METHODS OF PURE CULTURE STUDY 521 



method (Hucker, 1922) and the third that of Atkins (1920), 

 both of which are highly to be recommended on account of the 

 keeping quaUty of the solutions, but have not yet been com- 

 pared with the older methods sufficiently so that the committee 

 can be sure that they can replace them. 



Method I 



Ehrlich's anilin gentian violet' 



Gentian violet (saturated alcoholic solution) 6 cc. 



Alcohol 5 cc. 



Anilin water (98 cc. water to 2 cc. anilin oil) SO cc. 



Lugol's iodine solution 



Iodine 1 gram 



Potassium iodide 'i grams 



Water 300 cc. 



Method S (H-ucker's) 



Gentian violet (saturated alcoholic solution) 1 part 



.\mmonium oxalate (1 per cent aqueous solution) 4 parts 



Lugol's iodine solution (as usual) 



Method 3 (Atkins') 



Gentian violet (saturated alcoholic solution) 1 part 



Anilin sulphate (1 per cent aqueous solution) 3 parts 



.\tkins' iodine solution 



Iodine 2 grams 



Normal NaOH 10 cc. 



Water 90 cc. 



Technic. Stain one minute; treat in iodine solution one minute; 

 decolorize one minute with methods 1 and 2, five minutes with method 

 3; counterstain about ten seconds. For the counterstain use 1:10 

 safranin (i.e., 1 part saturated alcohol solution to 10 parts water) 

 1:10 cosine or 1:10 bismark brown or pyronin. With method 1 do 

 not wash between the different procedures; merely drain thoroughly 

 with the other two methods wash between each step. 



' The committee has found crystal violet to be a very satisfactory substitute 

 for gentian violet in all these formulae. It is of much more constant composition, 

 and therefore to be preferred to the very variable gentian violet. See the fol- 

 lowing paper, p. 533. 



JOUBNAL or BACTBBIOLOOT, TOL. Til, NO. 5 



