Microbiological Media for Bacteria and Wild Yeast Detection in the
Brewery.
Gary Spedding, BDAS, LLC (From a 2000 Seminar).
Abstract.
The brewery microbiologist, expert and novice alike, has been presented
with a very wide array of growth media within the past 50 years. The
choice of which media to choose for a particular purpose (yeast, wild
yeast, mould or bacterial strain selection, detection and identification)
has not always been an easy one. This paper will provide a listing of the
properties of a select few media useful for the brewing microbiologist.
The topic will be handled from the perspective of the novice
microbiologist in the craft brewery environment. In consequence of
this, the considerations for the selection and use of such media will be
discussed. Furthermore, as a prelude to the main topic, a discussion as
to the types of, and the properties of, various microbial contaminants
most commonly found in the brewery will be entertained.
Introduction.
The ideal situation for the lager and ale brewer is to have only one
species of organism ever present in the beer; namely the culture yeast
pitched into the hopped wort. However, by the very nature of the sheer
abundance of microorganisms present in the environment this ideal
situation is never realized. There are many bacterial species and wild
yeasts (also moulds) in the environment or in the brewers’ raw
materials, which can infect beer; some cause damage while others do
not. Every brewer must therefore be aware of the types of organisms,
the kinds of spoilage they can render on the final product, and how to
effectively remove them as potential contaminants. The detection of
contaminating bacteria and wild yeast strains is often very much easier
than their identification; wild yeast strains being much more difficult to
identify than individual bacterial strains. The purpose, therefore, of
this paper is not to describe in any detail how to detect and recognize
many individual species. Instead, the intention is to illustrate a few of
the most useful kinds of media available which will enable you as a
microbiologist to determine if you do actually have contamination by
wild yeast or beer spoiling bacteria. The mere presence of contaminants
1
is sufficient news to send most brewers back into the brewery in order
to locate and clean up the source of contamination.
Some History and Recent Developments.
In 1950 two microbiologists, Green and Grey, introduced the antibiotic
cycloheximide (actidione) as a very powerful inhibitor of culture yeast.
Through the use of cycloheximide the main organism found in
fermenting wort and beer (the culture yeast!) could be suppressed. This
suppression allowed most other contaminating organisms to grow and
to be visualized on/in the culture media. Cycloheximide was thus
introduced into many selective media from the 1950s through the 1980s.
In addition, many other types of growth media were also developed
during this period. Nine media are described in this paper (some
incorporating cycloheximide into the formula and some not) which can
be used to determine bacterial and wild yeast contamination. One
major problem has now, however, arisen for the microbiologist in that
the antibiotic cycloheximide is no longer available. Brewers will need to
be aware of this as they now select the kinds of media that they will use
in their own breweries. More importantly microbiologists now need to
go and find other antibiotics and inhibitors of culture yeast which will
prove as useful as cycloheximide.
Beer Spoilage.
The potential sources of beer spoilage organisms in the brewery include
air, soil, water, raw materials, grain/malt dust, pitching yeast,
processing aids, vermin, human skin, brewery equipment, plant and
machinery. The types of spoilage caused by microorganisms include the
following; turbidity, haze formation, ‘rope’ (slime) formation, overattenuation, gushing, souring of wort and beer and the production of
varied off-flavors. Some of the key organisms (yeast and bacteria) that
are responsible for beer spoilage are illustrated in Table 1. As a brewer,
a comprehensive knowledge of the types of organisms that can result in
beer spoilage is most important. An understanding of the sources of
infection, together with a knowledge of the kinds of damage (including
the aromas and flavors) caused by microorganisms, can provide a useful
baseline which can be used to guide the type of microbiology testing
needed when a contamination issue arises. Presented in Table 1 is a
listing of bacteria and wild yeast strains, along with key characteristics
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of morphology, behavior, and kinds of damage caused by them.
Following this, the article moves on into looking at several key media
that can be used in the detection, identification and enumeration of wort
and beer spoiling organisms.
The brewer can often gauge the type of damage caused in the infected
beer by simple visual inspection of the product and by tasting. This
preliminary testing is then followed by implementation of
microbiological procedures in order to identify the contaminating
organism(s). The location of the contamination in the brewery is also
determined via microbiology testing. For the expert, the colony
morphologies, sizes, and colors of the organisms growing on selected
media can be used to more specifically identify bacterial and yeast
strains.
