Microbiology

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DIFFERENTIAL CULTURE MEDIA

Differential media can provide a means of deter- Notes
mining the identification of a microbiological conta- 1. Cycloheximide may be obtained from Sigma
minant in the brewery. This can be achieved by Aldrich or equivalent suppliers. It may also be available
exploiting differential antibiotic action or by employing on order from local pharmaceutical suppliers.
a nutrient medium that suppresses the growth of one 2. A safer alternative to cycloheximide is nystatin
type of microorganism or enhances the growth of (Microbiological Control-5K).
another.
References
A. CYCLOHEXIMIDE MEDIUM 1. American Society of Brewing Chemists. Report of Subcom-
mittee on Microbiological Controls. Proc. 1957, p. 169; Proc.
Cycloheximide (also known as actidione) is an 1963, p. 221.
2. Green, S. R., and Gray, P. P. Am. Soc. Brew. Chem., Proc. 1950,
antibiotic used to suppress the growth of most brewing p. 19; Wallerstein Lab. Commun. 13:357, 1950.
yeasts without affecting the growth of common
brewery bacteria (2). When incorporated into Wal- 1963, rev. 1978, 2011
lerstein Laboratories Nutrient (WLN) medium as
Upjohn’s Actidione to give “WLD,” it was the standard B. LEE’S MULTI-DIFFERENTIAL AGAR
medium used in the brewing industry for many years (LMDA)
(1, 2).
This medium yields a recovery of brewery bacteria
Preparation of Cycloheximide Solution that is equal to, or in some instances better than,
Prepare a stock solution by dissolving either 40 or Universal Beer Agar (UBA). In addition, it provides, by
100 mg of cycloheximide (see Notes) in 100 mL water, visual observation of colonies, some identifiable
depending on whether “low” or “high” concentration is characteristics for the microorganisms (2). The medium
desired. Stock solutions should be maintained at 4°C. If uses cycloheximide for yeast inhibition, CaCO3 for
the cycloheximide to be added to the base medium be- assistance in identification of acid-producing micro-
fore autoclaving, use 1 mL of the higher concentration organisms, and Bromocresol green for developing color
per 100 mL medium, since cycloheximide is somewhat characteristics. Incubation is under anaerobic conditions,
thermolabile. If added after autoclaving, use 1 mL/100 except when acetic acid bacteria are of major interest,
mL of the lower concentration, after membrane ste- where aerobic incubation is used.
rilization, to give 0.4 mg/100 mL in the final culture The original formula for LMDA is described below;
medium. Irrespective of the timing of cycloheximide however, it should be noted that pre-formulated medium
addition, the growth medium should be autoclaved at is available (see Notes).
15 lb/in.2 and 121°C for 15 min prior to use.
Culture Medium
Apparatus g/L
See Microbiological Control-2A and B. Tomato juice solids 20.0
Peptonized milk 20.0
Base Medium Yeast extract 10.0
See Microbiological Control-4. Dextrose 10.0
Calcium pantothenate 2.0
Method Citric acid monohydrate 1.1
Using appropriate aseptic precautions, dilute a sample Calcium carbonate 5.0
of yeast slurry, beer, or process water based on the number Dipotassium phosphate, K2HPO4 0.5
of bacteria anticipated in the sample. Aseptically add the Monopotassium phosphate, KH2PO4 0.5
cooled (45–50°C), but still liquid, sterile medium to 1 mL Magnesium sulfate, MgSO4 0.2
of the diluted sample in a Petri dish and thoroughly mix. Manganese sulfate, MnSO4 0.01
Inoculate replicate plates and incubate one set aerobically Ferrous sulfate, FeSO4 0.01
at 30°C and the other anaerobically at 25°C. Adequate Sodium chloride 0.01
aerobic growth for counting purposes should occur in 2–3 Tween 80 0.5
days. The anaerobic plates should be incubated for 6–7 Bromocresol green 0.022
days. Cycloheximide 0.007
Report the number of colonies developing under aero- Agar 15.0
bic and anaerobic conditions separately. Distilled water to 1 L

doi: 10.1094/ASBCMOA-MicrobiologicalControl-5
Microbiology
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Reagents Lactobacillus colonies Colonies appear translucent to

(a) Dilution solution (see Microbiological Control-2B). greenish white with a dark
(b) Phenethyl alcohol, for isolation of Gram-positive green center and a halo zone.
organisms. The undersides of colonies
appear yellow.
Apparatus Acetic acid bacteria The undersides of colonies
See Microbiological Control-2A and B. appear greenish blue. A halo
zone is also typically
Method observed.
