206 STUDY OF INDIVIDUAL BACTERIA-STAINING 



It is also of great value in staining Gram-positive bac- 

 teria in tissues, but the sections should be stained about five 

 minutes in the anilin gentian violet and be left about two 

 minutes in the Gram's solution. Sections are to be counter- 

 stained in Bismarck brown, dilute eosin or safranin solutions 

 and cleared in oil of bergamot, lavender or origanum and 

 not in clove oil or carbol-xylol, as these latter dissolve out 

 the dye from the bacteria. 



Staining of Spores in the Rod.— Prepare the films as usual. 

 Cover with carbol-fuchsin, using plenty of stain so that it 

 will not dry on the slide; heat until vapor arises, not to boil- 

 ing; cool until the stain becomes cloudy and heat again 

 until the stain clears, and repeat once more; wash in tap 

 water and then wash in 1 per cent H2SO4 three times, 

 dropping on plenty of acid, tilting and running this over the 

 slide three times and then pour off and use fresh acid and 

 repeat this once. Wash thoroughly in distilled water, then 

 stain with Loffler's blue one to three minutes. Wash, dry 

 and examine. The spores should be bright red in a blue 

 rod. 



This method will give good results if care is taken to 

 secure cultures of the right age. If the culture is too old 

 the spores will all be free outside the rods, while if too young 

 they will decolorize with the acid. For Bacillus suhtilis and 

 Bacillus anthracis, cultures on agar slants forty-eight hours 

 in the 37° incubator are just right. For the spores of Clos- 

 tridium tetani the culture should be three days old, but may 

 be as old as a week. 



Staining of "Acid-fast" 'Bz.ciWi.— Mycobacterium tubercu- 

 losis, Mycobacterium of Johies disease, ''grass'' and "butter 

 bacilli,'^ Mycobacterium leproe, Mycobacterium smegmatis. 



Gabbefs method: 



1. Prepare the film as usual. 



2. Stain with carbol-fuchsin as given above for spores. 



3. Wash with tap water. 



4. Decolorize and stain at the same time with Gabbet's 



blue, two or three minutes. 



5. Wash, dry and examine. 