Table 1. Some of the main beer spoilage organisms and their
properties.
Organism
Properties
Bacteria
Lactobacillus
Produce ‘silky’ turbidity and makes beer sour
via lactic (and acetic) acid production. Some
strains produce diacetyl; some produce rope.
Gram positive, rod shaped cells. Catalase
negative. Facultative anaerobes. Hop tolerant.
Pediococcus
Makes beer sour (Lactic acid). Some strains
produce diacetyl. Gram positive, round
(coccus-shaped) cells frequently found as
tetrads but can appear as diplococci. Only
cocci to grow in beer. Beer “sarcina”
organisms. Catalase negative.
Enterobacteriaceae
A family that includes the coliform bacteria.
Often found in water supplies. Gram negative
with rod-shaped cells. Most are facultative
anaerobes. Grow in wort to produce off-flavors
(acetate, celery-like, parsnip, phenols, cooked
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cabbage and DMS) but do not usually grow in
beer. All catalase positive and oxidase negative.
Includes the genera, Escherichia, Aerobacter
and Klebsiella.
Acetobacter
Gram negative. These organisms are very small
and exhibit varied rod-shaped cells. Produce
acetic acid (vinegar) in beer and ropiness. An
overoxidizer. Aerobes. Catalase positive and
oxidase negative. Hop resistant
Gluconobacter
Formerly called Acetomonas. Gram negative.
These appear as very small rod-shaped cells.
Produce acetic acid in beer, sometimes giving a
cidery note. Aerobes. Catalase positive and
oxidase negative.
Zymomonas
Gram negative plump rod-shaped cells that can
grow in cask-conditioned beer. They are quite
common in water, soil and plant material. May
form clumps or rosettes. Prefer acidic to
neutral pH. Anaerobic. Produce off-flavors
(acetaldehyde; rotten apples! and hydrogen
sulfide) and turbidity.
Pectinatus
Gram negative, rod-shaped and strictly
anaerobic. Produce a range of off-flavors (acids
and organic sulfur compounds) and turbidity.
Yeast
Saccharomyces
Wild strains produce phenolic off-flavors and
can lead to over-carbonation of beer via overattenuation.
Brettanomyces
Produce large quantities of acids and are a
major contributor of other off-flavors (ethyl
acetate and ethyl lactate), especially in draft
beers. Aerobic. Very slow growing (detect
problem after 1- 2 months!)
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Dekkera
This yeast produces acetic acid.
Kloeckera
Produce acids and other off-flavors (acetic acid,
lactic acid, esters). Aerobic. Causes cloudiness
in beer.
Pichia*
Produces cloudiness and off-flavors. Aerobic.
Candida*
Produces cloudiness and off-flavors (esters,
acids, phenolics). Aerobic.
Hansenula*
Produces cloudiness and off-flavors.
*As these organisms are aerobes, their spoilage potential is limited to
beers stored in the presence of air. However, under suitable conditions
they grow rapidly and often give rise to films on the surface of the beer
as well as resulting in the production of hazes and off-flavors.
A Discussion of Brewery Bacteria.
The identification of beer spoilage bacteria is of primary importance to
the brewer. Bacterial contamination’s in the brewery of concern to the
brewer fall into two categories, beer spoilers and wort spoilers. The
most common and most troublesome of the beer spoilers are the lactic
acid bacteria (see Table 1). These organisms thrive during oxygen
deprivation in fermentation and storage. About 10000 lactic acid
bacteria per mL produce detectable spoilage of beer. Wort spoilers, on
the other hand, grow and spoil beer in the early stages of fermentation.
These typically belong to the family Enterobacteriaceae (Table 1).
Depending on the specific organism in the Enterobacteriaceae family
only 300-800 bacteria per mL can lead to the spoilage of beer. In
addition to these organisms there are indeed many others that can grow
in the rich substrate of beer, many being classified as non-beer spoilage
bacteria. These non-spoilage organisms will not be a focus of the
present report.
Six different types of bacterial growth media are discussed below. While
there are very many media available, the following represent a good
spectrum for the detection of most brewery microorganisms. The media
presented are; Wallerstein Laboratories Nutrient Medium, Universal
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Beer Agar, MacConkey Agar, Lee’s Multi-Differential Agar (a.k.a.
