AGf~ICULTURAL RESEARCH, PUSA.

158 PRACTICAL IJACTERlOLOGY
STAINING OF TUBERCLE AND OTHER ACID-FAST
BACILLI
ZIEHL-NEELSEN METHOD
The tnberc1e bacillus does not stain with the
ordinary aniline dyes, on account of a resistant outer
envelope of a fatty nature which prevents penetration
of the stain. The principle of staining this organism
is the following :-
13y the use of a powerful staining solution which
contains a mordant, and the application of heat, the
dye can he made to penetrate the bacillus. Just as the
organism is resistant to ordinary stains, it also tends
to resist decolorisation. Once stained, the tubercle
bacillus will withstand the action of powerful decolorising
agents for a considerable time and thus
still retains the stain when everything else has been
decolorised.
The stain used consists of basic fuchsin, with phenol
as the mordant. The dye is basic and i.ts combination
with an acid produces a compound which is
yellowish-brown in colom. Any strong acid can be
used as a decolOl'ising agent, but sulphuric acid in a
20 per cent. solution is usually employed.
In order to show what has been decolorised, and
to form a conh'ast with the red-stained bacilli, the
preparation is counter-stained with methylene blue. 1
Ziehl-Neelsen's (stTong) CaTbol Ji'ucli&in :
Basic fuchsin . 1 part
Absolute alcohol . .. 10 parts
Solution of carbolic acid (1 : 20). 100 partA
Dissolve the dye in the alcohol and add the solution
to the carbolic acid.
1 Malachite green is rccommended as a eouuter-stain in the
Ziehl-Neelscn method. A stock solution of 1 pel' cent. in distilled
water is made, and for use a small quantity is dilutcd with
distilled water in a drop bottle so that fifteen to twcnty seconds'
application of the weak stain gives the background a pale green
tint. Deep counter-staining must he avoided. '1'he palc green
background is pleasant for the eyes, and is thought by some
workers to make scanty organisms more easily noticcd.