by Louis K. Bonham (Brewing Techniques - Vol. 6, No.3)
Easy quality control tests let you identify the source of off-flavors and screen for bacterial infection at very little cost. They’re so easy, there’s no reason every brewer can’t do them.
In the first installment of The Experimental Brewer (1), I described several possible setups for a small-scale brewing laboratory. In this article, we’ll put some of this lab equipment to use with some simple lab tests that I have adapted from a number of sources. All of them are easy to run, require a minimum of equipment, and provide useful information on your beer and brewing procedures.
Some of these tests involve “force testing,” in which wort or beer is stressed by warming it to the optimal temperature for the growth of various microorganisms. I have adapted these tests from an excellent brewery laboratory manual by Dr. Paul Farnsworth (2), who calls these methods “no lab, no brain, no excuse tests” that any commercial brewer should run on every batch of beer and that are within reach of every home brewer. For those who want more data on their QA/QC work, I include some secondary tests than can be performed to identify the most common offending organisms found through the force tests. Finally, I include several easy tests for assaying whether your beer is subject to increased diacetyl levels, what your final gravity will be, whether your sweet wort is sufficiently clear, and whether your yeast has mutated to unacceptable levels.
Force Testing for Stability
Wort Stability Test
The wort stability test provides a simple but effective way to see if your sanitization and brewing procedures, from the end of the boil to the pitching of yeast, are up to snuff. This test is so ridiculously simple that you can (and should) run it every time you brew.
Materials: For this test, you need only a sterile container (50-mL centrifuge tube, small flask, baby food jar, or other suitable vessel), a wine thief or turkey baster (if needed to draw a sample), and a water bath or incubator (optional).
Procedure: Follow the basic procedure for obtaining samples for analysis (see box, “How to Take Samples for Force Tests,” opposite). After you’ve run the wort into the fermentor, but before you pitch the yeast, transfer about 40–100 mL of wort to your sample container, cover, and set it in a warm, dark place. Check it every 12 hours or so. If the wort remains clear and shows no signs of growth for more than 48 hours, your sanitization is probably good enough; whatever contaminants may exist in your wort are probably at such low levels that yeast pitched at a decent rate would probably overwhelm and outcompete the contaminants. If the sample stays clear for 72 hours or more, pat yourself on the back for excellent attention to cleaning and sanitization. On the other hand, if the wort turns cloudy or shows other signs of growth within 24 hours, you’ve got major problems with your sanitization methods.
Once the sample turns cloudy or shows growth, take a sniff or sip to gather a little data on what’s likely to be growing (does it smell or taste like vinegar? rotten eggs? fruit? vegetables?). If you want to identify any bacterial contaminants more precisely, aseptically transfer a bit of the sample to a plate of LMDA (described later in the article) and wait for colonies to grow. (If you intend to do an LMDA plate, do the aseptic transfer before you sniff or sip.)
Pitched Wort Stability Test
Yeast and pitching procedures can also be contamination vectors, particularly if you are repitching yeast from a previous batch. To test for contaminants in your slurry or pitching methods, you need to kill off the brewers yeast but leave the contaminants alive. Fortunately, cycloheximide (also known as Actidione) will do just that.
Materials: Same materials as in the wort stability test, plus cycloheximide solution in a syringe with 0.22-micron filter. You can prepare your own solution by adding 100 mg of cycloheximide to 100 mL distilled water, but you still need a syringe and filter. Cycloheximide is heat-sensitive, so store your solution in a refrigerator (but keep in mind that cycloheximide is nasty stuff — please keep it away from children!).
Procedure: This test is performed exactly the same as the wort stability test, except that you take the sample shortly after pitching the yeast. Using the syringe with the filter attached, add the cycloheximide solution to the sample at the rate of 1 mL per 100 mL of pitched wort. They syringe filter keeps the cycloheximide solution from being a potential source of contaminants. Mark this sample as poison — you don’t want to sip this by mistake. Incubate it the same way as in the wort stability test. Cycloheximide kills brewers yeast, so if anything grows in your sample it’s not something you typically want (unless you’re brewing lambic, of course). If the sample stays clear and shows no signs of growth for 72 hours or more, all is well.
