IV46-8 MANUAL OF METHODS FOR PURE CULTURE STUDY 



BENIANS' CONGO RED 



Congo red (80% dye content) 2 g. 



Distilled water 100 ml. 



Staining schedule: 



1. Place a drop of the above staining fluid on a slide. 



2. Mix culture with the drop and spread out into a rather thick film. 



3. After film has dried, wash with 1% HCl. 



4. Dry, either in the air or by blotting. 



Results: Cells unstained in a blue background. Good results are 

 not to be expected from broth cultures or from cultures in salt 

 solutions unless the cells are first removed by centrifuging. 



The Gram Stain — Recommended Procedures 



There are numerous modifications of the Gram stain, many of 

 which have been listed by Hucker and Conn (1923, 1927). The 

 two modifications given below have proved especially useful to the 

 Committee. The Hucker modification is valuable for staining smears 

 of pure cultures, that of Kopelofl^ and Beerman for preparations of 

 body discharges such as gonorrhoeal pus, also for pure cultures of 

 strongly acid-forming organisms. The latter is itself a variation 

 of the modification by Burke (1921). 



hucker modification 



AMMONIUM OXALATE CRYSTAL, VIOLET 



(See p. IV46-6) 



gram's MODIFICATION OF LUGOl's SOLUTION 



Iodine 1 g. 



KI 2g. 



Distilled water 300 ml. 



COUNTERSTAIN 



Safranin O (2.5% solution in 95% ethyl alcohol) 10 ml. 



Distilled water 100 ml. 



Staining schedule: 



1. Stain smears 1 min. with ammonium oxalate crystal violet. 



This formula has sometimes been found to give too intense 

 staining, so that certain Gram-negative organisms (e.g. the 

 gonococcus) do not properly decolorize. If this trouble is 

 encountered, it may be avoided by using less crystal violet. 



2. Wash in tap water. 



3. Immerse 1 min. in iodine solution. 



4. Wash in tap water and blot dry. 



5. Decolorize 30 sec. with gentle agitation, in 95% ethyl alcohol. 



Blot dry. 



6. Counterstain 10 sec. in the above safranin solution. 



7. Wash in tap water, 



8. Dry and examine. 



Results: Gram-positive organisms, blue; Gram-negative organisms, 

 red. 