Medium preparation Flavobacterium Colonies exhibit a rough surface
Suspend the ingredients, except for phenethyl alcohol and edge. The centers of
(reagent b), if used, in water and mix thoroughly. colonies are slightly darker
Autoclave at 15 lb/in.2 and 121°C for 10 min (excessive than the outer edges. Colony
heat is detrimental to the medium). The pH should be color can vary from whitish to
approximately 5.5 after autoclaving and does not require light or dark blue.
adjustment. Cool the medium to 45°C in a water bath. Coliforms Smooth, shiny, large, spreader-
Before pouring LMDA into sterile Petri dishes, agitate type colonies are produced.
the agar solution gently to obtain a homogeneous Colony color varies.
suspension of calcium carbonate. Dispense the medium Pediococcus Small colonies are produced
into sterile Petri dishes. After the agar has solidified, with a narrow halo zone. This
invert the plates and store at 30°C overnight. Excessive species is slow growing on
drying at higher temperatures should be avoided. Dried this medium.
plates are ready to use or can be stored inverted at 5°C
until required. Notes
1. The completely formulated LMDA medium can be
Plating technique purchased from Siebel Institute of Technology,
Samples of wort, beer, or water can be used. Prior to Chicago.
use, samples should be diluted with sterile standard 2. Current commercially available formulations may
saline solution (reagent a) to achieve 40–60 colonies per not exactly match the original composition.
plate. 3. A safer alternative to cycloheximide is nystatin,
Spread either 0.1 or 0.2 mL of a sample over the which can be substituted here, if desired (Micro-
surface of the medium using sterile bent glass rod (3), or biological Control-5K).
use the pour-plate procedure (1).
Incubate plates anaerobically at 30°C. If acetic acid References
bacteria are of interest, duplicate plates should be incu- 1. American Society of Brewing Chemists. Report of Subcommit-
bated aerobically at 30°C to detect these organisms tee on Microbiological Controls. Proc. 1975, p.75.
2. Lee, S. Y. U.S. patent 3,878,050, April 15, 1975. Assigned to
only. Adolph Coors Co., Golden, CO.
To isolate only Gram-positive organisms, membrane 3. Lee, S. Y., Jangaard, N. O., Coors, J. H., Hsu, W. P., Fuchs, C.
filter-sterilized phenethyl alcohol (0.3%) (reagent b) can M., and Brenner, M. W. Proc. Am. Soc. Brew. Chem. 33:18,
be added directly to the medium before pouring into 1975.
sterile plates. This may replace the cycloheximide 1975, rev. 1978, 2011
normally used in LMDA, if desired (3).
Report the number, color, and shape of colonies.
C. RAKA-RAY LACTIC ACID BACTERIA
Interpretation of results MEDIUM (RRLM)
Initial colonies of coliforms and flavobacterium will
appear within 2 days, and by the fourth day most lactic In general, this medium selectively detects lactic acid
acid bacteria can be observed. Additional time may be bacteria endemic to breweries by fostering larger colo-
required for Pediococcus species. nies of this bacterial group in a short period of time. The
presence of lactic acid bacteria is also emphasized by
suppressing, in varying degree, the growth of facultative
Organism Reaction with LMDA nonlactic acid (wort) bacteria, such as Aerobacter
Lactic acid, acetic acid Medium-yellow colored aerogenes or Flavobacterium proteus (1, 2).
bacteria colonies are produced with a It should be recognized that certain lactic acid bacte-
clear halo zone surrounding ria, such as Lactobacillus brevis and Pediococcus cere-
each colony. visiae, may not show this response (2).
 Microbiology
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The original formula for RRLM is described below; purchased from a number of suppliers, including BD
however, it should be noted that pre-formulated medium Biosciences, Oxoid, Sigma-Aldrich, and Siebel Institute
is available (see Notes). of Technology.
2. Current commercially available formulations may
Culture Medium not exactly match the original composition.
g/L
References
Yeast extract 5.0 1. American Society of Brewing Chemists. Report of Subcommit-
Trypticase 20.0 tee on Microbiological Controls. Proc. 1975, p. 75.
Liver concentrate 1.0 2. American Society of Brewing Chemists. Report of Subcommit-
Maltose 10.0 tee on Microbiological Controls. Journal 34:93, 1976.
3. Saha, R. B., Sondag, R. J., and Middlekauff, J. E. Am. Soc.
Fructose 10.0 Brew. Chem., Proc. 1974, p. 9.
Betaine HCl 2.0
Diammonium citrate 2.0 1976, rev. 1978, 2011
Potassium aspartate 2.5
Potassium glutamate 2.5 D. LYSINE MEDIUM
Magnesium sulfate heptahydrate, 2.0
MgSO4·7H2O Brewer’s yeast and most other yeasts of the genus
Manganese sulfate hydrate, MnSO4·H2O 0.5 Saccharomyces are lysine-negative, i.e., they cannot util-
Dipotassium phosphate, K2HPO4 2.0 ize lysine as the sole source of nitrogen. Many other
N-Acetyl glucosamine 0.5 yeasts, including wild yeasts that may occur as brewery
Agar 20.0 contaminants, can utilize lysine-N, grow on a lysine
Tween 80 10.0 mL medium, and are lysine-positive (2). When interpreting
Distilled water to 1 L results, absence of growth on a lysine medium does not
necessarily mean the absence of wild yeasts (1). With
The ingredients are dissolved in distilled water to a appropriate sample dilution, the procedure is useful for
final volume of 1 L and sterilized by autoclaving at 15 yeast slurries, process beer, and rinse water samples (1).
lb/in.2 and 121°C for 15 min. The final pH, after
autoclaving, is approximately 5.4 and does not require Culture Medium
adjusting. Yeast carbon base 2.35 g
Lysine monohydrochloride 0.46–0.5 g
Reagents Agar 4.0 g
See Microbiological Control-2B. Distilled water 200 mL
Apparatus Reagents
See Microbiological Control-2A and B. See Microbiological Control-2B.
