Microbiology
Microbiological Control–3
DIFFERENTIAL STAINING
Identification of bacteria may be facilitated by
differential staining. The Gram stain procedure, with an
air-dried mount fixed to a slide, has been found to be
useful in identifying brewery contaminants (1, 2).
Gram-positive bacteria will stain blue or violet;
Gram-negative bacteria will aqppear red or pink from
the safranin counterstain (3). If the microorganisms are
known to be from a pure culture, and both red and blue
forms are seen, they are described as Gram-variable.
This is more likely to be observed with old cultures;
accordingly, it is necessary to use fresh cultures to
obtain consistent results.
Gram Stain
Reagents1
(a) Ammonium oxalate-crystal violet.
Solution A: Dissolve 2 g crystal violet (Note 1) in
20 mL 95% ethyl alcohol.
Solution. B: Dissolve 0.8 g ammonium oxalate
(NH4)2C2O4·H2O in 80 mL distilled water.
Mix equal volumes of Solution A and Solution B.
(b) Iodine solution. Dissolve 2 g iodine crystals in 10
mL 1N NaOH and dilute to 100 mL with distilled
water. Store in brown dropper bottle away from
light.
(c) Aqueous Safranin-O. Add 10 mL 2.5% solution of
Safranin-O dissolved in 95% ethyl alcohol to 100
mL distilled water.
(d) Ethyl alcohol, 95%.
(e) Immersion oil, for microscope.
Apparatus
(a) Microscope, compound, with oil immersion objec-
tive (between 40 and 100× magnification) and
substage condenser.
(b) Microscope slides, standard 1 × 3 in.
(c) Bunsen burner (or similar type).
(d) Staining jars or dishes (optional).
(e) Blotting paper (or filter paper usable as blotters).
1A
commercial Gram stain set (Difco 3328) is available containing the
required ingredients: crystal violet, safranin, and decolorizer.
Page 1 of 1
Method
Spread a thin, uniform film of organisms on a thor-
oughly cleaned microscope slide and allow to air-dry
(Note 2). Fix the dried film by quickly passing the slide
through the flame of the Bunsen burner two or three
times. Flood the slide with ammonium oxalate-crystal
violet (reagent a) and allow to stand for 1 min. Wash the
stain from the slide under a gently flowing stream of tap
water.
Flood the slide with iodine solution (reagent b) and
allow to stand for 1 min (Note 3). Rinse the slide under
a gently flowing stream of tap water. Decolorize the
stained film on the slide by flowing 95% ethyl alcohol
(reagent d) over it drop by drop until the decolorizer
runs clear. Rinse the slide with tap water and blot dry.
Counterstain by flooding the slide with Safranin-O
(reagent c) for 10–20 sec. Wash the counterstain from
the slide using a gentle stream of tap water and blot dry.
Place a drop of immersion oil (reagent e) directly onto
the stained film and examine the slide using the oil
immersion objective.
Report the morphological characteristics of the organ-
isms and note the color of the Gram stain to determine
whether cells are Gram positive or Gram negative.
Notes
1. Any of the gentian violets (90% dye content) sold
under the Biological Stain Commission Certificate are
satisfactory. These may include the bluer grades of methyl
violet, such as methyl violet 2B, 6B, or 10B (1–3).
2. If organisms are suspended in wort or beer (do not
adhere to the slide), they should be collected by
centrifugation and resuspended in sterile distilled water
before making the smear.
3. An alternative iodine treatment is to immerse the
slide in the iodine solution for 1 min.
References
1. American Society of Brewing Chemists. Report of Subcommit-
tee on Bacteriology. Proc. 1949, p. 162; Proc. 1950, p. 150.
2. American Society of Brewing Chemists. Report of Subcommit-
tee on Microbiological Controls. Proc. 1958, p. 156; Proc. 1963,
p. 221; Proc. 1967, p. 273.
3. Society of American Bacteriologists. Manual of Micro-
biological Methods. McGraw-Hill, New York, 1957.
1967, rev. 1978, 2011
doi: 10.1094/ASBCMOA-MicrobiologicalControl-3