IV46-6 MANUAL OF METHODS FOR PURE CULTURE STUDY 



AMMONIUM OXALATE CRYSTAL VIOLET (hUCKER's) 



Solution A Solution B 



Crystal vioiet (90% dye content) 2 g. Ammonium oxalate 0-8 g. 



Etiiyl alcohol (95%) 20 ml. Distilled water 80 ml. 



Mix solutions A and B. 



CRYSTAL VIOLET IN DILUTE ALCOHOL 



Crystal violet (90% dye content) 2 g. 



Ethyl alcohol (95%) 20 ml. 



Distilled water 80 ml. 



LOEFFLEr's alkaline METHYLENE BLUE 



OBIGINAL STATEMENT OF FORMULA EMENDED STATEMENT 



Solution A 

 Cone. sol. methylene blue in al- Methylene blue (90% dye con- 



cohol 30 ml. tent) 0.3 g. 



Sol. KOH in distilled water Ethyl alcohol (95%) 30 ml. 



(1 :10,000) 100 ml. Solution B 



Dilute KOH (0.01 % by weight) 100 ml. 

 Mix Solutions A and B. 



METHYLENE BLUE IN DILUTE ALCOHOL 



Methylene blue (90% dye content) 0.3 g. 



Ethyl alcohol (95%,) 30 ml. 



Distilled water 100 ml. 



CARBOL ROSE BENGAL 



Rose Bengal (80% dye content) 1 g. 



Phenol (5% aqueous solution) 100 ml. 



CaCla . 0.01-0.03 g. 



(The amount of CaCl2 added determines the intensity of staining.) 



Staining schedule: Follow the general procedure given under "Pre- 

 paration of Smears", p. 3-4 above, allowing 5-60 seconds for ap- 

 plication of the stain. Overstaining rarely occurs except with 

 carbol fuchsin; understaining does not have to be feared except 

 with rose Bengal. 



Results: The results depend on which of the above staining fluids is 

 selected. They are listed in the order of intensity of action; 

 i.e. carbol fuchsin gives the most intense stain, and is not indicated 

 when selective staining is desired or when much debris is present 

 on the slide. The crystal violet solutions are very good for 

 routine purposes. The methylene blue solutions are much more 

 selective, with special affinity for metachromatic granules. The 

 rose Bengal solution is much less commonly used; it is specially 

 valuable when mucus or colloidal organic material is present, as 

 such material is not ordinarily stained by it. 



General Bacterial Stains — ^Alternate Procedures 



Kinyoun's Carbol Fuchsin 



Basic fuchsin (dye content not specified; probably 90%) 4 g. 



Phenol crystals 8 g. 



Ethyl alcohol (95%o) 20 ml. 



Distilled water • 100 ml. 



This formula is preferred in some quarters to the Ziehl carbol fuchsin. It is attri- 

 buted to Kinyoun, but the reference to its original publication has not been located. 



