204 MICROSCOPIC AND CULTURAL STUDY OF BACTERIA 
The above solutions are unstable, but retain their tinctorial value 
for two or three days if they are kept stoppered. 
Solution Bi— Distilled water 300 cc. 
Potassium iodide 2 gm. 
Iodine crystals 1 gm. 
Solution C — Bismarck brown, saturated aqueous solution ... 10 cc. 
Distilled water 90 cc. 
Procedure. — (a) Prepare and fix film of bacteria in the usual manner. 
(h) Flood with anilin-oil gentian violet (or carbolic gentian violet) 
and stain five minutes. 
(c) Pour off excess of stain and flood with iodine solution. 
(d) Decolorize with 96 per cent alcohol until no more stain can be 
removed. 
(e) Wash thoroughly in water. 
(/) Counterstain with Bismarck brown- for two minutes. 
(g) Wash in water, dry and mount. 
Bacteria which retain the initial stain — Gram-positive bacteria— 
are colored dark purple or blue; those which fail to retain the initial 
stain— Gram-negative bacteria— are brown, or bright pink if fuchsin 
is used as a counterstain. 
Alternative Differential Stain of Jensen.^— Jensen has show^n that 
the addition of mordants, as anilin oil, carbolic acid or formalin, to 
the pararoseanilin dyes is unnecessary. Simple aqueous solutions of 
the dyes are sufficient. These solutions are stable for long periods. 
It is found advantageous to use stronger iodine solutions, however, 
and to decolorize with 98 to 100 per cent ethyl alcohol. Neutral red 
is an excellent counterstain. 
The staining reagents: 
1. 0.5 per cent aqueous solution of methyl violet (6B). 
2. Strong iodine solution. Iodine, 1 part; potassium iodide, 2 
parts; distilled water, 100 parts. 
3. Neutral red solution: 
Neutral red 1 part 
Distilled water 1000 parts 
1 per cent acetic acid 2 cc. 
Procedure : 
1. Prepare thin, uniform film; fix in flame, avoiding overheating. 
2. Add methyl violet, and allow to stand thirty to sixty seconds. 
3. Pour oft' stain and wash with strong iodine solution. 
4. Replace spent iodine solution with a fresh quantity, and allow 
to stand two minutes or until film turns nearly black. 
5. Wash off iodine solution with 98 to 100 per cent alcohol. Repeat 
process with gentle agitation for two minutes. 
> This iodine solution is variously known as Gram's iodine solution or Lugol's solution. 
2 Dilute aqueous fuchsin, 1 to 10, may be used in place of Bismarck brown. 
' Berliner kHn. Wchnschr., 1912, 49, 1663. 