Method
Apparatus
Culturing should be performed using solid autoclaved
See Microbiological Control-2A and B.
medium in sterile Petri dishes. Any standard method of
inoculation is acceptable, but for quantitative work, the Method
overlay technique is recommended (3). If serial dilutions Medium preparation
are required to effect a suitable concentration of micro- Prepare the medium by adding 0.46–0.5 g of lysine
organisms, for example 300–500 cells/mL for a 0.1-mL monohydrochloride and 4.0 g agar to 100 mL distilled
inoculum, sterile saline (NaCl, 8.5 g/L) should be water and sterilize by autoclaving at 15 lb/in.2 and 121°C
utilized. Organisms should be incubated anaerobically at for 15 min. Separately, dissolve 2.35 g of the yeast carbon
28°C. For most species of lactic acid bacteria, plate base in 100 mL distilled water and sterilize by membrane
counts can be performed after 3 days of incubation. For filtration. While the autoclaved lysine-agar mixture is still
Pediococcus cerevisiae, however, plates should be liquid, aseptically add the sterile yeast carbon base and
scored after 5–7 days of incubation (2). cool to 45–50°C before using.
Five replicates of each inoculum are recommended.
Suitable blanks or controls, in which sterile saline is Evaluation
substituted for inocula, should be included with each Using aseptic technique, dilute the sample based on
run. the number of wild yeasts anticipated in the sample of
yeast slurry, process beer, or rinse water. Thoroughly
Note mix 1 mL of the diluted sample with liquid medium
1. The completely formulated RRLM medium can be (45–50°C) in a Petri dish and allow to set. Incubate
Microbiology
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microbes aerobically at 25°C for 2–6 days. Enumerate in 0.5 mL 95% ethyl alcohol and dilute to 100 mL with
colonies periodically and report the number of wild distilled water. Store the stock solution at 4°C. For use,
yeasts per mL of the original undiluted sample. dilute the stock solution to the desired concentration (as
described below) with distilled water.
References
1. American Society of Brewing Chemists. Report of Subcommit- Preparation of fuchsin-sulfite mixture (2)
tee on Microbiological Controls. Proc. 1962, p. 173; Proc. 1963, Grind and mix with a mortar 4 g basic fuchsin, 25 g
p. 221.
2. Walters, L. S., and Thiselton, M. R. J. Inst. Brew. 59:401, 1953.
anhydrous sodium sulfite, and 1 g dextrin. Store the
mixture in a desiccator for 3 days to remove moisture,
1963, rev. 1978, 2011 transfer to a tightly capped dark brown bottle, and store
at room temperature.
E. LIN’S WILD YEAST DIFFERENTIAL
MEDIUM (LWYM) Determination of crystal violet concentration for LWYM
Introduce into each of a series of Erlenmeyer flasks
This medium inhibits, or markedly restricts, growth of 100 mL of a well-mixed suspension of medium contain-
brewery culture yeasts while permitting growth of many ing all the ingredients specified for LWYM except crys-
wild yeasts. It incorporates the known effects of fuchsin- tal violet. Add different concentrations of crystal violet
sulfite (2) and crystal violet (3) into one medium, along (from 0.1 to 6.0 mg/L) into each flask and heat to boil-
with adjustments in preparation and use of the medium. ing. Sterilize the medium by autoclaving at 15 lb/in.2
The limitations of the medium should be recognized and 121°C for 15 min. Cool the medium and dispense
(1, 4). On occasion, a specific strain of brewer’s culture into sterile Petri dishes. Maintain prepared at 4°C for
yeast may show weak growth on LWYM; however, such 24–48 hr prior to use. Aseptically add 0.2 mL of
growth is typically markedly restricted relative to the inoculum containing a brewer’s culture yeast (approx.-
vigorous growth of wild yeasts. A few wild yeasts, such imately 1 million cells/mL) onto an agar plate. Onto a
as Torulospora fermentatii and Torulopsis kefyr, cannot separate plate, a known LWYM detectable wild yeast
be detected using LWYM. These yeasts, however, are (200 cells per plate), or a mixture of both wild yeast and
lysine-positive and may be detected by using lysine culture yeast should be cultivated. Disperse the inocula
medium (Microbiological Control-5D). This emphasizes over the surface of the medium with a sterile bent glass
the desirability of using more than one testing procedure rod. At least two sets of plates should be prepared.
for evaluation of the presence of any wild yeasts. Incubate one set at 25°C and the other at 30°C for 4–6
The original formula for LWYM is described below; days. Select the most effective concentration of crystal
however, it should be noted that pre-formulated medium violet that inhibits culture yeast growth and supports the
is available (see Note). growth of wild yeast (see Note).
Culture Medium Detection of wild yeasts
g/L Prepare LWYM plates containing the most effective
Yeast extract 4.0 concentration of crystal violet as described above, and
Malt extract 2.0 use for wild yeast detection as directed below.
