ZOOLOGY AND BOTANY, MICKOSCOPY, ETC. 431 



degrees. Heat very slowly to 95° C, keeping the mixture at this 

 tem])erature for about an hour or longer ; filter through cotton-wool and 

 muslin. Divide the tubes and autoclave at 115° C. for twenty minutes. 

 This egg fluid may be added to ordinary nutrient broth in the 

 proportions of about 1 to 5. The egsr fluid must be added to the 

 broth when both the fluids are cool. The egg fluid may be added to 

 agar at 50° C. All the sugar media should be made up with nutrient 

 broth instead of pepton water. The anaerobes do not grow sufficiently 

 well upon pepton water. 



f4) staining- and Injecting. 



Staining of Bacterial Capsules.* — R. Muir claims that by this 

 method the capsules of bacteria can be stained differentially, while a 

 Gram-positive reaction is also obtained. A thin film of the material is 

 made on a cover-glass, and after drying in the air is fixed for one 

 minute in a saturated watery solution of mercuric chloride. The 

 preparation is then washed in water and in methylated spirit. With 

 wet films the cover-slip is placed film-side downwards in 10 p.c. formalin 

 for two to five minutes. It is then gently washed with water and 

 methylated spirit. The film is covered with freshly prepared Gram- 

 stain of the following composition : Saturated alcoholic gentian violet, 

 1 part : 5 p.c. watery carbolic acid, 5 parts. The preparation is heated 

 over a Bunsen for a few minutes ; when cool, the stain is washed off with 

 Gram's solution, and a little fresh iodine solution is added. After two 

 or three minutes the iodine is washed off with methylated spirit and the 

 washing with spirit repeated. A few drops of clove oil are then placed 

 on the film and the warming repeated. Wash with spirit and then with 

 water. Filter on the film a few drops of solution containing one part 

 each of a saturated watery solution of mercuric chloride, a saturated 

 watery solution of potash-alum, and a 20 p.c. solution of tannic acid. 

 This is allowed to act for five minutes ; wash in w-ater and counter-stain 

 for one or two minutes with a saturated watery solution of eosin. 

 Wash in water. Filter on a few drops of saturated watery solution of 

 potash-alum and allow to act for a minute. Then wash, dry and mount, 

 or, in the case of wet fixed films, dehydrate, clear in benzol and mount. 



For sections, small pieces of tissue should be fixed in 5 to 10 p.c. 

 formalin for two or three days, and after a few minutes' washing should 

 be transferred to methylated spirit for two or three days to complete 

 the hardening. For removal of the precipitate, which forms in the 

 material thus fixed, place the pieces in a solution containing one part 

 of 1 p.c. aqueous potash solution and twenty parts of 80 p.c. alcohol. 

 The precipitate takes about a week to become removed. Wash thoroughly 

 for several hours in running water, replace in methylated spirit, and 

 keep till required. Embed the tissues in the usual way. The method 

 of staining sections is the same as that described for films, except that 

 the specimen may not require heating after the clove oil is placed on it, 

 and in any event heating need not be prolonged beyond a few seconds 



* Journ. Path, and Bact., xx. (1916) pp. 257-9. 



