652 M E D I C A L M I C R O B I O L O GY Methylated spirit (64 O.P.) . . .

Procedure for films

These are made, dried and fixed in the usual manner. Ammonium oxalate, 1 per cent. in water .
(1)Stain with carbol gentian violet for 2-3 min. Z I E H L - N E E L S E N S TAI N 653
(2)Pour off stain, replace with Gram's iodine solution and allow to act for 1
mm. (2)Iodine solution Iodine . .
(3)Dry thoroughly by blotting. . 10 g.
. 20
(4)Decolourise with aniline-xylol, using several changes until the stain Potassium iodide . Distilled water . . 1000g.
ceases to be removed. ml.
Now examine at this stage under, the low power of the microscope; the (3)Liquor iodi fortis (BP)
nuclei of the pus cells should be of a pale-violet colour; if the nuclei are deeply Iodine . . . 10 g.
stained, then decolourisation is incomplete. . 6.
Potassium iodide . .
(5)Wash with several changes of xylol and dry. . 90 g ml. .
Methylated spirit (74 O.P.) . D istille d wa te r .
(6)Counterstain with dilute carbol fuchsin for 10-25 sec. Wash with . 10 ml. .
water and dry.
(4)Iodine-acetone. 35 ml. .
Procedure for sections Liquor iodi fortis Acetone .
965 ml.
Weigert's modification is specially recommended for the staining of (5)Dilute carbol fuchsin.
sections. . .
After removing the paraffin with xylol or benzol, treating with alcohol and Ziehl-Neelsen's (strong) carbol fuchsin (p. 654) 50 ml.
washing with water, counterstain first with carmalum for 10 min. and then Distilled water . . 950 ml.
proceed as above. After (5) the sections will be cleared and can at once be
mounted in Canada balsam or DPX. Procedure for films
(1)Cover slide with ammonium oxalate-crystal violet and allow to act for about
30 sec.
CLALTDIUS'S (1897) MODIFICATION OF GRAM'S METHOD (2)Pour off and wash freely with iodine solution . Cover with fresh
iodine solution and allow to act for about 30 sec.
(3)Pour off iodine solution and wash freely with iodine-acetone. Cover
(Counterstaining with picric acid) with fresh iodine-acetone and allow to act for about 30 sec.
(4)Wash thoroughly with water.
This method, which gives very pale, "transparent" counterstaining, is (5)Counterstain with dilute carbol fuchsin for about 30 sec.
recommended for the demonstration of fungi in tissue sections. (6)Wash with water, blot and dry.
(1)Stain with 1 per cent. aqueous methyl violet for 1-2 min. It is essential that the whole slide is flooded with each reagent in turn, and that
(2)Wash in water. the previous reagent is thoroughly removed at each step.
(3)Flood slide with a half-saturated watery solution of picric acid (i.e. 0.6 per
cent.). Cover slide with fresh picric acid and allow to act for 1-2 min.
(4)Remove excess picric acid solution with blotting paper.
(5)Decolourise with-Chloroform (or, preferably, aniline containing 0.1 per
cent. picric acid) until no more violet comes away in the decolouriser.
5 STAINING OF TUBERCLE AND OTHER ACID-FAST
BACILLI
(6)Rinse with xylol and mount in Canada balsam. 4. ZIEHL-NEELSEN METHOD
This method is a modification of Ehrlich's (1882) original method for the
differential staining of acid-fast bacilli with aniline-gentian violet followed by
PRESTON AND MORRELL'S (1962) MODIFICATION OF strong nitric acid. It incorporates improvements suggested, successively, by
Ziehl and Neelsen, and is described in a footnote in a paper by Johne (1885).
The ordinary aniline dye solutions do not readily penetrate the substance of
GRAM'S METHOD the tubercle bacillus and are therefore unsuitable for staining it. However, by
the use of a powerful staining solution that contains phenol, and the
(Decolourisation with iodine-acetone) application of heat, the dye can be made to penetrate the bacillus. Once
stained, the tubercle bacillus will withstand the action of powerful decolour ising
This method is recommended as giving reliable results without the need for agents for a considerable time and thus still retains the stain when everything
taking great care in adjusting the duration of decolourisation. else in the microscopic preparation has been decolourised.
The stain used consists of basic fuchsin, with phenol added. The dye is
Solutions required basic and its combination with a mineral acid produces a compound that is
(1) Ammonium oxalate-crystal violet. yellowish brown in colour and is readily dissolved out of all structures except
Crystal violet . . . . 20. acid-fast bacteria. Any strong acid can be used as a decolourising agent, but
. 200 g ml .
. 800 ml.