BrewingTechniques
The Microbrewery Laboratory Manual-
Part III: Wild Yeast Detection and Remediation

By Fal Allen
Republished from BrewingTechniques' November/December 1994.

Last issue, Part II of this four-part series on laboratory techniques for quality control discussed bacteria detection, enumeration, and identification. This installment extends the basic principles behind the detection of infections by considering unwelcome wild yeasts. The mantra rejoins: clean, clean, clean, and always test.

Like bacteria, wild yeast can infect your beer and cause off flavors. Wild yeast can be defined as any yeast not deliberately used in your beer and not under your control. They can be harder to detect and harder to control than bacteria.

Some brewers will pitch a culture of yeast that consists of two or three strains, as is done in some British breweries (as a rule, multistrain cultures will produce more-complex beers). When you don't intend to pitch a multistrain culture or when you pick up a noncultured yeast, then you have a wild yeast infection.

Not all wild yeast will harm your beer, but their presence indicates an infection and nonsanitary procedures, which is never good.

EFFECTS

Wild yeast can produce a number of problems in your beer.

Haze or turbidity: Wild yeast often flocculate and sediment poorly. They may also be less sensitive to fining because they lack a strong negative charge.

Surface film or pellicle formation: In the presence of air, some wild yeast can grow rapidly and form a film on the surface of the beer. They can also cause haze.

Superattenuation: Wild yeast may be able to ferment sugars that normal cultured yeast cannot (like maltotetraose and dextrins), which can lead to significantly lower terminal gravities, higher alcohol content, and in some cases off flavors.

Off flavors: Each yeast produces a different flavor profile because of differences in ester, fusel alcohol, and diketone production and because of other metabolic processes. Thus, any yeast not intended to be in your beer can produce unintended flavors. Non-Saccharomyces yeast may produce radically different flavors in beer, and even very low concentrations of some yeast may have dramatic effects on beer flavor. Also, some wild yeast strains, such as Brettanomyces, may take as long as six to eight weeks before they begin to produce off flavors. This can be a serious problem in packaged beer, which may be out in the market for 30-90 days before consumption.

DETECTION

Wild yeast can be detected in several ways. It is usually not necessary to identify wild yeast after detection. Identification or accurate classification, if necessary, should be done by a professional lab. The methods of detection are as follows:

Heat resistance: Suspend your yeast in sterile water and heat to 53 °C (127 °F) for 10 min., and then take a viability test to measure the survival rate. Normal cultured yeast will not survive this test. Table I identifies the wild yeasts that are heat resistant.

Microscopic examination: This can be of limited use because a very high rate of contamination must be present for you to be able to see the offending yeast and because the infecting yeast must be morphologically different from the cultured yeast. You must also be familiar with the morphology of your cultured yeast. This method does have the advantage of being quick and easy. You can also examine the sample for spore formation. Most cultured Saccharomyces yeast do not readily form spores under normal conditions.

Selective media: Selective media are widely used in the brewing industry. They offer a simple and effective way for you to check for wild yeast contamination. Several types of media are used to detect wild yeasts.

Selective sugars. These media are prepared using a single, specific type of sugar as a nutrient for yeast growth during incubation. Only certain types of yeast will be able to grow on these specific sugars. Maltotetraose, for example, will not be fermented by most strains of Saccharomyces cerevisiae and Saccharomyces uvarum, but it will be fermented by Saccharomyces diastaticus and some other strains of wild yeast. If you plate out a sample of your beer on a medium with maltotetraose as the only sugar and you see growth, wild yeast is indicated. A strain that is more fermentive than your culture yeast will cause your beer to be overattenuated and probably cause off flavors.

Actidione. Actidione (cycloheximide) is an antibiotic that may be mixed with culture media. Many wild yeast strains have a stronger resistance to higher levels of Actidione than do cultured yeast, so normal cultured yeast will be killed off at higher concentrations (~0.2 ppm or higher).

Lysine. In normal fermentation, yeast need to take in nitrogen. They do this by using amino acids in the wort. Lysine, an amino acid, can be used only by certain strains of wild yeast. If lysine is the sole source of nitrogen in a medium and growth appears on your plates, non-Saccharomyces wild yeast contamination is indicated. No Saccharomyces yeast can grow on such a medium.

