104 MICKOSCOPIC METHODS 



2. Without washing in water, now treat the section or film with 

 repeated doses of Gram's solution till its colour becomes a purplish 

 black, and allow the solution to act for 1 minute. 



3. Again without washing with water, decolorise with absolute alcohol 

 or methylated spirit till the colour has almost entirely disappeared, the 

 tissues having only a faint violet tint. The period of time for which the 

 alcohol is allowed to act varies in different laboratories. The best period 

 is probably about three minutes. 



4. For sections dehydrate completely, clear with xylol, and mount. 

 In the case of film preparations of Gram -positive organisms, the specimen 

 is simply washed in water', dried, and mounted. With films of 

 organisms, whose reaction towards the Gram stain is unknown, a contrast 

 stain (vide infra) should be used. 



In stage (3) the process of decolorisation is more satisfactoi fly per- 

 formed by using clove oil after sufficient dehydration with spirit, the 

 clove oil being afterwards removed by xylol. As elove oil is a powerful 

 decoloriser care is necessary in fts use. 



As a contrast stain for the tissues, carmalum or lithia carmine is used 

 before staining with gentian-violet (1), As a contrast stain for bacteria 

 which are decolorised by Gram's method, carbol-fuchsin diluted with 

 twenty volumes of water or a, saturated watery solution of Bismarck- 

 brown may be used before stage (4) ; the former should not be applied 

 for longer than a few seconds. 



The following modifications of Gram's method may be given : — 



1. Weigert's Modification. — The contrast staining of the tissues and 

 stages (1) and (2) are performed as above. 



(3) After using the iodine solution the preparation is dried byblotting 

 and then decolorised by aniline-xylol (aniline oil 2, xylol 1). 



(4) Wash well in xylol, and mount in xylol balsam. Film preparations 

 after being washed in xylol may be dried, and thereafter dilute carbol- 

 fuchsin may be used to stain bacteria which have been decolorised. 



This modification probably gives the most uniformly successful Tesults 

 in the case of sections, but decolorisation by alcohol is preferable in the 

 case of films of pus, etc. 



2. Nicolle's Modification. — Carbol-gentian-violet is used as the stain. 

 Treatment with iodine is carried out as above, and decolorisation is 

 effected with a mixture of acetone (1 part) and alcohol (2 parts), or by 

 the other methods mentioned above. 



3. Jensen's Modification. — For this there are required : (a) a 0'5 per cent, 

 solution of methyl -violet (tj B) in water ; (b) a solution of iodine, 1 gramme ; 

 potassium iodide, 2 grammes ; water, 100 c.c. ; (c) a solution of neutral-red, 

 1 gramme in 1000 c.c. water, to which are added 2 c.c. of 1 per cent, 

 acetic acid. Thin films are fixed by heat and allowed to cool ; treat these 

 with the methyl-violet for ^-J minute ; wash the stain oft" with the iodine 

 solution, and allow this to act for \-\ minute ; wash off with absolute 

 alcohol, and treat with fresh alcohol till the necessary decolorisation is 

 complete ; wash off the alcohol with the neutral-red, and allow the 

 counter-stain to act for J-£ minute ; wash with water ; dry with filter 

 paper, and mount. 



There is great variability in the avidity with which organ- 

 isms stained by Gram retain the dye when washed with alcohol, 