Schwarz Differential Agar), Hsu’s Lactobacillus/Pediococcus Medium
and Carr’s Bromocresol Green Medium.
Media Specific or Useful for Bacteria.
1.
Wallerstein Laboratories Nutrient Medium (WLN)
This is a complex growth medium with an early and interesting
history. It is defined as a medium for the differentiation of wild
yeasts, culture yeasts and bacteria. Originally as WLN, Green
and Grey (see “History”) added cycloheximide (actidione) at 4
ppm. and with this addition the medium became known as
Wallerstein Laboratories Differential Agar (WLD). As defined
above, it allows for the growth and enumeration of many brewery
organisms, both yeast and bacteria. See the section on yeast for
further details concerning the use of this medium for yeast
microbiology. For bacteria, WLN picks up most of the wort
spoilers but not the species known as Obesumbacteria.
Disadvantage:- 7 to 14 days are required for full growth of, for
example, fastidious lactic acid bacteria.
The plates with this medium are incubated aerobically or
anaerobically (3-16 days) at 25–30 oC, depending upon the types
of bacteria suspected to be present in the sample. Under
anaerobic conditions it is good for beer cocci and lactic rods.
Under an aerobic environment it is a good medium for acetic
rods. The pH should be at 5.5 for brewery work. The
microbiologist needs to be very well aware of brewery
microorganisms and laboratory practice to work with this culture
medium. Furthermore, this medium has seen some modifications
over the years and the interested reader is referred to the
reference by Casey and Ingledew (Feb., 1981), as cited in the
reference section, for further information on the development and
use of this media.
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2.
Universal Beer Agar (UBA)
Since it’s development at the Jos. Schlitz Brewing Company this
has been a favorite medium for brewery microbiologists. This
medium may be described as a universal medium that will enable
the growth of yeasts, wort spoiling bacteria, common
environmental bacteria (coliforms), and beer spoilers that require
oxygen. As a general-purpose microbiological medium it is
therefore used for the detection of many brewery bacteria. It has
similar components to those found in Lee’s Multi-Differential
Agar (LMDA, discussed in detail below) with the addition of beer,
and originally with the addition of 1 ppm of cycloheximide
(actidione). [It can of course be used without cycloheximide and,
in this case, is suitable for the detection of most yeasts.]
Satisfactory recovery is attained for wort and beer
microorganisms but this is not a differential medium; it only gives
total counts! Compared to WLN (see above) it was found to be
better for the recovery of acetic acid and lactic acid bacteria.
Plates are generally incubated at 28 oC for about 3 days. Aerobic
incubation is needed for measuring overall sanitation
(Enterobacteria and acetic acid bacteria) and for the detection of
yeasts (and molds!). For confirmation of Pediococcus or
Lactobacilli, plates should obviously be examined for growth
under anaerobic conditions.
The recovery of organisms is purported to be better on LMDA
than on UBA (see below). LMDA may therefore now be the
better choice. The product profile supplied with the UBA media
from the manufacturer can be consulted, as well as the references
cited in Casey and Ingledew (Feb., 1981), for further information
on the use of this product. It should be stated, as for WLN, that
the microbiologist needs to be well versed in the field and to really
understand the different yeast strains and bacteria that can be
found in the brewery in order to make best use of this growth
medium.
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3.
MacConkey Agar
A medium used for the detection, isolation and enumeration of
Enterobacteriaceae. This differential medium, for the detection of
most coliforms, has lactose, crystal violet and a neutral red
indicator as part of the formula. The crystal violet inhibits the
growth of Gram positive organisms.
The differential nature of this medium is also based upon the
production of acids from lactose fermentation by various
organisms (especially coliforms). Acid is produced and the
neutral red is absorbed to give rise to brick-red colonies.
Incubation (for brewery work) is typically allowed for four days
at 30 oC prior to examination of the plates. Coliforms develop as
dark pink to red colonies. Other Gram negative species form
translucent almost colorless colonies. Gram positives are
suppressed. Note, some yeast strains will grow on this medium!
4.
Lee’s Multi-Differential Agar (LMDA, a.k.a. Schwarz Differential
Agar, SDA)
Used for the detection and enumeration of yeast and bacteria in
the brewery. Allows for the growth of organisms including wortspoiling bacteria and beer spoiling bacteria. Lactobacilli,
Pediococci and Enterobacteria grow well, as do yeasts in the
absence of cycloheximide.
