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, over-attenuation, 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

2

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

3

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 over-attenuation.

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!)

4

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 contaminations 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

5

Beer Agar, MacConkey Agar, Lees Multi-Differential Agar (a.k.a. Schwarz Differential Agar), Hsus Lactobacillus/Pediococcus Medium and Carrs 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 2530 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.

6

2. Universal Beer Agar (UBA)

Since its 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 Lees 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.

7

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. Lees 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 wort-spoiling 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. Hsus 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).

9

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. Carrs 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 yeast-selective 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, Lins Wild Yeast Medium and Lins 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 bluish-green 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. Lins 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 (non-Saccharomyces) 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. Lins 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 Lins Copper Sulfate Medium detect non-Saccharomyces wild yeast strains. Lins Wild Yeast Medium, on the other hand, is capable of capturing both non-Saccharomyces 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 Lins 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; 1-22. 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) Lees 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.

18

Yeast