AGf~ICULTURAL RESEARCH, PUSA.

170 PRACTICAL BACTERIOLOGY
preparation is best purchased madc up by Griibler
01' Gurl', but may be prepared as follows :- '
Azul' II-eosin a grams
Azul' II . O'S grams
Glycerin (chemically pure) 250 grams
Methyl alcohol (acetone-free) 250 gralllH
The stain may be used in a manner similar to Leishman's
preparation (the "rapid mcthod "), or prolonged
staining may be carried out, as, for example, in
staining spirochaetes (the" slow method "). In hoth
cases the preparation must be fixed prior to staining,
either by methyl alcohol for three minutcs, or by
absolute alcohol for fifteen minutes.
RAPID METHOD
(1) Fix films in methyl alcohol-thrce minutes.
(2) Pour on a mixture of 1 part stain and 2 parts
distilled water (see p. IuS) and allow to aet lor
five minutes.
(3) Wash with distilled water, allowing the preparation
to differentiate for about half-a-minutc.
( 4) Blot and allow to dry in the t1ir.
A wpid method with the application q/, heat is lIseful for
demonstrating spirocllaetes :--
Fix preparations with absolute alcohol (fiftecu minutes) 01'
by drawing three times through u flame. Prepare a fresh Rolution
of 10 drops of Giemsa's solution with 10 e.c, of distilled
water of correct pH (p. lOS), shake gently, and cover the fixed
film with the diluted stain. 'Warm till :;tcHm rises, allow to
cool for fifteen seconds, then pour 011' and replace by fresh
stain aml heat again, Rcpc\\t the procedure four or live tiIncK,
wash in distilled water, dry and mount.
SLOW M'E'l'HOD
A specially valuable method for demonstrating
objects difficult to stain in the ordinary way, such as
the pathogenic spil'ochaetes. 'l'hc principle is to allow
the diluted stain to act for a considerable period.
As the mixture of stain and water cnuses a finc prccipitate,
care has to be taken that this cloes not
deposit on the film.