Peptone 2.0 1. For samples containing high populations of yeast
Dextrose 10.0 cells (such as pitching yeast or yeast in storage tank):
Dipotassium phosphate, K2HPO4 1.0 Wash the yeast cells centrifugally three times with
Ammonium chloride 0.5 sterile saline. Inoculate 0.2 mL of each washed sample,
Crystal violet (Prepare as below) See Notes containing approximately 1 million yeast cells, onto
Fuchsin-sulfite mixture (Prepare as each LWYM plate. Disperse the inoculum over the
below) 1.0 surface of the medium using a sterile bent glass rod.
Agar 20.0 Incubate a set of plates aerobically at 25°C and another
Distilled water to 1 L set at 30°C. Examine the plates for growth after 4–6
days. Distinct colonies observed on the medium may be
Reagents considered to be wild yeasts.
See Microbiological Control-2B. 2. For samples containing low populations of yeast
Apparatus cells (such as unpasteurized beer, draft beer, or water):
See Microbiological Control-2A and B. Filter each sample through a membrane filter
(recommended pore size < 4 m) followed by 500–
Method 1,000 mL sterile distilled water to wash and remove
Preparation of crystal violet stock solution extraneous material from the filter. Place the membrane
Dissolve 0.1136 g crystal violet with 88% dye content filter onto the surface of the growth medium in a Petri
 Microbiology
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dish. Incubate one set of plates at 25°C and another set Autoclave at 15 lb/in.2 and 121°C for 15 min.
at 30°C for 4–6 days. The distinct colonies that develop
on the membrane filter may be considered to be wild Reagents
yeast. See Microbiological Control-2B.
Report the number of wild yeast colonies found per unit
of starting material: pitching yeast, process beer, or water. Apparatus
See Microbiological Control-2A and B.
Notes
Method
1. Agar purchased at different times from the same
Dilution of samples with sterile saline solution or sterile
supplier can have a different effect on LWYM (4).
water may be necessary to achieve approximately 100
Therefore, the effective concentration of crystal violet in
colonies per plate. Either spread 0.1 mL of the sample over
LWYM should be determined with every agar purchase.
the surface of the medium, using a sterile bent glass rod or
2. The effective concentration of crystal violet in
other nonabsorbing substance (4), or use a pour-plate
LWYM should be determined to suit each laboratory’s
procedure (2).
requirements.
Incubate plates anaerobically at 28°C. Plate counts
3. The completely formulated LWYM can be
can be performed as early as 2 days but should be
purchased from Siebel Institute of Technology, Chicago.
repeated at 7 days for slow-growing lactic acid bacteria.
4. Current commercially available formulations may
Three replicates of each inoculum are recommended,
not exactly match the original composition.
along with suitable blanks or controls of dilution solution.
References References
1. American Society of Brewing Chemists. Report of Subcommit-
1. American Society of Brewing Chemists. Report of Subcommit-
tee on Microbiological Controls. Proc. 1975, p. 75.
tee on Media for Lactobacilli. Journal 49:174, 1991.
2. Brenner, M. W., Karpiscak, M., Stern, H., and Hsu, W. P. Am.
2. American Society of Brewing Chemists. Report of Subcommit-
Soc. Brew. Chem., Proc. 1970, p. 79.
tee on Microbiological Controls. Proc. Am. Soc. Brew. Chem.
3. Kato, S. Bull. Brew. Sci. (Japan) 13:19, 1967.
33:75, 1975.
4. Lin, Y. Am. Soc. Brew. Chem., Proc. 1974, p. 69.
3. Barney, M. C., Kot, E. J., and Chicoye, E. Culture medium for
detection of beer spoilage microorganisms. U.S. patent
1975, rev. 1978, 2011
4,906,573, 1990.
4. Lee, S. Y., Jangaard, N. O., Coors, J. H., Hsu, W. P., Fuchs, C. M.,
and Brenner, M. W. Proc. Am. Soc. Brew. Chem. 33:18, 1975.
F. BARNEY-MILLER BREWERY
MEDIUM (BMB) 1991, rev. 2011

This medium detects beer spoilage microorganisms G. DE MAN ROGOSA SHARPE

such as Lactobacillus sp. and Pediococcus sp. (3). The MEDIUM (MRS)
time required for incubation has been reduced (1). It is
illegal to produce this medium without a license or This medium detects beer spoilage microorganisms
approval from the patent holder. such as Lactobacillus sp. and Pediococcus sp. (1–3).
The original formula for MRS medium is described
below; however, it should be noted that pre-formulated
g/L medium is available (see Notes).
Tomato juice broth 15.0
Maltose 15.0 Culture Medium
Dextrose (glucose) 10.0 g/L
Polypeptone 5.0 Peptone 10.0
Potassium acetate 3.0 Lab-Lemco Powder 10.0
Beef extract 2.0 Yeast extract 5.0
L-Malic acid 0.5 Dextrose 20.0
Tween 80 0.5 Tween 80 1.0 mL
L-Cysteine hydrocholoride 0.2 Dipotassium phosphate, K2HPO4 2.0
Agar 15.0 Sodium acetate trihydrate,
Distilled water 750 mL CH3COONa·3H2O 5.0
Beer (typically lager style) 250 mL Triammonium citrate 2.0
Magnesium sulfate heptahydrate,
Dissolve individual components in 750 mL of distilled MgSO4·7H2O 0.2
water and 250 mL of beer, typically lager style. The final Manganese sulfate tetrahydrate,
pH is 5.5–5.7 and usually does not require adjustment. MnSO4·4H2O 0.05
Microbiology
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Agar 15.0
Distilled water to 1 L
Dissolve the ingredients in 1 L of distilled water and
autoclave at 15 lb/in.2 and 121°C for 15 min. The pH is
6.2–6.6 before autoclaving and 6.0–6.5 after autoclaving
and does not require adjustment.