Crystal violet. By using a medium that contains crystal violet you will inhibit the growth of cultured yeast but allow the growth of Saccharomyces spp. that will not be detectable on lysine medium. The absence of growth on a lysine medium and the presence of growth on a crystal violent medium indicates that you have a noncultured Saccharomyces spp. wild yeast infection. Examples include Saccharomyces diastaticus, Saccharomyces pastorianus, and Saccharomyces cerevisiae var. ellipsoideus, all of which can have negative effects on your beer.

Use of selective media. Each of the selective media are delivered from a supplier with instructions on methods of use, applications, and material safety data sheets (often called simply MSDSs). If they do not, you should request each of these. It is important to follow all instructions and to follow good lab procedures and techniques. Always use clean and sterile equipment. Always attempt to collect samples and perform the work as quickly as possible to minimize contamination of your samples. (See Table II for further information on individual media.)

Differential media: By using a medium that contains an indicator dye, brom cresol green, you can observe differences in the way yeast use the dye. Different yeast will metabolize the dye to different degrees. This differentiation will be readily distinguishable as colonies on your plate.

By using a combination of these methods you should be able to detect wild yeast in your beer. Two other tests are worth mentioning. The first is a serological test that uses an immunofluorescence technique. The procedure for this test lies outside the scope of this article. The second is a test for respiratory deficient mutants (RDM). RDMs are cultured yeast that have mutated and are no longer able to carry out normal respiration. They are unable to oxidize glucose. The cells are usually smaller and produce a different balance of metabolic products and thus will influence the flavor of your beer. The most common test for RDMs is the triphenyltetrazolium chloride overlay (TTC overlay) technique.

TTC overlay: TTC is a colorless salt that will form a red precipitate when reduced. The accompanying box lists the equipment needed for this procedure. Aseptically collect a few milliliters of beer or yeast and, if necessary, serially dilute to ~500-1000 cells/mL for use in a 0.1-mL inoculum per plate for guidelines for counting cells, see reference1). Because you will need to count a minimum of 500 colonies to get a statistically accurate sampling, make up multiple plates. Prepare general growth agar plates in sterile petri dishes. Place 0.1 mL of sample on the agar of each petri dish by spreading the sample over the surface of the plate. Incubate for three days at 28-30 °C (82-86 °F) under aerobic conditions. Remove from incubation and overlay each plate with 20 mL of 50 °C (122 °F) TTC overlay agar. Incubate an additional 3 h at room temperature. Observe the yeast colonies. Pink to red colonies are respiratory sufficient. Colorless colonies are respiratory deficient and should be reported as a percentage. You would like to see less than 10% RDMs.

Laboratory Equipment for TTC Overlay

Petri dishes                  Test tube
Glass rod                     Pipette
General growth agar     Flask
TTC overlay agar         Incubator

HOW TO DEAL WITH WILD YEAST

Cleaning: If you find wild yeast or bacteria in your beer or on what should be sanitary surfaces (for example, inside clean fermentors or hoses), several methods can be used to combat them.

The first thing to do is to determine at what stage you are picking up the infection. You can do this by sampling at different points during the production process. For example, if you detect wild yeast (or bacteria) in the fermentor but not in the lines leading from the wort chiller to the fermentor, then you can assume it is being introduced during fermentation, either from the yeast stock or from uncleaned areas in the fermentor. Dirty equipment can account for many infections. Wild yeast and bacteria often live in areas that have not been properly cleaned and/or sanitized.

To properly clean equipment, first remove the biological matter. This is usually done using caustic soda (sodium hydroxide), chlorine, or a combination of both. Then the mineral deposits formed by beer - calcium oxalate, or beer stone, which often forms on the inside of equipment - must be removed. It is usually a brownish color and is often gritty to the touch. If you remove the biological matter but do not remove the mineral deposit, microorganisms will hide, live, and grow inside these areas. I have visited breweries that do not properly clean equipment and have seen deposits on the inside of postfermentation hard piping that was so thick it was dark brown. At this level it was not only harboring microorganisms, it was creating friction in the lines and causing the pump to work harder.

The most common way to reduce or remove beer stone is with a fairly strong hot acid or chlorine wash - though never together. Never mix chlorine and acid. Caustic soda will not remove beer stone. Acid or chlorine will. Sometimes cleaning in place will not be enough, and physical action (scrubbing) is necessary. At the Pike Place Brewery, we alternate using chlorinated caustic for one month and then regular caustic followed by a hot phosphoric acid wash the following month. To sanitize, we always use an acid blend sanitizer, which also helps reduce beer stone buildup. Chlorine used at too high of a concentration will damage stainless steel (ask your equipment manufacturer for recommended chlorine concentrations in cleaning solutions). We do not use chlorine in any form on our plate heat exchanger. The metal of the plates is very thin and can easily be damaged (especially if metal-to-metal contact is present).

