STAINING OF FLAGELLA 85 
STAINING OF SpoRES 
Neisser’s Method. Flood the smear with hot aniline fuchsin for 
about an hour. Wash in water and decolorize with acid alcohol (1 part 
HCl and 3 parts alcohol). Too long decolorization will remove the 
stain from the spores also. Counterstain with methylene blue if 
desired. 
Chromic Acid Method. Place the smear in 1 to 20 aqueous chromic 
acid for five minutes. Wash in water and stain with carbol fuchsin 
for thirty minutes. Wash and examine. 
STAINING OF FLAGELLA 
This has always been a difficult procedure for many bacteriologists. 
It may be due to lack of care in some of the preparatory procedures. 
Smith (1905) has mentioned some of the common errors. 
. Oily or otherwise dirty cover glasses. 
. Unsuitable cultures. 
. Breaking off the flagella. 
Uneven or too copious distribution of the bacteria. 
. Imperfect mordanting. 
. Excessive mordanting. 
. Understaining. 
. Overstaining. 
. Precipitates on the cover glass during some stage of the process. 
The greatest error probably lies in dirty slides and cover glasses. These 
should be cleaned in strong alkali in order to saponify any animal 
grease which may be on them. Otherwise, the smear will “ roll” 
and not spread evenly. The culture which is stamed must be young 
and vigorous. A small amount of this growth should be transferred 
to a test tube of sterile water and incubated at 37° C. After several 
hours, some of this suspension should be put on slides and allowed to 
dry by placing the slides in the 37° C. incubator. The smear should 
then be fixed and stained by any of the many processes. 
The author has secured the best results with Loeffler’s method. 
A double boiler is prepared from two beakers. The inner is supported 
by pieces of cork. The staining solution should be put into the inner 
one and the temperature raised to about 65° C. or until the stain steams. 
The slides are immersed in this solution and stained as long as required. 
CONOR WN ES 
