Microbiology
Microbiological Control–2
DETECTION OF MICROORGANISMS
Detection, enumeration, and partial characterization
of viable microorganisms in liquid samples are usually
done by plating on suitable nutrient agar media. A small
aliquot of sample, containing an appropriate number of
microorganisms, is either spread evenly over the
surface of the solidified medium or mixed with the
medium before it solidifies. Incubation may be either
aerobic or anaerobic, depending on the growth
requirements of the organisms of interest. After a
suitable incubation period, the Petri dish is examined
for the presence of microbial colonies. If the con-
centration of microorganisms in the test sample is low,
a suitable volume of sample can be filtered through a
sterile membrane filter of the proper porosity (Micro-
biological Control-2C). The membrane filter is then
placed on the surface of a nutrient agar plate and
incubated as desired.
The type of medium, plating technique, and incuba-
tion parameters will determine which microorganisms
are able to proliferate; thus, the plating procedure and
the medium used are critical for selection and partial
characterization of the microorganisms.
A. INCUBATION
The incubation conditions of time, temperature, and
the presence or absence of oxygen must be controlled
for the growth of the organisms of interest on the
nutrient medium.
Apparatus
(a) Incubators, adjustable for 25 and 30°C.
(b) Anaerobic incubators, or anaerobic jars (BD BBL
Gas Pak jars or equivalent). Carbon dioxide
generators, with oxygen absorbers and indicators,
are available commercially. Oxygen indicators can
also be prepared as follows:
Method
Make up the following stock solutions:
1. 6.0 mL 10N sodium hydroxide diluted to 100 mL
with distilled water.
2. 3.0 mL 0.5% methylene blue diluted to 100 mL
with distilled water.
3. 6.0 g glucose in 100 mL distilled water to which
has been added a small crystal of thymol.
Mix equal portions of the three solutions in a test tube
each time the indicator is needed. Place tube in boiling
water until the blue color disappears and then place the
open tube into an anaerobic jar. If the indicator turns
blue and stays blue, the jar atmosphere is not
satisfactory for anaerobic incubation.
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B. PLATING
The following general plating procedures are useful
for samples of yeast, beer, and ingredients such as proc-
ess water (1).
Reagents
(a) Media. See Microbiological Control-4.
(b) Saline solution, sterile, 0.85% NaCl. Dissolve 8.5 g
reagent-grade sodium chloride in 1 L distilled water.
Dispense as required into dilution bottles. Sterilize
by autoclaving at 15 lb/in.2 and 121°C for 15 min.
(c) Antifoam, silicone type (bulk or spray). Sterilize
bulk antifoam by autoclaving at 15 lb/in.2 and
121°C for 15 min. Do not autoclave pressure spray
can.
Apparatus
(a) Serological pipets, or equivalent, sterile, 1-, 2-, and
10-mL.
(b) Dilution bottles, assorted sizes as convenient.
(c) Petri dishes. May be pre-poured with desired agar
medium, or sterile 47-mm disposable dishes, with
pads, containing desired medium.
(d) Erlenmeyer flasks, 1-L, sterile (plugged with foam
bungs or nonabsorbent cotton wool), or sterile
dispensing bottles for media.
(e) Incubators, adjustable for 25 and 30°C.
(f) Autoclave.
(g) Water bath, about 45–50°C.
(h) Hot air oven, 170°C (optional).
(i) Colony counter, Quebec type, or equivalent
equipped with lens system magnifying 1.5 times and
illumination equivalent to that of Quebec counter.
(j) Stereoscopic microscope, magnification between
30× and 100× for counting colonies (optional).
(k) Membrane filters and apparatus, sterile, 0.45-μm
porosity, for filtration sterilization and for isolating
small numbers of organisms from large volumes of
beer or water.
(l) Forceps.
Sterilization
Sterilize glassware in a hot air oven at 170°C for 2 hr
or by autoclaving followed by drying. Sterilize liquids in
an autoclave at 15 lb/in.2 and 121°C for 15 min.
Depressurize gradually to avoid explosive boiling of
liquids.
Method
Yeast
Yeast samples (for sampling, see Yeast-1) should be
diluted with sterile 0.85% saline solution (reagent b) to
facilitate plating and counting. To plate for establishing
doi: 10.1094/ASBCMOA-MicrobiologicalControl-2
Microbiology
Microbiological Control–2
Page 2 of 3
the pitching rate equivalent (PRE), which is optional,
dilute 4 g (or mL) liquid yeast in 96 mL 0.85% saline
buffer. An aliquot of 0.1 mL of this dilution should
contain the same number of yeast as would be present in
1 mL wort as a result of pitching at a rate of 1 lb yeast
per bbl wort. The sample should be plated within 20 min
from time it is added to the sterile saline solution. When
several dilutions are made, each should be thoroughly
mixed to assure homogeneous sampling.
For detection of contaminants in culture yeast, prepare
appropriate medium containing cycloheximide (see
Microbiological Control-5A) or nystatin
(Microbiological Control-5K).
Transfer 0.1 mL or more of diluted sample to each of
four Petri dishes. Pour about 15–20 mL attemperated
(about 45–50°C) of the desired medium into each plate,
swirl to mix thoroughly, and allow to solidify. Incubate
two plates aerobically at 30°C for 48 h and two plates
anaerobically at 25°C for 6–7 days.
Count the number of colonies, using stereoscopic
microscope if necessary. Report aerobic and anaerobic
counts separately and in PRE units if desired. For a
partial identification, perform a wet mount of typical
colonies, examine microscopically, and report the
morphology of the organisms.