Reagents
See Microbiological Control-2B.
Apparatus
See Microbiological Control-2A and B.
Method
Dilution of samples with sterile saline solution or ster-
ile water may be necessary to achieve approximately
100 colonies per plate. Either spread 0.1 mL of sample
over the surface of the medium, using a sterile bent glass
rod or other nonabsorbing substance (4), or use a pour-
plate procedure (2).
Incubate plates anaerobically at 28°C. Plate counts
can be conducted after 7 days. Three replicates of each
inoculum are recommended, along with suitable blanks
or controls of dilution solution.
Note
1. The completely formulated MRS medium can be
purchased from a number of suppliers including BD
biosciences, Oxoid, and Sigma-Aldrich.
2. Current commercially available formulations may
not exactly match the original composition.
References
1. American Society of Brewing Chemists. Report of Subcommit-
tee on Media for Lactobacilli. Journal 49:174, 1991.
2. American Society of Brewing Chemists. Report of Subcommit-
tee on Microbiological Controls. Proc. Am. Soc. Brew. Chem.
33:75, 1975.
3. De Man, J. C., Rogosa, M., and Sharpe, M. E. A medium for the
cultivation of Lactobacilli. J. Appl. Bacteriol. 23:130, 1960.
4. Lee, S. Y., Jangaard, N. O., Coors, J. H., Hsu, W. P., Fuchs, C.
M., and Brenner, M. W. Proc. Am. Soc. Brew. Chem. 33:18,
1975.
1991, rev. 2011
H. MYGP + COPPER MEDIUM
This medium detects certain wild yeast species, Sac-
charomyces and non-Saccharomyces (1, 3).
Culture Medium
YM agar 41 g/L
Cupric sulfate solution, CuSO4·5H2O 20 mL
Distilled water to 1 L
Dissolve YM agar in 1,000 mL of distilled water and
autoclave at 15 lb/in.2 and 121°C for 15 min. Add 20 mL
cupric sulfate solution, produced by dissolving 1.56 g
CuSO4·5H2O in 100 mL water (sterilized at 15 lb/in.2 and
121°C for 15 min), to the cooled agar before pouring
plates.
Reagents
See Microbiological Control-2B.
Apparatus
See Microbiological Control-2A and B.
Method
Prepare MYGP + copper medium (MYGP + Cu)
plates as described above for wild yeast detection.
Samples containing a high population of yeast cells
(e.g., pitching yeast) should be washed 3 times with ster-
ile saline. Inoculate 0.1 or 0.2 mL of washed, diluted
sample, containing approximately 1 million yeast cells,
on duplicate MYGP + Cu plates. Disperse the inoculum
over the surface of the medium, using a sterile bent glass
rod (2). Incubate the plates at 27°C and examine after 4
days. Distinct colonies developed on MYGP + Cu may
be considered wild yeasts.
The guidelines described in Microbiological
Control-5E for analyzing samples with low and high
concentrations of yeast can also be applied here.
Samples containing low populations of yeast cells may
also be concentrated by centrifugation, suspended in
sterile saline, and treated as described above.
References
1. American Society of Brewing Chemists. Report of Subcommittee
on Copper Media for Wild Yeast Detection. Journal 50:153, 1992.
2. Lee, S., Jangaard, N., Coors, J., Hsu, W., Fuchs, C., and
Brenner, M. Proc. Am. Soc. Brew. Chem. 33:18, 1975.
3. Taylor, G., and Marsh, A. J. Inst. Brew. 90:135, 1984.
1992, rev. 2011
I. CLEN MEDIUM FOR WILD YEAST
CLEN medium contains cadaverine, lysine,
ethylamine, and nitrate as nitrogen sources (3). This
medium is suitable for the detection of some wild yeasts
and is intended to be used in combination with other
wild yeast media for thorough examination and optimal
detection of wild yeasts (1–3).
Culture Medium
Yeast carbon base 11.7g
Agar 15 g
Cadaverine dihydrochloride (Sigma C-8561, or
equivalent) 2.4 g
Ethylamine, 70% (v/v) solution 0.9 g
Lysine monohydrochloride 2.5 g
Potassium nitrate 1.4 g
 Microbiology
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Distilled water to 1 L References

Hydrochloric acid, concentrated 1. American Society of Brewing Chemists. Report of the Subcommittee
on CLEN Medium for the Detection of Wild Yeast. Journal 55:185,
1997.
Reagents 2. American Society of Brewing Chemists. Report of the Subcommit-
See Microbiological Control-2B. tee on CLEN Medium for the Detection of Wild Yeast. Journal
56:202, 1998.
