South Bay, Los Angeles County, California

At NHC 2001, Louis Bonham discussed "Guerilla Lab Techniques," a set of procedures and practices that you can do at home without the need for a true brewing laboratory. Most of these techniques are independent of each other, so you can choose to conduct some of them but not others. Whether you work only one of them or all of them into your brew day, they can help you wage the war against the undesirable microorganisms encountered throughout the brewing process.

Louis discussed four types of laboratory tests that can be done in the home brewery: (A) Force testing; (B) Using prepared media; (C) Basic brewery microscopy; and (D) Specific equipment techniques.

A. Force Testing:

Louis calls force testing the "No lab, no brain, no excuse tests," and believes that they should be run on every batch of beer. Only a few basic pieces of equipment are needed: sterile containers 50-500 ml in size, a warm place, and a 0.1% cyclohexamide solution (preferable in a luer-lok syringe with a 0.22 micron filter). The use of an incubator and a stirred plate in these tests is optional. Cyclohexamide is a selective poison that kills yeast without killing bacteria. It can be purchased from White Labs through some homebrew retailers.

To sterilize his flasks, Louis covers them with aluminum foil and bakes them in the oven at 350 F. He turns on the oven some time during the mash, bakes the flasks until mash out, and allows them to cool until the end of the boil.

A1. Wort Stability Test (WST)

To test for contamination present in your cooled wort, aseptically collect a sample of unpitched wort either directly from the fermenter or as it is run into the fermenter from the kettle. Incubate the sample at 80-90 F. Periodically inspect the wort for haze and bubbles, and at the end of the test, check for off-odors and tastes.

The length of time it takes for the wort to show signs of infection is a qualitative measure of your contamination level. If the sample made it less than 24 hours before showing signs of infection, you probably have major problems with your brew house cleanliness. Going 48 hours with a clear sample is OK, but you probably don't want to repitch the yeast from this batch. If the sample survives for 72 hours, you can consider your cleanliness excellent.

A2. Pitched Wort Stability Test (PWST)

To test for contamination present in your yeast starter, aseptically collect a sample of pitched wort. Add cyclohexamide solution at the rate of 1 ml per 100 ml of sample, and put a POISON sticker on the flask to clearly identify samples that should never be ingested. Incubate the sample at 80-90 F. Periodically inspect the wort for haze, bubbles, and off-odors. Compare the results from this test with those from the WST. If this test shows signs contamination while the WST remains clear, then you've shown that your yeast starter was contaminated when it was pitched into the wort.

A3. Forced Fermentation Test

This test can determine the fermentation end point of your wort. Aseptically collect a 150 ml sample of your pitched wort. Incubate the sample and either shake the flask periodically or use a stirred plate (which is highly recommended) to force the fermentation to completion. Check the gravity after activity ceases, usually within 24 hours with a stirred plate, or 48 to 72 hours otherwise. This reading is the minimum gravity for that yeast and wort. The FG of the beer is usually a bit higher than that from the forced fermentation test, especially for big beers and less attenuative yeast strains.

A4. Beer Stability Test

You can test your beer's stability by running the WST and PWST on finished beer. For the easy method, just bottle a few sterile screw-top culture tubes (half with cyclohexamide, and half without) and store with your beer. For draft beer, just dispense the beer to sterile tubes when you want to run the test. To run the test, just crack the seal on the tube to release the pressure, then reseal and incubate. If the beer tastes OK but fails the tests, expect shorter shelf life.

Louis quotes Farnworth's rule of thumb: Beer lasts ten times longer at 38 F than at 80 F. Therefore, if your stability test goes bad in two days, you've got three weeks to finish that keg on tap.

A5: Forced Diacetyl Test

Bacterial infections can produce diacetyl, especially those caused by pediococcus. Yeast, though, produce alpha-acetolactate from acetaldehyde and pyruvic acid. Over time, the alpha-acetolactate oxidizes (oxidative decarbolization) to form diacetyl. Heat speeds this reaction. Yeast can reabsorb diacetyl and convert it to butanediol, which is essentially tasteless. If your beer has high levels of alpha-acetolactate remaining after yeast decline, it can develop increasing amounts of diacetyl as it ages, even without an infection.

To run a forced diacetyl test, take two samples of green beer, preferably at the end of primary fermentation. Heat one sample to about 140 F for 30-60 minutes, and then cool it to the same temperature as the control sample. If the heated sample tastes more buttery than the control sample, your beer has excess alpha-acetolactate. Solutions to this condition are to conduct a diacetyl rest, or possibly krausening the beer with fresh yeast.

B. Using Prepared Media:

By purchasing prepared media and pouring your own petri dishes, you can conduct "no microscope microbiology" at home. These techniques are useful for identifying contaminants in your beer (especially from force tests), assessing the health and condition of your yeast before pitching, and checking your sanitation procedures.

To use prepared media, you will have to equip your Guerilla lab with the equipment necessary to inoculate the plates under sterile conditions. Basic equipment includes a butane torch, inoculating loops, sterile transfer pipettes, sterile swabs, tubes of prepared media, sterile petri dishes, ethanol, hydrogen peroxide, potassium hydroxide solution, and electrician's tape.

