to employ different stains 

 according to the species of bacteria to be demonstrated. These 

 stains will, therefore, be given in the chapters on these special bac- 

 teria. However, there is one method of staining bacteria known as 

 Mallory's eosin-methylene blue stain, which has a wide range of use- 

 fulness and which furnishes excellent results. To get the best results 

 tissues should first be fixed in Zenker's solution. It is necessary to 

 remove from sections so fixed the precipitated mercury sulphite. 

 The steps of the method are the following: 



1. After removal of the paraffin and xylol from the sections, place 

 them into Gram's decolorizing fluid (iodine 1 part, iodide of potash 

 2 parts, water 300 parts) for twenty minutes. 



2. Wash out the iodine solution in 95 per cent, alcohol for ten 

 minutes. 



3. Wash in water and stain sections for twenty to thirty minutes 

 in a 10 per cent, watery eosin solution. 



4. Wash rapidly in water to get rid of the excess of eosin. 



5. Stain in Unna's alkaline methylene-blue solution diluted with 

 four to five times its bulk of distilled water for ten to fifteen minutes. 

 Formula for Unna's alkaline methylene-blue solution: 



Methylene blue (Koch's) 1 gram 



Carbonate of potassium 1 gram 



Distilled water 100 c.c. 



This solution keeps several months, but it then loses in staining 

 power, because much of the methylene blue is oxidized into methyl 

 violet and methylene red. 



6. Wash in water. 



7. Wash, for the purpose of decolorizing, in 95 per cent, alcohol, 

 keeping the slide constantly in motion, so that the decolorizing will 

 go on uniformly. When the pink color of the eosin has returned, 

 dehydrate in absolute alcohol and clear in xylol. Then, without 

 mounting in Canada balsam, look at the sections with the low power 

 of the microscope. If the nuclei stand out well differentiated in blue 

 from the eosin-stained protoplasm the section has been decolorized 

 enough. If there is still too much blue present wash in 95 per cent, 

 alcohol again until the differentiation is sufficient. 



8. Finally dry and mount in Canada balsam in the usual manner. 

 Gram's Staining Method for Paraffin Sections. 1. Stain section 



in warm anilin-water gentian-violet solution for twenty minutes. 



2. Wash in normal salt solution. 



3. Decolorize for one minute in Gram's decolorizing fluid (iodin 

 1 part, iodide of potash 2 parts, water 300 parts). 



