50 LABORATORY MANUAL OF MICROBIOLOGY 



Ordinary India ink cannot be used because of the bacteria which 

 it contains. 



Spore Stain (Dorner Modified) 



1. Spread a loopful of culture, suspension or liquid culture, 

 on a clean glass slide ; dry and fix by heat. 



2. Cover with Ziehl's carbol fuchsin. Steam over water 

 bath for 10 to 20 minutes. Do not allow fuchsin to dry on smear. 

 Add more fuchsin if needed. Wash in water. 



3. Spread thinly over the smear a loopful of saturated aqueous 

 nigrosin solution. Spread either with wire loop or with a glass 

 slide as for blood smears. If the nigrosin is not well spread the 

 first time it may be washed off with water and more nigrosin 

 applied. 



4. Dry without heating and examine in oil, or mount in Canada 

 balsam. 



Spore Stain (Lagerberg's Modified) 



1. Make film, fix by heat, and cover with small amount of 

 5 per cent chromic acid. 



2. After 30 seconds, add about twice as much concentrated 

 ammonia as there is chromic acid on the slide. Allow to act 

 about 2 minutes. 



3. Rinse with tap water and steam with carbol fuchsin for 2 

 or 3 minutes. 



4. Rinse and destain with 1 per cent sulphuric acid for 15 to 

 30 seconds. 



5. Rinse again and flood slide with the tap water. 



6. Add to this a few drops of Loeffler's methylene blue and 

 allow to stain for 10 to 30 seconds. 



7. Rinse, blot, dry, and examine. 



Nigrosin for Negative Mounts 



1. Place a loopful of culture on a clean glass sfide, spread, and 

 allow it to air dry. 



2. Spread thinly over the smear a loopful of saturated aqueous 

 nigrosin solution. Spread either with the wire loop or with a 

 glass sHde (as for blood smears). Dry and examine in oil, or 

 mount in Canada balsam. 