LMDA confers differential properties due to the presence of
bromocresol green and insoluble calcium carbonate (powdered
chalk). The bromocresol green turns yellow in the presence of
acid and the acid dissolves the calcium carbonate. Lactic acid
bacteria, Pediococcus species, and acetic acid bacteria that
produce acid, dissolve the calcium carbonate, and yield a clear
yellow halo around them distinguishable from the rest of the
medium.
Organisms exhibit markedly different colony morphologies (and
colony sizes) on LMDA making it useful as an identification
medium. Colonies also take on different colors (as also seen
8
above) according to genus or species (ranging from colorless/white
to yellow, to green to blue, with varying shades inbetween).
Media is prepared and poured into plates. Incubation is usually
at 30 oC for 2 days if aerobic, and 5 days for anaerobic conditions.
For the identification of possible Pediococcus species or
Lactobacillus species incubate under 5% CO2. (Hint, place plates
in an air-tight jar with a lighted candle! or use commercially
available anaerobic jars). To detect acetic acid bacteria (and wort
spoilers) incubate the plates aerobically at 30 oC.
The aim is for a maximum of 25-30 colonies per plate using a
suitable dilution of the suspect/test sample. To suppress yeast,
cycloheximide (actidione) is added at 7-10 ppm final
concentration.
For the differentiation of the acid-producing bacteria the plates
may also be viewed from below. Lactic acid producing colonies
are generally yellow underneath whereas acetic acid producing
colonies are blue-green. If in doubt about an identity, the
catalase test-reaction can also be performed. Lactobacilli give a
negative result and Acetobacteria and Gluconobacteria give rise to
a positive result. (See the Appendix for details of the catalase
reaction.)
5.
Hsu’s Lactobacillus-Pediococcus Medium (HLPM)
This is a selective medium for the detection and enumeration of
Lactic acid bacteria. Enterobacteria and yeast do not grow on this
medium. [The medium has cycloheximide (actidione) as part of
the formula; this is now a concern as the production of this drug
has been discontinued.]
This media is used typically in culture tubes rather than in plates.
Moreover, it was also designed to be a user friendly medium.
Autoclaving is not necessary for sterilization in the preparation
(boiling for 2-3 minutes suffices!). In addition, no external
anaerobic growth apparatus is needed either (sodium
thioglycolate is incorporated in the formula in order to maintain a
reducing environment).
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Typically 0.1-1.0 mL of test sample is added to a tube of cooled
HLP and incubation allowed at 30 oC for 40-48 hours
(preliminary count) and 64-72 hours (final count). Acetic acid
bacteria can be suppressed by an overlay of 2-4 mL of sterile
paraffin on the surface to block air access. The media can be used
on plates for membrane filtration work via the addition of extra
agar.
Lactobacillus colonies are identified as light/white with inverted
teardrop shapes. Pediococci are identified as light/white
circular/spherical/round colonies. Any growth on the surface of
the tube should be disregarded.
6.
Carr’s Bromocresol Green Medium
This is a simple medium for the detection of acetic acid bacteria.
The medium consists of 3% yeast extract, 2% agar, 1 mL of a
2.2% solution of bromocresol green/L and 2% ethanol. Further
details are cited in Casey and Ingledew (1981).
Gluconobacter (Acetomonas) colonies change the indicator from
blue-green to yellow. Acetobacter species do the same initially but
then revert back to blue-green as acetic acid is used up.
Summary: Bacterial growth media.
As seen above, several media are available for a fairly comprehensive
evaluation of relevant brewery bacteria. Some are differential in nature
and some are more for simple detection of contamination and/or the
enumeration of the organisms. Many other media are available (consult
the references for further details) and all have their advantages and
disadvantages. The use of any media and the interpretation of growth
upon them does, however, require practice and expertise. The
information in this presentation should give you some idea as to how the
various media discussed herein were designed and developed and which
could be right for your own use. The recipes for most of the media cited
above are also available and can again be found by consulting the
references in the bibliography. As mentioned previously one of the
biggest concerns at the moment, for the use of selective media, is the
ending of the production of cycloheximide (actidione). This drug has
10
been the only real choice for suppressing the growth of culture yeast in
media used for the detection of bacteria (see the section on yeastselective media below for alternative strategies for culture yeast
suppression). Microbiologists must now very quickly find a
replacement for this very useful (but dangerous) antibiotic if HLP and
related media are to continue to be a useful tool for the brewery
microbiologist.
