120 A MANUAL OF BACTERIOLOGY 



Absolute alcohol 2 minutes 



Counter-stain 30 seconds 



(10 c.c. of saturated alcoholic sal'ranin and 

 90 c.c. of water.) 



METHOD 2. 



Gentian violet solution . . . . .3 minutes 



(Anilin oil 3 c.c., absolute alcohol 7 c.c., 

 water 90 c.c. Shake and filter through moist 

 filter paper. Add 2 grm. of gentian violet 

 and allow the mixture to stand for 24 hours. ) 

 Iodine solution (Lugol's, as above) . . .2 minutes- 

 Absolute alcohol . . . . . .2 minutes. 



Counter-stain : 



Either Fuchsin (one part saturated alcoholic solu- 

 tion to nine parts of water) . . . .30 seconds 



or 



Bismarck brown (2 per cent, dissolved in hot 



water and filtered) . . . . .2 minutes 



There is not much to choose between these methods : the first 

 one is shorter and the gentian violet solution will keep for 3-4 

 months. Only young cultures should be used and the films 

 made on cover-glasses with distilled water and, in the portions 

 examined, organisms should be only one layer thick. The gentian 

 violet and iodine solutions should each be removed by merely 

 draining and blotting, before applying the next solution. Cover- 

 glasses should be treated in watch-glasses with the alcohol and 

 kept in constant agitation and be transferred without washing 

 to the counter-stain. 



Another method is given under the " Gonococcus " (clinical 

 diagnosis). 



Weigert's modification of Gram's method. In this process the 

 sections, whether frozen or paraffin ones, should be manipulated 

 on the slide. They are stained with anilin gentian violet or with 

 the solution of Method 2 above and treated with Weigert's iodine 

 solution (iodine 4-5 per cent., potassium iodide 6 per cent.), 

 The iodine is then removed with filter-paper and the sections are 

 flooded with anilin oil two or three times. This removes the 

 colour and dehydrates. The anilin oil is removed by flooding 

 two or three times with xylol, 