Sanitizing: After the equipment is cleaned it must be sanitized. Many methods of sanitization are available. The best is heat - high temperatures for long periods of time. Heat can be expensive and is sometimes impractical. A plethora of sanitizing agents are available. You must decide which one works best for you in your brewery (we like to use a phosphoric acid blend). The important thing to remember is that for the sanitizing agent to work properly the proper concentrations (ppm) must be used for the proper contact time.

Hosing: Even when your equipment has been properly cleaned and sanitized, your brewery may still harbor microorganisms in beer hoses and rubber gaskets. When hosing gets old it starts to crack as a result of heat, stretching, wear, internal pressure during use, and harsh chemicals. When transferring beer under pressure, internal pressure within the hose causes it to expand. Beer and microorganisms may get into these cracks. If you do not clean your hoses under pressure the beer (and microorganisms) will remain in those cracks and will come out during subsequent transfers. For this reason, you must always clean hoses under pressure between uses. We clean all of our hoses in place between uses. After cleaning in place, we store them clamped together (end to end) with sanitizing solution inside. We also replace all hose once a year.

To determine whether hosing is too old, you can cut off an end section and turn it inside out and look for cracks (see Figure 1). The cracks will be much more visible when the hose is turned inside out.

Dust: Airborne dust can vector microorganisms. Excessive amounts of dust are created during construction or remodeling and especially during the milling of grain. Malt mills should always be located in an area separate from the fermentation area. Keep the amount of dust created to a minimum, and regularly clean the fermentation area so that it is free of any dust that does occur.

The floor: Floors are the greatest source of bacteria (besides the floor drain) in the brewery. Nothing that touches your beer should ever touch the floor. When visiting other breweries I am often amazed to see hose ends or gaskets laid on the floor. Even if these are "resanitized," they may still harbor live bacteria. Some sanitizers require long exposure times to work properly (10-15 min), and a casual dip in any sanitizer may not be enough to resanitize an item's surface. Experiments have shown that some spore-forming bacteria can survive 5-10 min of autoclaving. A 60% solution of ethanol is the fastest-acting sanitizer and can be handy to have ready-made in a spray bottle. For a good example of what grows on the floor, plate out 0.1 mL of liquid from the floor on a petri dish with general growth agar, or put an item on the floor and then gently place it on the agar and remove it. You will be surprised to see how much lives down there.

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PURE CULTURES AND CLEAN BREWERIES

If you do all the right things, your brewery should be free from infection. If you do pick up an infection, replace your yeast stock immediately. That is the only cure for a wild yeast infection.

But that's only the first step. Reevaluate your cleaning and sanitation regimen. Is all your equipment getting really clean? Is it being sanitized properly with the proper contact time? Review your yeast handling procedures. Could you be picking up airborne contamination from excessive grain dust? If other people work in your brewery, do they all understand proper procedures and the importance of keeping things clean and sanitary?p In the words of Malting and Brewing Science (pp. 772), "Elimination of wild yeast [and bacteria] or at least holding their numbers low, is principally a matter of using pitching yeast free of wild yeast [and bacteria] and keeping equipment sterile" (2).

ACKNOWLEDGEMENTS

I would like to extend special thanks to Dave Logsden of Wyeast Laboratories (Hood River, Oregon) and to the Siebel Institute (Chicago). Special thanks also to Toby Malina of ParaGraphics (Seattle) for assistance with the technical drawings.

REFERENCES

(1) Fal Allen, "The Microbrewery Laboratory Manual - Part I: Yeast Management," BrewingTechniques 2 (4), 28-35 (1994).

(2) Malting & Brewing Science, D.E. Briggs, J.S. Hough, R. Stevens, and T.W. Young (Chapman and Hall, London, 1981).

Further Reading

ASBC Methods of Analysis, 8th ed. (American Society of Brewing Chemists, Egan, Minnesota, 1992).

Malting & Brewing Science, D.E. Briggs, J.S. Hough, R. Stevens, and T.W. Young (Chapman and Hall, London, 1981).

Microbiological Methods (Siebel Institute, Chicago,1994).

The Practical Brewer, M. Meilgaard (Master Brewers Association of the Americas, Madison, Wisconsin, 1977).

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