A Discussion of Brewery Yeast Strains.
Wild yeast strains come in two groups, Saccharomyces wild yeast and
Non-Saccharomyces wild yeast. Wild yeast in fermenting and primary
aging tanks usually comes from infected pitching yeast. Wild yeast in
bottled product is also a major issue. High wild yeast numbers in a beer
is usually a result of poor sanitizing in the bottling/storage area. Wild
yeast if found to be growing in the product will lead to major off-flavor
issues and spoilage. Again, as for bacteria, a number of types of yeast
culture media have been developed especially within the past 50 years in
order to detect and enumerate spoilage yeast strains. Three types of
selective media are advocated for the suppression of brewing yeast
strains (ale and lager) and the subsequent detection of wild yeasts.
These are Lysine Media, Lin’s Wild Yeast Medium and Lin’s Copper
Sulfate Medium. A discussion of each is presented below. These media
were originally developed to test the quality of brewers yeast but can
also be used to test wort and beer. In addition, Wallerstein
Laboratories Nutrient Medium is also useful for evaluation of yeast
strains; a brief discussion follows.
Media Specific or Useful for Yeast.
1.
Wallerstein Laboratories Nutrient Medium (WLN)
Allows the growth of many brewery organisms, yeast and bacteria
(see also the section on bacterial media). All yeasts will grow on
this media with differing colony morphology. The medium
contains bromocresol green which makes it a differential media.
Different species of yeast vary in their ability to utilize this dye
and appear as readily distinguishable colonies on this medium.
Saccharomyces cerevisiae grow as white, pale green or pale bluishgreen raised colonies. Other Saccharomyces species and other
11
general wild yeast can be distinguished from culture yeast due to
distinguishing morphology (e.g., wrinkled, raised, rough or
powdery characteristics). Color (as stated above) also differs
(ranging from white through shades of blue, to green). Quite
specifically, it can be used for the detection of “phenolic” wild
yeasts. These are differentiated from normal S. cerevisiae because
they fail to reduce the bromocresol green dye. As a result they
appear as fairly flat, large dark/bottle green colonies.
Aerobic incubation for this medium is at 25-30 oC. As the
medium also supports bacterial growth the microbiologist must
really know yeast characteristics for successful detection of
contaminating wild yeast and bacteria. The pH should be at 5.5
for the reliable count of brewers yeast.
2.
Lysine Medium (LYS)
This is a medium, containing lysine as the sole source of nitrogen,
and is used for the differentiation of Saccharomyces species from
non-Saccharomyces species of yeast. Saccharomyces yeast (culture
and wild) cannot utilize lysine as a sole source of nitrogen but
non-Saccharomyces yeast can grow on a medium incorporating
lysine. Any yeast found growing on lysine media is therefore
usually non-Saccharomyces (Candida, Pichia, Dekkera, etc.)
Disadvantages: Yeast cultures must be rinsed free of the original
cultivation medium (using saline washes) in order to remove
exogenous nitrogen. Some expertise is also required in the
enumeration and recognition of growth on lysine agar because of
the possible growth of bacteria (e.g., Escherichia coli and acetic
acid bacteria) or growth of culture yeast on trace nitrogen
impurities, especially if less than 104 viable culture yeast cells are
plated. Also, as wild yeast grow and die they autolyse releasing
nitrogen-containing nutrients, which then allow culture yeast to
grow.
Incubation of plates is at 25 oC for about 5 days prior to
examination for growth. Discreet colonies within a background of
haze of culture yeast that have not developed are indicative of
non-Saccharomyces species. (Plate 0.2 mL of a sample containing
12
106 cells/mL.) Count the number of colonies which develop and
express the degree of contamination as the number of wild yeast
cells per million cells of the original inoculum.
3.
Lin’s Wild Yeast Medium (LWYM)
A medium for the detection of most wild Saccharomyces yeasts.
Crystal violet and a fuchsin-sulfite mixture suppress the brewers’
yeast and supports wild yeast growth into distinct colonies.
