146 SPORE STAINING 



All these facts can be observed under the microscope in a hanging drop 

 culture of the anthrax bacillus (p. 134). When it is desired merely to deter- 

 mine the presence of spores in bacteria an ordinary film is made on a slide 

 as described on pp. 140 and 141. 



2. The staining of spores. 



When spore-bearing organisms are stained with the basic aniline dyes the 

 spores do not take up the dye and appear as unstained spots in the stained 

 bacilli. To stain the spore it is necessary therefore to apply special methods 

 which have been designed to overcome its resistance to staining reagents. 



(i) Simple staining. 



This method stains both the bacilli and the spores. 



A. Method recommended. 1. Prepare a cover-glass film of the culture to 

 be examined and dry it. 



2. Pass the cover-glass, film side uppermost, ten times through the heating 

 flame of the Bunsen, but sufficiently quickly to prevent the preparation being 

 scorched. 



3. Stain with carbol-violet for 15 to 30 minutes. 



4. Wash, dry, mount in balsam. Examine. The bacteria and the spores 

 are both stained violet. 



B. Chromic acid method. 1. Make a film on a cover-glass and dry it. 



2. Drop a large drop of a 1 in 20 aqueous solution of chromic acid on the 

 film, and leave it for 4 or 5 minutes. 



3. Wash in water. 



4. Stain in carbol-violet for 15 minutes to half an hour. 



5. Wash. Mount. Examine. 



(ii) Double staining. 



The object of double staining is to differentiate the spore from the bacillus 

 by staining the bacillus one colour and the spore a different colour. 



Principle of the method. Spores stain with difficulty, but once stained 

 they retain the dye with more tenacity than the bacillary protoplasm, so 

 that decolourizing agents will decolourize the latter before they take the 

 stain out of the spores. 



A. Method recommended. 1. Prepare a cover-glass film, dry and fix it by 

 passing it rapidly through the flame two or three times. 



2. Drop a large drop of carbol-fuchsin on the film and warm it over a 

 small flame until steam just begins to rise, then keep the solution warm for 

 4 or 5 minutes by moving it about over the flame. 1 Both the bacilli and 

 the spores are now stained an intense red. 



3. Wash in water. 



4. Decolourize for a few seconds in a solution of nitric acid : 



Pure nitric acid, - 1 part. 



Distilled water, - 3 parts. 



The bacilli should be decolourized while the spores are still stained red. 



5. Wash well in water. 



6. Counterstain with a drop of diluted alcoholic solution of methylene 

 blue for 30 to 60 seconds. The decolourized bacilli take up the blue stain. 



7. Wash. Dry. Mount in balsam. 



The bacilli are stained blue ; the spores red. 



[ x This may be done on a warm stage (p. 141, fig. 127) taking care to select a place where 

 the metal is not too hot.] 



