512
Bacterial Cell Culture Methods
primarily to detect
Mycobacterium tubercu-
losis
, but other species of
Mycobacterium
are acid-fast to some extent and can be
destained by following the acid treatment
with 95% alcohol.
The dried Flm is stained with Ziehl’s
carbol
fuchsin
for
5 min,
applying
enough heat to give steam. Do not let
the slide dry out. Ziehl’s carbol fuchsin
is prepared by dissolving basic fuchsin
(0.3 g) in 10 mL of ethanol. This is then
added to phenol (heat melted crystals),
5 mL dissolved in 95 mL of distilled
water. The solution must be mixed,
allowed to stand for a few days, and
Fltered prior to use.
The slide is washed.
After removing the excess water, the
slide
is
treated
with
a
decolorizing
solution
(hydrochloric
acid,
3.0 mL;
ethyl alcohol, (95% v/v) 97 mL).
The Flm is immediately washed with
water and the decolorization repeated
until the Flm appears faintly pink.
The organisms are counterstained with
0.3% aqueous methylene blue for 20
to 30 s, washed and blotted dry. Acid-
fast organisms retain the red stain while
others are stained blue. Endospores of
Bacillus
are also acid-fast (see below).
Spore staining (Conklin’s method).
Spores
are not so readily stained by Gram’s stain
as is the rest of the cell. Thus, frequently
they are observed as clear areas in the
center of otherwise stained cells. This is
b
e
c
au
s
eth
es
p
o
r
ei
sr
a
th
e
rm
o
r
ea
c
i
d
-
fast than the rest of the cell, that is, it
does not stain easily, but once stained does
not release the stain readily. Perhaps the
easiest method of staining spores is by
Conklin’s method.
Smears
are
prepared
and
Fxed
in
the normal way. The slide is stained
with 5% aqueous malachite green for
5 min, heating the slide gently over
a low flame until it steams. Aqueous
malachite green is produced by adding
5 g
of
malachite
green
to
100 mL
distilled water.
After washing the slide for 30 s, the
smear is counterstained with 5% aque-
ous mercurochrome or 0.25% aqueous
safranin (10 mL 2.5% safranin solution
in 95% ethanol added to 100 mL of dis-
tilled water) for 30 s.
The slide is then washed and blotted dry.
Under oil, the spores are stained green
and the rest of the cell is stained red.
4
Estimation of Population Sizes of Bacteria
4.1
Bacterial Biomass Measurement
Biomass is the dry weight of bacterial
substance per mL of a suspension and can
be determined directly provided that the
suspension contains no foreign matter.
A sample of culture is treated with
formalin (to give a Fnal concentration of
1% v/v) and centrifuged in a weighed tube.
The deposit is washed with 0.85% w/v
saline
+
1% formalin and then in 0.05%
saline and Fnally in distilled water. The
deposit is dried to constant weight at 105
,
placed in a desiccator over phosphorus
pentoxide and allowed to cool, and then
weighed in a balance (previously dried
atmosphere with silica gel).
4.2
Turbidometric Estimations
Where large numbers of determinations
are to be made, for example, in making
growth curves, turbidometric estimations
can be made. These are meaningless,
previous page 512 Encyclopedia of Molecular Cell Biology and Molecular Medicine read online next page 514 Encyclopedia of Molecular Cell Biology and Molecular Medicine read online Home Toggle text on/off