Culture yeast strains will appear as micro-colonies but a wide
range of wild yeasts (Saccharomyces, Candida, Oospora,
Rhodotorula, and other species) grow as sizable colonies.
Caution: - some Lysine positive wild yeast strains (nonSaccharomyces) do not grow on LWYM and so it is recommended
to use Lysine medium and LWYM together in order to capture
most wild yeast types.
Incubation under aerobic conditions is for 4-6 days at 28 oC.
Distinct colonies on the medium may be considered as wild yeast.
(Plate 1 x 106 cells of culture yeast.) Plates should be used within
five days of preparation as wild yeasts are inhibited after
prolonged standing on this medium.
4.
Lin’s Copper Sulfate Medium (LCSM)
A medium also formulated to detect primarily non-Saccharomyces
wild yeast whilst suppressing culture yeast. Copper in low
concentrations increases the growth of yeast cells. At higher
concentrations it inhibits yeast growth. Copper sulfate is adjusted
to give optimum conditions for wild yeast growth and culture
yeast inhibition. (Note some Saccharomyces wild yeast will also
grow on this medium.) This medium does not allow for
identification.
Plates are incubated at 28 oC aerobically for 2-6 days and
approximately 1 x 106 culture yeast cells are plated when testing
for pitching yeast quality. Distinct colonies represent wild yeast.
Plates should be used within 3 days of preparation or else
inhibition of wild yeast also occurs.
13
Summary of Wild Yeast media.
Wild yeast detection in pitching yeast or in wort and beer can only be
determined when the culture yeast is suppressed. A number of ways to
achieve this have been built into various types of “yeast specific”
microbiological media. Lysine medium and Lin’s Copper Sulfate
Medium detect non-Saccharomyces wild yeast strains. Lins’ Wild Yeast
Medium, on the other hand, is capable of capturing both nonSaccharomyces and Saccharomyces wild yeast strains. Wallerstein
Laboratories Nutrient Medium can also be used to detect most yeast
strains and has some differential capabilities. It is typically
recommended, however, that both of the Lin’s media types be used
alongside Lysine medium in routine practice in order to capture the full
spectrum of wild yeasts which might be present in the brewhouse.
Concluding Statement.
It is hoped that the information provided herein will enable you to get
started, and to feel comfortable, in the brewery microbiology lab. The
primary consideration here has been the simple detection of
contamination. In some cases we touched upon further identification.
Definitive identification of brewery bacteria or brewery yeast strains is
not an easy task, nor always a necessary one. Furthermore, there is no
readily apparent “perfect” medium for the detection and enumeration
of all microbial floras of wort, beer or brewing ingredients. You will
need to work with several different media and become competent in
handling and detecting specific microorganisms on the various media if
you are to be successful in detecting microbial contaminants. The
interpretation of results will also involve the use of specific chemical
tests and the efficient use of the microscope. The catalase and oxidase
tests, which are quite useful, are presented in the Appendix to this
paper.
Normally, as stated above, a simple determination that there are
contaminants in the brewery or the final product (beer) is sufficient for
most small-brewery microbiologists. If, however, you are really keen to
definitively identify specific contaminants you will need a very well
equipped laboratory. You should also acquire and maintain a culture
collection of pure known species of bacteria and yeast strains for
comparison purposes; but beware not to let them become the sources of
14
new contamination! A good manual showing microscopic images
(preferably in color) of brewery microorganisms, together with full
descriptions of their growth characteristics and properties, would also
be a worthwhile investment. Alternatively of course, you can always
send out your samples to one of the reputable microbiology testing
facilities. With your knowledge of brewery bacteria/wild yeast in hand
the interpretation of external laboratory reports will be simplified.
No matter which route you take, learning about the microorganisms
that invade your brewery will help you to be a better brewer. Details of
when, where, and how to sample for organisms in the brewery can be
obtained by seeking out other references listed in the bibliography. The
author would also be happy to supply additional information upon
request. Seeking out potential contaminants in the brewhouse can be a
rewarding experience in many ways and not just in protecting your
brews!
References.
Material utilized in the compilation of this article includes the following
references. Some of these references are useful for quality control
information and in dealing with brewery hygiene; particularly useful if
you do find contamination in your brewery!
Brewers Laboratory Handbook. Brewing Science Institute, Colorado
Springs, CO. www.brewingscience.com.