3. Martin, C. P., and Siebert, K. J. J. Am. Soc. Brew. Chem.
Apparatus 50:134, 1992.
(a) pH meter.
(b) Hemocytometer. 1998, rev. 2011
(c) See Microbiological Control-2A and B.

Method J. SELECTIVE MEDIUM FOR MEGASPHAERA

Preparation of yeast carbon base-CLEN stock solution AND PECTINATUS (SMMP)
Prepare a 10× yeast carbon base (YCB) by dissolving
11.7 g YCB in 100 mL of distilled water (with warming, This medium detects the anaerobic beer spoilage
if necessary) and filter-sterilize through a 0.45-m mem- organisms Megasphaera and Pectinatus. A reduced-
brane filter. Store at 2–8°C until use. In 100 mL of 10× incubation environment is obtained through the inclu-
concentrated solution of YCB, dissolve 2.5 g lysine sion of sodium thioglycollate and L-cysteine HCL, while
monohydrochloride, 1.4 g potassium nitrate, and 2.4 g the reagents in the selective stock solution inhibit, or
cadaverine dihydrochloride, and add 0.9 g of ethylamine markedly restrict, the growth of other species of anaero-
solution. Adjust the solution pH to 5.8  0.2 with bic strains of spoilage bacteria and yeasts (4).
concentrated HCl. Filter-sterilize the solution.
Culture Medium
Preparation of CLEN medium Basal medium
Add 15 g agar to 900 mL distilled water and autoclave Yeast extract 75 g
at 15 lb/in.2 and 121°C for 15 min. Cool agar slightly. Bacto-Peptone 75 g
Aseptically add 100 mL of sterile YCB-CLEN stock DL-Lactic acid sodium salt (60% syrup) 75 mL
solution to the molten agar and swirl before pouring into Sodium thioglycolate 0.75 g
sterile Petri dishes. L-Cysteine HCL 0.75 g
Dipotassium phosphate, K2HPO4·3H2O 7.5 g
Inoculation of CLEN medium
Monopotassium phosphate, KH2PO4 7.5 g
Samples containing a high population of yeast cells
Sodium chloride 7.5 g
(such as pitching yeast) may require dilution with sterile
Ammonium phosphate 7.5 g
saline solution to achieve 50–100 colony-forming units
Sodium acetate, NaC2H3O2·3H2O 7.5 g
(cfu) per plate. Wash the yeast cells to remove any beer
Distilled water 736 mL
or wort, as follows: Centrifuge the yeast culture at 4700
Selective stock solution
rpm or higher for 10 min to pelletize the cells. Decant
Sodium fusidate 0.75 g
the supernatant. Aseptically add 10 mL of fresh sterile
Cycloheximide 0.6 g
saline solution. Resuspend the cell pellet, ensuring that
Crystal violet 0.15 g
cells are well mixed in the saline. Repeat the washing
Absolute ethanol 100 mL
procedure twice. Adjust the cell concentration to
approximately 500–1,000 cells per mL (of suspect wild Reagents
yeast), as determined by hemocytometry (Yeast-4). Plate See Microbiological Control-2B.
0.1 mL of the cell suspension onto duplicate plates.
Additionally, conduct the above washing procedure Apparatus
using a brewery culture lager yeast (using a cell See Microbiological Control-2A and B.
concentration of 107 cells per milliliter) as a control to
help determine the extent of background growth by Method
brewery lager yeast on CLEN medium. Incubate plates Preparation of SMMP medium
aerobically at 27°C for 4 days or longer, if necessary. Prepare the basal medium by adding all of the
Distinct cfu that develop on CLEN may be considered to specified components to 736 mL of distilled water in a
be wild yeast. 2-L Erlenmeyer flask and heating to boiling with
Samples containing a low population of yeast cells constant agitation, using either a hot plate stirrer or
may require concentration by centrifugation before the Bunsen burner. While hot, dispense 20-mL aliquots of
wash treatment and plating procedure described above. medium into 150-mL medium bottles. Sterilize the media
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by autoclaving at 15 lb/in.2 and 121°C for 10 min. After
cooling the medium to room temperature, aseptically add
0.5 mL of the selective stock solution, tighten cap, mix by
inverting, and store in the dark at room temperature.
Inoculation of SMMP medium
Add 130 mL of the beer to be tested to the medium
bottle containing SMMP medium, recap, and mix by
inversion. Prepare duplicate samples. Incubate the sam-
ple for 14 days at 28–30°C.
Detection of Megasphaera and Pectinatus
Report visible turbidity of the sample as presumptive
positive. This should be followed by microscopic exami-
nation to determine the morphology of the organisms
involved. Pectinatus are rod-shaped and Megasphaera
are coccoid (3). In addition, the SMMP medium will
exhibit color changes. For Pectinatus, the medium will
remain purple with sediment; for Megasphaera, the
medium may exhibit a yellow color with prolonged
incubation (1, 2).
Final confirmation is obtained by identification of
volatile organic acids via gas chromatography. Pectina-
tus is identified by the production of acetic and propi-
onic acids, while Megasphaera is identified by the pro-
duction of butyric, isovaleric, valeric, caproic, and
caprylic acids (4).
