48 GENERAL BACTERIOLOGY 



c. Simple stain for spores. 



1. Prepare film of B. subtilis. 



2. Fix by passing through flame 10 or 12 times instead of 3 times. 

 (This prevents the vegetative portion from taking the stain). 



3. Stain 2-5 minutes in hot carbol-fuchsin. 



4. Mount and examine. 



d. Double stain for spores (Hauser's method). 



1. Prepare a film of any of the above organisms (providing a 

 previous examination has shown that the spores are fully developed 

 and the mother-cells have not disintegrated). 



2. Fix, three times through the flame. 



3. Stain with hot (steaming) carbol-fuchsin for 5 minutes. 



4. Cautiously decolorize with 5 per cent, acetic acid until the 

 pink color is nearly removed from the film. 



5. Wash thoroughly in water. 



6. Dry (blot). 



7. Stain with Loeffler's methylen blue, 3 minutes. 



8. Mount and examine. The spores should appear crimson in 

 blue bacilli. 



KEFERENCES. A. 171 ; H. 98 ; M. & R. 106 ; McF. 154 ; P. 46 & 

 203 ; P. B. C. 15. 



EXERCISE 26. FLAGELLA STAIN (BUKGE). 



GENERAL DIRECTIONS. 



a. Make an agar streak of the organism to be stained. 



I). After 18 to 24 hours, by means of the platinum needle re- 

 move a portion of the growth (being careful to avoid the culture 

 medium) to a large drop of tap water on a perfectly clean cover- 

 glass (16). Allow this to stand 5 minutes rather than spread, as 

 there is less danger of breaking off the flagella. 



c. Spread carefully 2 or 3 loopfuls of this drop on each of sev- 

 eral clean cover-glasses and dry at room temperature. 



d. Fix by passing the cover-glass while it is held in the hand, 

 (not in the forceps, as over-heating will injure the preparation) 

 through the top of the flame. 



e. Flood the cover-glasses thus prepared with the following solu- 

 tion (mordant) : Liquor ferri sesquiMoridi diluted with distilled 

 water 1 : 20, 1 part ; saturated aqueous solution of tannic acid, 3 

 parts. This mixture improves with age but should be filtered before 

 using. Allow to act 1 minute. 



f. Wash in water and dry between filter paper. 