Casey, G. P. and Ingledew, W. M. (1981) The Use and Understanding of
Media Used in Brewing Bacteriology: I.
Early History and Development of General Purpose Media.
Brewers Digest, February; 26-32.
II.
Selective Media for the Isolation of Lactic Acid Bacteria. Brewers
Digest, March; 38-45
III. Selective Media for Gram-Negative Bacteria. Brewers Digest,
April; 24-35.
Dowhanick, T. M. (1990) Is There a Need for Microbiological Quality
Control in a Microbrewery? Brewers Digest (December); 14-17, 34.
15
Gillet, Ir. A. and Sopora, S. A. (1989) The Practical Identification of
Beer-Spoiling Bacteria. Monatsschrift fur Brauwissenschaft. No., 12; 122.
Ingledew, W. M. and Casey, G. P. (1982) The Use and Understanding of
Media Used in Brewing Mycology: I.
Media for Wild Yeast. Brewers Digest, March; 18-21.
II.
Media for Moulds/Rapid Methods. Brewers Digest, April; 22-26,
50.
Priest, F. G. (1990) “Contamination” in, An Introduction to Brewing
Science and Technology (Series II) Vol., 3, “Quality”, Chapter 1; 1-15.
The Institute of Brewing.
Lee, S. Y. et al. (1975) Lee’s Multi-Differential Agar (LMDA); A
Culture Medium for Enumeration and Identification of Brewery
Bacteria. ASBC Proc., 33 (1); 1-42.
Lin, Y. (1974) Detection of Wild Yeasts in the Brewery III. A New
Differential Medium. ASBC Proc., 69-76.
Lin, Y. (1981) Formulation and Testing of Cupric Sulphate Medium for
Wild Yeast Detection. J. Inst. Brew., 87; 151-154.
Middlekauf, J. E. et al. (1983) Microbiology Subcommittee. ASBC Proc.,
41 (3); 100-103.
Morris, E. O. and Eddy, A. A. (1957) Method for the Measurement of
Wild Yeast Infection in Pitching Yeast. J. Inst. Brew., 63; 34-35.
Scheer, F. M. (1996) Microbiological Control in a Micro-Brewery.
MBAA Tech. Q., 33 (2); 87-90.
Simpson, W. J. (1991) Shedding Light On Brewery Hygiene. J. Biol.
Education. 25 (4); 257-262.
Simpson, W. J. (1995; 1996) Microbiology for the small brewer (Parts I
and II). Brewers Guardian, Dec ’95; 33-37 and March ’96; 21-25.
16
Smith, C. E., Casey, G. P., and Ingledew. W. M. (1987) The Use and
Understanding of Media Used in Brewing Microbiology. Update 1987.
Brewers Digest (October); 12-16, 43.
Van Keer, C., Van Melkebeke, L., Vertriest, W., Hoozee, G. and
Schoonenberghe, E. Van. (1983) Growth of Lactobacillus Species on
Different Media. J. Inst. Brew. 89; 361-363.
Walters, L. S. and Thiselton, M. R. (1953) Utilization of Lysine By
Yeasts. J. Inst. Brew. 59; 401-404.
17
Appendix: - Catalase and Oxidase Tests.
Information is taken from the Brewers Laboratory Handbook provided
by the Brewing Science Institute (BSI). See the bibliography for citation
details.
The Catalase Test:
Drop a small quantity of 3% hydrogen peroxide
onto a colony on a plate, or onto bacteria
smeared onto a microscope slide. A positive
result is indicated by the formation of bubbles.
The Oxidase Test:
Smear a colony of interest onto a Dryslide
oxidase test slide (obtainable from Difco). A
positive result is indicated by the bacterial
smear turning a very dark purple color within a
couple of minutes.
These tests and the Gram staining test are useful sources of information
together with that obtained by the culturing of organisms. The
Gram staining test is discussed in the BSI manual and in many other
microbiology methods manuals.
If an organism proves to be Gram positive go to the catalase test.
If catalase negative, the organism is likely a lactic acid bacterium.
If catalase positive, the organism is likely a non-beer spoiler.
If an organism is Gram negative go to the oxidase test.
If oxidase negative, the organism is likely a wort spoiler.
If oxidase positive, the organism is likely a non-beer spoiler.
End.
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