650 MED ICAL MICROBIO LO GY (3)Wash off the iodine with absolute alcohol (ethanol) and treat with

(3)Wash off the iodine with absolute alcohol (ethanol) and treat with fresh
(6)At once blot slide and allow to dry in air. alcohol, tilting the slide from side to side until colour ceases to come out of the
(7)Counterstain for 10 sec. or longer with a 2 per cent. aqueous solution of preparation. This is easily seen by holding the slide against a white back-
safranine (or neutral red). ground.
(8)Wash off counterstain by flushing with water for a few seconds, blot (4)Wash with water.
and allow to dry in air. (5)Apply the counterstain for 1-2 min.
(6)Wash with water and dry between blotting paper.
This method is very simple and gives excellent results with freedom from
deposit. Safranine, 0.5 per cent. in distilled water, may be substituted for
JENSEN'S MODIFICATION OF GRAM'S METHOD neutral red as counterstain. Basic fuchsin, 0.05 per cent., may be used for up to
30 sec., but dilute carbol fuchsin should not be used because it tends to stain
Gram-negative bacteria so intensely that they may appear Gram-positive.
(Decolourisation with alcohol) For the gonococcus and meningococcus in films, Sandiford's counterstain is
useful, particularly when the organisms are scanty.
This modification can be recommended for routine bacteriological work. It Malachite green . . 0.05 g.
has been used especially in examination of exudates for gonococci and
meningococci. Pyronine . . 015 g.
D i s t i l l e d w a t e r . t o 1 0 0 m l .
Solutions required (The stain keeps for about a month only.) Apply the counterstain for 2 min.,
(1) Methyl violet stain: flood off with water (but do not wash) and blot. Cells and nuclei stain bluish
Methyl violet 6B (or crystal violet) . 5 g. green. Gram-positive organisms are purple-black and gonococci red. It
. . . 1000 ml. should be noted that not all samples of pyronine are gatisfactory for this stain,
Distilled water . . . .
The solution should be made up in bulk and filtered. It keeps indefinitely, so that with each new purchase of pyronine the made-up stain should be tested
and does not precipitate, but should be filtered again before use. on a film known to contain gonococci or meningococci.
(2) Iodine solution' : WEIGERT'S MODIFICATION OF GRAM'S METHOD
Iodine . 10 g.
Potassium iod ide . . 20 g.
. 1000 ml. (Decolourisation with aniline-xylol)
Distilled water . . . .
Dissolve 20 g. potassium iodide in 250 ml. water, and then add 10 g. Solutions required
iodine; when dissolved, make up to 1000 ml. with water. (1)Carbol gentian violet.
Saturated alcoholic solution of gentian violet . . 1 part
(3) CounterstainNeutral red solution: . 2 ml. 5 per cent. solution of phenol in distilled water . '10 parts
Neutral red (This mixture should be made up each day, as it tends to precipitate.)
1 per cent. acetic acid Distilled (2)Gram's (Lugol's) iodine.
water . . 1000 ml.
Iodine . . 1 g.
Potassium iodide . 2 g.
Procedure for films Distilled water . 300 ml.
These are made, dried and fixed in the usual way. Aniline-xylol.
(1)Cover the slide with methyl violet solution and allow to act for about 30 (3) Aniline . . 2 pa r t s
sec.
(2)Pour off stain and, holding the slide at an angle downwards, pour on the Xylol . . . 1 part
iodine solution so that it washes away the methyl violet. Cover the slide with
fresh iodine and allow to act for about 30 sec. (4) Dilute carbol fuchsin.
Ziehl-Neelsen's carbol fuchsin (p. 654) . . 1 part
1
Iodine solution does not keep well and it is convenient, especially where stains are Distilled water . . 9 pa r t s
distributed from a central source, to have potassium iodide and iodine mixed ready for
solution when required. Potassium iodide tends to be hygroscopic and must be dried,
otherwise the mixture becomes sticky and lumpy. Place the potassium iodide in a thin Carmalum solution.
layer in a Petri dish overnight in a desiccator over calcium chloride. Mix two parts of (5) Carminic acid 1 g.
potassium iodide by weight with one part of iodine in a mortar. Weigh out at once
amounts of 7.5 grams and place them in 1-oz. screw-capped bottles (p. 725) and screw Potassium alum 10 g.
down the caps. This is sufficient for 250 m l . of solution. The mixture keeps Distilled water . . . . 200 ml.
indefinitely and easily "pours" from the bottle. For use, place the contents of one Dissolve with gentle heat; filter and add 1 ml. formalin as
bottle into an empty 10-oz. screw-capped bottle. Add about 50 ml. distilled water and
agitate until the iodine is dissolved. Make up to 250 ml. with distilled water.
preservative.
GRAM'S STAIN 651