166 PUBLIC HEALTH BACTERIOLOGY 



Granules. Diphtheria bacilli when stained show oval 

 bodies, which stain more deeply than the rest of the cell. 

 Loeffler's methylene-blue (page 161) shows them well, but 

 a contrast stain is often used, such as that of Neisser. 



Neisser's Method. Two solutions are used : Solution i : 

 methylene-blue I grm. + 20 c.c. alcohol (96 per cent) + 

 50 c.c. glacial acetic acid + 950 c.c. aq. Solution 2 : 

 Bismarck-brown 2 grm. dissolved in 1 litre of boiling 

 distilled water. Make a film, fix, stain with Solution 1 

 for 30 to 60 seconds. Wash, and pour on Solution 2, and 

 after 30 seconds wash off with water. Dry, and mount. 

 Bodies of the bacilli are brown, and the granules are blue. 



Paraffin- section Staining. Sections must first be 

 fixed on slides by one of two modes : 



1. Float section on warm water (under 40 C.J, insert 

 slide underneath, with a needle fix one corner, and withdraw 

 slide. Dry for 24 hours in incubator at 37 C. 



2. Place a drop of solution of egg-white (10 per cent in 

 aq.) on a slide, draw on section as before, and incubate at 

 37 C. for 30 minutes ; or, remove excess of moisture, heat 

 over small flame until paraffin melts, and then until 

 vapour arises. 



Staining. General Method. 



1. Preparation : Remove paraffin with xylol, and 



xylol with absolute alcohol. Wash in water 

 (unless alcoholic solution of stain is used). 



2. Staining : Use methylene-blue for 15 minutes ; 



carbol-thionin-blue (5 minutes), or aniline-oil- 

 gentian violet, carbol-fuchsin, etc. For over- 

 staining reduce with very weak acid for 5 to 30 

 seconds. This also decolorizes the tissues. 



3. Counter-staining : W T ash in water, stain with \ 



per cent eosin for 30 seconds, and wash in water. 



4. Dehydration and Clearing : Remove water with 



absolute alcohol (some organisms are decolorized 

 very easily at this stage, and hence treatment 

 must be rapid). Remove alcohol with xylol, 

 and mount in Canada balsam. 

 Weigert advises aniline oil, aniline-xylol, xylol, and 

 balsam. 