Running both the wort and pitched wort stability tests in tandem is easy to do — just take one sample before pitching and the other right after — and gives the most meaningful results. If your wort stability test comes out OK but the pitched wort stability test fails, then it’s likely that your yeast is infected or you otherwise introduced an infection at pitching. If the wort stability test fails but the pitched wort test comes out OK, then the contaminant is something sensitive to cycloheximide — such as brewers yeast.
Bright Beer Test
Even if you have done everything correctly up front, your beer can still pick up an infection during racking, filtering, or packaging. This force test can alert you to such problems many days or weeks before they are noticeable in the beer.
Materials: Same as for the pitched wort stability test, except that you need two sterile containers.
Procedure: Follow the same procedure as for the wort and pitched wort stability tests, only use bright beer. If you plan to sample beer in kegs or serving tanks, sanitize the sample port or serving hose first, then take two samples. If testing bottled beer, wipe the crown and neck down with alcohol, uncap, flame the bottle mouth, and quickly pour samples into the two sterile containers. In either case, add cycloheximide to one sample and mark it, and incubate both samples in a warm, dark place.
If either sample shows signs of growth within seven days, you’ve got a problem. If the plain sample shows growth but the cycloheximide sample does not, your contamination is an organism that’s sensitive to cycloheximide — such as brewers yeast. In that case, either the beer wasn’t quite finished fermenting before going to the bright tank, or you have some sort of mutant or wild yeast that’s eating dextrins in the beer. If both samples show growth, you’ve probably got a bacterial infection.
Interpreting Force Test Results with LMDA
Lee’s Multi-Differential Agar (LMDA) is really cool stuff. It is a special growth medium developed and patented by S.Y. Lee of Coors Brewing Company in the 1970s (3). It contains cycloheximide and therefore will not support the growth of brewers yeast, but will support and encourage the growth of most common brewery bacteria better than Universal Beer Agar (UBA). I’ve discovered that it can also be used to test for certain black molds endemic to the Texas Gulf Coast (a common contaminant in my brewery).
What makes LMDA particularly neat is that besides being very user-friendly, it differentiates between the various genera of common bacteria. For example, a colony of Lactobacillus will look different from one of Pediococcus, and both will look different from acetic acid bacteria colonies, based on color and texture (see box, “Identifying Common Brewery Bacteria,” opposite). Further, while anaerobic (oxygenless) incubation is needed to grow colonies of Zymomonas, Megasphaera, and other true anaerobes, most of the bacteria of interest to brewers — including facultative anaerobes such as Lactobacillus and Pediococcus — will grow on LMDA under aerobic conditions. If you want to be really thorough, you can inoculate two plates of LMDA and incubate one under normal aerobic conditions and the other in a sealed container with an activated GasPak Plus envelope to create an anaerobic environment.
While the recipe for LMDA is readily available (4), it is easiest for most of us to buy it from brewing consultants or brewing laboratory supply houses; it can be found as prepared plates for as little as $0.80 each (Brewing-Science Institute), or 1 L for $25 if you want to pour your own plates. (The Brewers’ Market Guide, published by Brewing Techniques, provides exhaustive listings of laboratory equipment and consumables suppliers.) You can use LMDA to test for bacterial contamination in a number of ways (see the Brewing-Science Institute’s lab manual in the Further Reading section), but I’ll just focus on how to use it for two tests that help identify offending bacteria found through the force tests described above.
Basic Application of LMDA
Materials: To grow up colonies on LMDA you need an LMDA plate, a sterile transfer pipette, and a water bath or incubator (optional but recommended).
Procedure: Set up your work space as of sediment, additional recirculation is needed. If there is significantly less than 5 mL after 5–10 minutes, you’re probably OK, and you can simply empty the Imhoff cone into the kettle if you wish. To be completely thorough, let the wort stand for 2 hours to assess the true amount of sediment. Of course, this is rarely practical for 5-gallon batches, but it will give you a good data point about your procedures for future batches.
Diacetyl Force Test
Have you ever sent a prize lager off to a competition in what you thought was prime condition, only to get the score sheets back to find that it had been marked down for elevated diacetyl levels (buttery flavors)? It’s possible your beer had a Pediococcus infection, but it might have fallen victim to a much simpler problem instead.