Beer in process or packages
Sample beer tanks or lines according to the procedure
outlined in Beer-1B. If the sample is from a bottle or a
can, open aseptically, and carefully transfer the entire
contents to a sterile 1-L plugged Erlenmeyer flask.
Ensure that the contents of the bottle or can are mixed
thoroughly during transfer to ensure all possible
microorganisms are collected. Swirl the sample in the
flask to remove excess carbon dioxide and to ensure an
even distribution of microorganisms in the beer.
Plate appropriate volumes of sample in duplicate,
using the desired medium, and incubate aerobically at
30°C for 48 h and anaerobically at 25°C for 6–7 days.
If the total number of organisms per mL is expected to
be very low, analysis should be made using membrane
filtration procedure (Microbiological Control-2C).
Process water
For water samples, such as rinse water, follow the
procedure described for beer, above. Sample as
described in Microbiological Control-1C.
Brewery air (see Microbiological Control-1D).
Process equipment and surfaces (see Microbiological
Control-1E).
Reference
1. American Society of Brewing Chemists. Report of Subcom-
mittee on Microbiological Controls. Proc. 1957, p. 169; Proc.
1958, p. 156; Proc. 1963, p. 221; Proc. 1966, p. 255; Proc.
1967, p. 273.
C. MEMBRANE FILTRATION
The membrane filtration technique is recommended
for examination of beer, rinse water, or similar samples
that contain relatively few bacteria, yeast, or molds (1).
It uses cellulose acetate or cellulose nitrate membranes
of 0.45-µm porosity with grid design on the membrane
surface. Nonsterile membranes should be sterilized at 15
lb/in.2 and 121°C for 10 min, taking care to avoid
membrane shrinkage. Presterilized membranes are also
available for purchase.
Reagents
(a) Media. See Microbiological Control-4.
(b) Saline solution, sterile, 0.85% NaCl. Dissolve 8.5 g
reagent-grade sodium chloride in 1 L distilled water.
Dispense as required into dilution bottles. Sterilize
by autoclaving at 15 lb/in.2 and 121°C for 15 min.
(c) Antifoam, silicone type (bulk or spray). Sterilize
bulk antifoam by autoclaving at 15 lb/in.2 and
121°C for 15 min. Do not autoclave pressure spray
can.
Apparatus
(a) Incubators, adjustable for 25 and 30°C.
(b) Anaerobic incubators,or anaerobic jars (BD BBL
Gas Pak jars or equivalent). Carbon dioxide
generators, with oxygen absorbers and indicators,
are available commercially. Oxygen indicators can
also be prepared as in Microbiological Control-2A.
(c) Serological pipets, or equivalent, sterile, 1-, 2-, and
10-mL.
(d) Dilution bottles, assorted sizes as convenient.
(e) Petri dishes. May be pre-poured with desired agar
medium, or sterile 47-mm disposable dishes, with
pads, containing desired medium.
(f) Erlenmeyer flasks, 1-L, sterile (plugged with foam
bungs or nonabsorbent cotton wool), or sterile
dispensing bottles for media.
(g) Water bath, about 45–50°C.
(h) Hot air oven, 170°C (optional).
(i) Colony counter, Quebec type, or equivalent
equipped with lens system magnifying 1.5 times and
illumination equivalent to that of Quebec counter.
(j) Stereoscopic microscope, magnification between
30× and 100× for counting colonies (optional).
(k) Membrane filters and apparatus, sterile, 0.45-μm
porosity, for filtration sterilization and for isolating
small numbers of organisms from large volumes of
beer or water.
(l) Forceps.

Method
Place a drop of sterile antifoam into the vacuum flask.
Aseptically remove the filter assembly from its sterile
wrappings and insert into a vacuum flask (see Note).
Place the sterile air filter into the funnel. Dip the forceps
into alcohol, flame sterilize, and aseptically place the
sterile membrane filter onto the filter base, grid side up,
and secure the funnel to the base.
Take samples as described in Beer-1B and Beer-1C. To
prevent foaming, add 0.05–0.10 mL sterile antifoam to
each sample before pouring, or spray the inside of the
filter funnel with antifoam before sterilization.
Remove the air filter and aseptically transfer the sample
to the filter funnel, taking care not to overfill the funnel,
so that the air filter stopper does not become contaminated
with the sample when it is replaced in the funnel. If beer is
at 3°C, filtration will be faster with less foaming.
Turn on the vacuum system and filter 100 mL or the
entire contents of the package. Rinse the funnel three
times with 20–30 mL of sterile saline solution (reagent
c), maintaining the vacuum until all of the foam on the
filter is gone. Flame sterilize the forceps and aseptically
transfer the filter membrane, grid side up, to a sterile
Petri dish containing the desired medium.
Incubate plate at 25 or 30°C, or both, for 2–3 days
Microbiology
Microbiological Control-2
Page 3 of 3
aerobically or 6–7 days anaerobically as desired. Count
the number of colonies on the filter, using stereoscopic
microscope if necessary.
Report the total numbers of organisms per 100 mL or
on a per-package basis.
Note
Sterile equipment is always recommended for each
test. If this is not available, equipment should be cleaned
before reuse according to the following procedure:
Disassemble the filter, wash with water, and place in
boiling water for 20 min. Reassemble while hot and place
a sterile air filter-stopper into the funnel. When the funnel
is cool, proceed as previously described. If three units are
available, one can be in operation, one cooling, and one
heating.
Reference
1. American Society of Brewing Chemists. Report of Subcommit-
tee on Microbiological Controls. Proc. 1959, p. 193; Proc. 1960,
p. 201; Proc. 1961, p. 144; Proc. 1962, p. 173; Proc. 1966, p.
255.
1966, rev. 1977, 2011