IV46-12 MANUAL OF METHODS FOR PURE CULTURE STUDY 



Note: If even backgrounds for exhibiting or photographing are re- 

 quired, especially in the case of slime-producing bacteria, the 

 following procedure is recommended: 



1. Make the suspension in 0.5 ml. nutrient broth or water. 



2. Add 1 ml. of 10% gelatin solution. 



3. Add 1 ml. of carbol fuchsin and stain as in (1) and (2) above. 



4. Wash out the colloids with warm tap water, with the help of 



centrifuge or sedimentation. 



5. Mix with nigrosin and proceed as above. 



Results: Spores, red; vegetative cells, unstained; background, gray. 



DORNER METHOD — SNYDER MODIFICATION 



Snyder (1934) 

 Staining schedule: 



1. Prepare a dried smear on a slide and cover with a small piece of 



blotting paper. 



2. Saturate blotting paper with freshly filtered Ziehl's carbol 



fuchsin (p 5). 



3. Allow to steam 5-10 min., keeping paper moist by adding more 



staining fluid. 



4. For neat preparations, decolorize instantaneously with 95% 



ethyl alcohol (but omit this step if the organisms do not hold 

 color well.) 



5. Wash with tap water. 



6. Apply a drop of saturated acjueous nigrosin (or Dorner's fluid) 



and spread evenly. 



7. Allow slide to dry quickly with gentle heat, without prior 



washing. 

 Results: Same as with original method; but this modification proves 

 applicable to some bacteria (e.g. Bacillus subtilis) that are difficult 

 to stain by Dorner's technic. 



conklin's modification of wirtz method 

 Wirtz (1908); Conklin (1934) 



Staining schedule: 



1. Make smears as usual and fix by heat. 



2. Flood slide with 5% aqueous malachite green, and steam for 10 



minutes, keeping slide flooded by addition of fresh staining 

 fluid. 



3. Wash 30 sec. in running water. 



4. Counterstain 1 min. with 5% aqueous mercurochrome. 



5. Wash in running water. 



6. Blot dry and examine. 



Results: Spores, green; rest of cell, red. Trouble is sometimes 

 experienced with the green fading after the slides have stood a few 

 days. Apparently this is due to an alkaline reaction and can be 

 prevented by treating the slides in acid before making the smears. 

 (The alkalinity may be due to an invisible film of soap or washing 

 powder.) 