When working with the plates, set up a clean area and work in and under a hot air plume from the torch. Flame your loops often, and keep plates open as little as possible. You should seal the cooled plates after pouring, don't reseal them after inoculation, and incubate them upside down.

Louis covered six types of prepared media:

B1. Lee's Multi Differential Agar-Permits visual differentiation of major brewery bacteria.
B2. Hsu's Lactobacillus Pediococcus Medium-Specific test for lactos and pedios; a good screen for yeast.
B3. Universal Beer Agar-Specific test for beer-loving microorganisms, great for swab tests of sanitized equipment.
B4. Respiratory Deficient Mutant Agar-Yeast screen for common mutants.
B5. Lin's Cupric Sulfate Medium-Screens for non-saccharomyces wild yeast.
B6. Lin's Wild Yeast Medium-Tests for saccharomyces wild yeast.

Louis was generous enough to distribute his presentations in electronic format to the conference attendees. If you're interested in these tests, let me know and I'll send you a copy of his presentation with the details.

C. Brewery Microscopy

Conducting basic brewery microscopy at home requires a significant addition to the Guerilla lab: a microscope. The main use of a microscope in the Guerilla Lab is to assess your pitching rate and yeast vitality. Additionally, you will be able to do gram staining and gross bacterial identification. You will not, though, be able to identify or differentiate yeast, or definitely identify bacteria.

A basic brewing microscope should have the following features: 5x, 10x, 40x and 100x (oil) objectives; 10x or 16x eyepieces; an Abbe condenser and aperture; and x/y stage control. Great brands to look for on the used market are Zeiss, Leica, Olympus, and Nikon. For older used models, look for Bausch & Lomb, Swift and American Optical. Additional equipment includes lens paper and cleaning solution; slides and coverslips; a hemacytometer kit; methylene blue solution; immersion oil; and a gram staining kit.

C1. Viability Testing

Put one drop of yeast starter on a slide, add one drop of methylene blue, and cover with a coverslip. Examine the sample at 400x. The methylene blue will stain the dead cells blue, leaving the live cells clear. Determine the viability of your starter by counting the percentage of cells that are live. Don't count the trub particles, which will appear as fluffy blue clumps. A viability greater than 95% is desirable, and a value greater than 98% is preferred.

C2. Cell Counting

George Fix refers to cell counting with a microscope and hemacytometer the "most important lab technique." A hemacytometer is a specialized glass slide for counting cells. A tiny drop of pitch wort or diluted starter is transferred using a pipette to a notch in the hemacytometer. Capillary action will pull the drop under the coverslip into a tiny chamber of known volume. Using 400x and the rectangular grid on the coverslip, the number of cells per milliliter of solution can be calculated by counting the cells that appear in the little blocks of the grid. When counting cells, buds don't count, and you should be consistent on how you count cells that are on a line. For example, count cells on the left and top lines, and ignore those on the right and bottom lines.

Various sources quote quite a range for the ideal pitching rate. Compare your calculated pitching rate to the ideal levels.

D. Specific Equipment Techniques

D1. Sweet Wort Clarity Test

The purpose of recirculating the runoff from the lauter tun before filling the kettle is to clarify the wort of unwanted particles. As Louis says, "Sweet wort good; starch bad." An Imhoff cone (or sedimentation cone) can be used to quantify the clarity of the sweet wort after recirculation. A 1-liter sample is collected in the cone, and the particles are allowed to settle for 5 to 10 minutes. If less than 5 ml of sediment drops to the bottom of the cone, the clarity is good and the wort can be run into the kettle, otherwise the recirculation should continue. This is the least expensive of the specific equipment test discussed here.

D2. CO2 and Headspace Air Test

Using a Zahm & Nagel Tester, you can measure CO2 pressure and headspace air in bottles. Less than 1 ml of air is desired in the headspace.

D3. Fun with Spectrophotometers

Equipping your lab with a spectrophotometer opens up a wide range of additional tests that you can do in your home brewery: IBU assays; alpha and beta acid assays of hops; tests for beer color; yeast counts; and with some additional reagents and equipment, precision alcohol determination, tests for protein levels, and diacetyl assays.

A spectrophotometer measures the intensity of a known wavelength (measured in nanometers, nm) of light passed through a sample. Since Beer's Law states that the fraction of light absorbed is proportional to the concentration of the absorbing solute, we can determine the concentration of the solute in the sample.

The biggest drawback to the spectrophotometer is the cost. Louis states that new ones that provide the necessary UV and visible capability start at $5000, however used ones can often be found under $500. A caveat on the used machines, though, is that the replacement Deuterium bulbs are between $250 and $400 each, so determining the need for a replacement bulb in a used machine seems a necessity before purchase.

Finally, Louis gives sources for equipping your small lab. These include: www.labx.com for used lab equipment; www.cynmar.com for discount lab equipment; www.brewingscience.com for prepared media; and www.FungiPerfecti.com for big pressure cookers and other culturing equipment.

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