Yeast convert acetaldehyde and pyruvic acid into alpha-acetolactate, which leaks out of the yeast cell and spontaneously oxidizes to form diacetyl. The yeast can, over time, reabsorb the diacetyl and reduce it to an almost flavorless compound, butanediol (5). The oxidation (oxidative decarboxylation) of alpha-acetolactate to diacetyl takes place more rapidly at higher temperatures, which is one reason why a diacetyl rest works — it accelerates this reaction at a time when lots of active yeast are available to reabsorb and reduce the diacetyl. If a significant amount of alpha-acetolactate is present after primary fermentation, however, it can remain in the beer and oxidize into diacetyl. This happens rapidly if the beer is ever subjected to warm temperatures (such as might be encountered in shipping), and more slowly as the beer ages.
The diacetyl force test test lets you determine whether an excessive amount of alpha-acetolactate is present near the end of primary fermentation. Armed with this information, you will know whether to conduct (or continue) a diacetyl rest to facilitate its elimination before racking the beer off the yeast. (Thanks again to George DePiro for this test.)
Materials: Two small flasks or glasses.
Procedure: At the end of primary fermentation (or whenever you are considering doing a diacetyl rest), draw off a 100-mL sample of the beer. If you take a sample to assess the beer’s gravity at this point, you can just use the beer from the hydrometer jar; this test doesn’t require aseptic sampling. Pour the sample into two flasks or glasses. Heat one to about 140 °F (60 °C) for 30–60 minutes, then chill it to approximately the same temperature as the other. Taste each one.
Heating the sample forces the alpha-acetolactate to oxidize into diacetyl. If the heated sample tastes significantly more “buttery” than the other, then your beer contains a substantial amount of alpha-acetolactate and a diacetyl rest may be prudent. If the two samples are roughly equivalent and the diacetyl level is not objectionable, then no diacetyl rest is needed.
You can also run this test on your finished beer to see whether it would be susceptible to increased diacetyl formation if it were subjected to warm temperatures or prolonged aging.
Testing Yeast Viability with RDMA
If you repitch your yeast repeatedly, you should periodically check it for petite mutants, also known as respiratory-deficient mutants. At elevated populations, these mutated yeast cells can cause increased levels of diacetyl and fusel alcohols, among other problems (6). One of the most common off-flavors is caused by mutants’ production of 4-vinyl guaiacol, a compound that imparts a phenolic, Weizenbier-like flavor.
The traditional method of screening for petite mutants is either to grow up several hundred yeast colonies on standard wort agar and then cover them with a special overlay agar containing tri-phenyl tetrazolium chloride (TTC) (2), or to grow them on a special filter membrane, which is then removed and soaked in a solution containing TTC. While not especially difficult, these methods are sufficiently inconvenient to discourage many brewers from using them. Fortunately, an easier way exists (courtesy of the Brewing-Science Institute): a special prepared media known as Respiratory Deficient Mutant Agar (RDMA), which allows “one-step” screening for petite mutants. The only minor drawback is that RDMA is heat- and light-sensitive (as is TTC), but this is no real problem if you keep your RDMA plates wrapped in foil and refrigerated until needed.
Materials: RDMA plate, inoculation loop or sterile transfer pipette, and a water bath or incubator (optional).
Procedure: After preparing a clean work space (see the procedure for LMDA, starting on page 37), sterilize your innoculation loop by heating it until it’s red hot. Scoop up a very tiny bit of yeast slurry and streak it out on the RMDA plate. (The procedure is exactly the same as streaking an agar plate with a yeast sample from a slant. Many books and articles describe the technique [see the Further Reading section], so I’ll spare you the details.) Alternatively, use a sterile pipette to transfer a few drops of fermenting beer or starter to the plate, and spread them out as in the LMDA test described above. Incubate until yeast colonies are visible (usually about 72 hours).
Colonies of normal yeast will be red or pink; mutants will be white. If you have less than 1% mutants, things are fine; if the mutant count exceeds 10%, you have probably just identified the source of the problems you are having.
All contents copyright 2019 by MoreFlavor Inc. All rights reserved. No part of this document or the related files may be reproduced or transmitted in any form, by any means (electronic, photocopying, recording, or otherwise) without the prior written permission of the publisher.