Note
1. A safer alternative to cycloheximide is nystatin,
which can be substituted here, if desired (Microbio-
logical Control-5K).
References
1. American Society of Brewing Chemists. Report of Subcommit-
tee on SMMP Medium for Selective Isolation of Megasphaera
and Pectinatus. Journal 55:197, 1997.
2. American Society of Brewing Chemists. Report of Subcommit-
tee on SMMP Medium for Selective Isolation of Megasphaera
and Pectinatus. Journal 56:212, 1998.
3. Chelack, B., and Ingledew, W. M. J. Am. Soc. Brew. Chem.
45:123, 1987.
4. Lee, S. Y. J. Am. Soc. Brew. Chem. 52:11, 1994.
1998, rev. 2011
K. NYSTATIN IN SELECTIVE MEDIA
Cylcoheximide (trade name Actidione) has tradition-
ally been used in brewing selective culture media to
inhibit the growth of culture yeast while allowing bacte-
ria and some wild yeast to grow. However, cyclo-
heximide has been shown to be a teratogen, and there
has been increased concern that it may be a health haz-
ard for laboratory personnel. Therefore, a suitable non-
(or less) toxic alternative has been sought to replace
cycloheximide for inhibiting culture yeast. Nystatin, a
polyene antibiotic (3), has been shown to be effective at
inhibiting yeast in high concentration without affecting
the growth of bacteria (1, 2, 4). Two potential drawbacks
to using nystatin as a replacement for cycloheximide are,
first, nystatin tends to inhibit all yeast species found in
the brewery and will not allow for the detection of wild
yeasts (some wild yeasts are cycloheximide-resistant
while brewer’s yeasts are usually inhibited) and, second,
nystatin is more expensive and difficult to dissolve at the
concentrations needed to inhibit brewer’s yeast. How-
ever, nystatin is not regarded as a health hazard.
Reagents
(a) Ethanol, 95%.
(b) Nystatin, five million units (Sigma-Aldrich or
equivalent).
(c) Microbiological medium (any commonly used
medium for detecting brewery bacteria, e.g., UBA,
NBB, SDA, BMB, Raka Ray, MRS, WLN, etc.).
Apparatus
(a) Test tube with cap, 10-mL sterile, disposable
(b) Water bath, 47°C
(a) Autoclavable media-dispensing bottle with cap, 2-L
(b) Autoclave
Method
Media preparation
Prepare a 1-L batch of a brewing microbiological
medium according to the manufacturer’s directions in a
suitable dispensing bottle. Sterilize the medium accord-
ing to its individual requirements, and cool to 47°C in a
water bath.
Preparation of nystatin solution
The amount of nystatin needed to provide 250,000
units/L can be calculated from the activity given on the
manufacturer’s label by the following formula:
Desired level per liter
 mg/L
Manufacturer's units per mg
For example:
250,000 units/liter
 41.4 mg/L
6,038 units/mg
Weigh (transferring the nystatin with a sterile spatula)
the calculated amount of nystatin directly into a sterile
10-mL capped test tube and add 5 mL of 95% alcohol.
Thoroughly mix the contents to dissolve the nystatin and
to ensure that the contents have been sterilized. Nystatin
powder is not commonly supplied as sterile. Add the
contents of the tube to the cooled agar and thoroughly
mix to disperse the nystatin.
Agar plates should be poured immediately. After the
plates have solidified, media should be used as soon as
possible. If plates need to be stored, they should be
refrigerated at 4°C and kept in the dark.
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Notes g/L
1. It is recommended that a concentration of 250,000 Sodium acetate anhydrous 3.0
units/L (or a concentration that is adequate to inhibit Citric acid 1.25
normal fermentation concentrations of ale and lager Pantothenic acid 1.0
yeasts) can be used as a suitable replacement for cyclo- Cycloheximide 0.01
heximide in selective culture media for detecting brew- Tomato juice broth 19.0
ery bacteria in process. Agar powder (A&C American Chemicals
2. It is recommended that nystatin-containing media Ltd., Prod. # A17971 or equiv.) 1.6
should be used as fresh as possible. Refrigerated (4°C) FNI 100 dried yeast extract (Lallemand Bio
storage in the dark for as long as 2 weeks is suitable (at Ingredients or equiv.) 5.0
250,000 units/L) for use with lager yeast but is marginal Corn syrup solids (Maltrin 200,
for use with ale yeast. Media with 500,000 units/L is Grain Processing Corp. or equiv.) 29.2
effective for inhibiting ale yeast after 2 weeks of Malt extract 5.0
refrigerated storage. Sodium sulfate 4.4
3. To ensure that the addition of nystatin be as aseptic Mercaptoacetic acid 0.55
as possible, the method has been amended to include the
step of dissolving nystatin powder in 95% alcohol in a (b) Distilled water.
sterile test tube. Powdered nystatin is usually supplied (c) Agar. (Optional: 1.5–2.0% agar can be added prior
nonsterile, but the product is available in sterile solution. to boiling to produce a solid medium capable of
It is recommended to weigh the powdered nystatin into a supporting growth of Lactobacillus and Pediococcus
sterile test tube and to add 5 mL of 95% alcohol species).
followed by a thorough mixing of the contents to ensure
Apparatus
sterility of the solution.
