DIPHTHERIA STAINING 43 



To stain: Fix the preparation. Pour on the dilute acetic acid methylene blue 

 solution and allow to act from thirty to sixty seconds. Wash. Then pour on the 

 Bismark-brown solution, and after thirty seconds wash off with water. Dry and 

 mount. The bodies of the bacilli are brown with dark blue dots at either end. 



Neisser recommends only five seconds as the time of application of each solution. 

 He also recommends that the culture be only nine to eighteen hours old and that the 

 temperature of the incubator shall not exceed 36C. Incubation at 37C. gives 

 satisfactory results. 



Ponder's Stain for Diphtheria Bacilli 



Toluidin blue (Grubler) o. 02 gram. 



Glacial acetic acid i c.c. 



Absolute alcohol 2 c.c. 



Distilled water to 100 c.c. 



The film is made on a cover-glass and fixed in the usual way. A small quantity 

 of the stain is spread on the film and the cover-glass is turned over and mounted as 

 a hanging-drop preparation. The metachromatic granules of the diphtheria bacilli 

 stain with striking intensity. With diphtheroids, the more intense staining sharply 

 differentiates from ordinary cocci and bacilli, which show in the preparation only as 

 faint light blue bodies. It is a most excellent stain for bringing out the ascopores of 

 yeasts. In my opinion the stain is more valuable than the Neisser method. 



Capsule Staining. The best method for studying bacteria, as to 

 presence of capsules, is in the hanging drop, with the greater part of the 

 light shut off by the diaphragm. 



In material where capsules are well developed, as in pneumonic sputum, the 

 Gram method of staining brings out the capsule perfectly. This is of diagnostic 

 value, as the more or less nonpathogenic pneumococci common about the mouth 

 do not seem to show a capsule when stained in this way. The India ink method of 

 staining gives good results for capsules. 



The most beautiful method of staining capsules is the latest one pro- 

 posed by Muir. 



1. Prepare thin film, dry and stain in carbol fuchsin one-half minute; the prepa- 

 ration being gently heated (steamed). 



2. Wash slightly in 95% alcohol, then wash well afterward in water. 



3. Flood preparation in mordant for five to ten seconds. 



Mordant. Sat. aqueous sol. mercuric chloride 2 parts. 



Tannic acid (20% aqueous sol.) 2 parts. 



Sat. aqueous sol. potash alum 5 parts. 



4. Wash in water thoroughly. 



5. Treat with 95% alcohol for one minute. (The preparation should have a pale 

 red color.) 



6. Wash well in water. 



7. Counterstain with methylene blue one-half minute. 