(a) Balance.
4. It is also recommended that all nystatin-containing
(b) Measuring cylinder.
media be kept in the dark.
(c) Erlenmeyer flasks, 500-mL, with foam or cotton
References wool bung or media flask with screw cap.
1. American Society of Brewing Chemists. Report of Subcommittee (d) Microwave oven, or device capable of attaining
on Evaluation of Nystatin as an Alternative to Cycloheximide in boiling temperature.
Selective Culture Media. Journal 59:220, 2001.
2. American Society of Brewing Chemists. Report of Subcommittee
(e) Sterile tubes, 15-mL, fitted with screw caps (e.g.,
on Evaluation of Nystatin as an Alternative to Cycloheximide in centrifuge tubes or Falcon tubes).
Selective Culture Media. Journal 61:235, 2003. (f) Eppendorf pipette, 1,000-L (or equivalent) with
3. West, A., and Lewis, M. Proc. Am. Soc. Brew. Chem. 1967, pp. fitting sterile pipette tips.
129-137.
4. Sobczak, J., Russell, I., Laidlaw, L., and Stewart, R. Brew. Dig. (g) Incubator, set at 30°C.
75(5):33, 2000.
Method
2003, rev. 2011 Dissolve ingredients in 1 L of distilled water by
bringing to a boil for 2–3 min. using a microwave oven
or similar device. If a direct flame is applied, swirl the
L. HSU’S LACTOBACILLUS medium to avoid sticking or scorching. Once the
AND PEDIOCOCCUS MEDIUM medium has cooled slightly but is still hot (above 50°C),
(International Method) transfer aliquots to sterile tubes, leaving approximately 1
cm of headspace (or test sample volume). Apply caps
This medium allows for the detection of Lactobacillus
and screw tightly. If agar has been added to produce
and Pediococcus species, while restricting the growth of
solid media, add aliquots (10–15 mL) to sterile Petri
culture yeast. Hsu’s Lactobacillus and Pediococcus
dishes at this stage.
(HLP) medium (2) can be prepared as a solid or a
Freshly prepared HLP tubes may be used immediately
semisolid. However, in semisolid form, it offers a means
after cooling to approximately 40°C. Alternatively, HLP
of identifying lactic acid bacteria to the genus level on
tubes may be stored at 0–5°C prior to use. Tubes should
the basis of colony morphology.
not be stored longer than 2 weeks. Prior to use, stored
Reagents tubes should be placed in a boiling water bath or
(a) HLP medium. The formula is outlined below. How- equivalent to liquefy the medium. Care should be taken
ever, it is recommended that preformulated medium to loosen lids at this point. Once the medium is
be purchased (available from Siebel Institute of liquefied, caps should be closed tightly and medium
Technology, Chicago, IL). cooled to 40°C for inoculation.
Microbiology
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Fig. 1. Colony morphology of Pediococcus damnosus (A) and Lacto-
bacillus brevis (B) species cultivated on semisolid HLP medium.
Introduce a 0.1- to 1-mL aliquot of the test sample (or
diluted sample if heavy microbial loading is expected) to
the sterile HLP medium using a pipette. It is not
important to completely fill tubes at this stage, although
a large headspace should be avoided. Recap tubes, and
gently invert twice to distribute any microorganisms
uniformly throughout the medium. Incubate closed tubes
at 28–30°C (anaerobic environment is not required), and
analyze after 48 h for growth. Tubes that do not show
visible growth should be examined daily for a further 72
h to ensure the absence of microbes. It is recommended
that an uninoculated tube of HLP medium be maintained
in parallel with test samples to act as a negative control.
The morphology of colonies present in semisolid
media can be used to indicate the genus of the organism
present. Lactobacillus species produce cream or white
inverted teardrop-shaped colonies, while Pediococcus
species produce cream or white comet-shaped colonies
(Fig. 1).
Notes
1. To facilitate collaborative testing, only a limited
number of yeast and bacterial strains were assessed
during the collaborative study (1). While the medium is
known to be specific to lactic acid bacteria, some
untested microbes may yield variable results.
2. Description of colonies on semisolid medium may
vary according to the operator. However, a clear differ-
ence should be observed between Lactobacillus and
Pediococcus species.
3. If it is suspected that the sample may be heavily
contaminated with acetic acid bacteria, 2–4 mL of sterile
paraffin may be used to overlay the surface of the
medium after inoculation to provide a strictly anaerobic
environment and suppress the growth of these bacteria.
4. Tubes >15 mL volume can be used. However, in all
instances, narrow tubes are recommended to facilitate
observation of colonies.
5. The use of semisolid HLP media provides a quali-
tative assessment of brewery samples. For a quantitative
estimation of microbial loading, solid medium should be
utilized by the addition of agar as described.
References
1. American Society of Brewing Chemists. Report of Subcom-
mittee on HLP Medium for the Detection of Lactic Acid
Bacteria. Journal 67:247, 2009.
2. Hsu, W. P., Taparowsky, J. A., and Brenner, M. W. Two new
media for culturing of brewery organisms. Brew. Dig. 50:52
(1975).
2009