BrewingTechniques
The Microbrewery Laboratory Manual- A Practical Guide to Laboratory Techniques and Quality Control Procedures for Small-Scale Brewers,
Part 1: Yeast Management

By Fal Allen
Republished from BrewingTechniques' July/August 1994.

The first of a four-part series on laboratory techniques for quality control in the brewery, this article describes basic practices for yeast management, including yeast tracking, cell counting, yeast viability testing, and acid washing. Future installments will cover bacteria and wild yeast detection and enumeration and the use of simple analytical instruments.

As a small-scale brewer, you probably have other things than expensive lab equipment at the top of your expenditure list. You would probably rather buy another clean-in-place (CIP) pump or an additional fermentor. Lab equipment, you say, will have to wait. And in most cases, wait it does.

I often hear brewers and owners say things like, "We don't need a lab because we are really clean and have never had an infection." "Our beer is never around long enough for laboratory procedures to matter; it's consumed within a week or two," "Lab equipment is expensive, and we can't afford it right now," and "I've brewed good beer for years without a lab."

If any of these statements offered a good reason not to do lab work, I probably would not be writing this article. In our second year of operation, during a sizable remodel of the brewery, we came down with a fairly bad infection and ended up having to dump three or four batches of beer (I can't remember the exact number; I have mentally blocked out those few days from my life). Our yeast stock had been infected by Pediococcus bacteria vectored into our fermentors through tile dust created during the remodel. At least, that's what we hope had vectored it. It caused us to reevaluate all our brewery procedures and adopt a new quality control program.

When I started to put together the new quality control program, I wanted to find some basic laboratory techniques, simple stuff I could do easily in our crowded brewery. I could find nothing at all. I called my friends for advice, I scrounged literature wherever I could find it, and we paid a consultant to show us a few things.

To document the results of my research and its application to our brewery, I decided to write a laboratory manual that spells out our basic quality control practices. This article is the first of four installments of that manual to be published in BrewingTechniques. I hope the information will be helpful for those who want to set up a useful laboratory and quality control program.

I now believe there are only two types of breweries; those that have had an infection and those that will. Doing regular lab work will enable you to detect that infection while it is still low grade, long before it can affect your beer. You will be able to take corrective measures, and no one will ever taste the difference. No beer will have to be dumped. That, if nothing else, adequately justifies the expense of some basic lab equipment and the time taken to apply it properly.

THE IMPORTANCE OF GOOD YEAST MANAGEMENT PRACTICES

Bacteria and wild yeast are all around us - in the air, on the floor, on our hands. Some of that bacteria will get into your beer. It is unavoidable. Part of our job as brewers is to minimize the amount of bacteria that come into contact with our beer. One of the best ways to do this is to have a strong, clean, active yeast slurry to pitch.

When given the chance, yeast crowds out bacteria. During an active fermentation, the yeast quickly uses up all the oxygen, making it impossible for aerobic organisms to survive. It lowers the pH, making it hard for other anaerobic organisms to survive. Finally, it produces alcohol, which creates an even more hostile environment for foreign organisms.

Because strong, healthy yeast are critical to successful brewing, it is important to set up some method for monitoring and tracking the quality of yeast cultures.

A YEAST TRACKING SYSTEM

Managing your data: The easiest and least expensive part of a yeast management program is a good yeast tracking system. Keeping good records on your yeast will translate into better beer. At our brewery, we have incorporated a tracking system into our brew sheet, which now includes space for 12 items to be filled in for every batch of beer we brew (Figure 1).

Harvesting yeast: Keep in mind that when you harvest the yeast, you always want to crop from the center (in terms of both space and time). If you harvest too early, or from the bottom of the cone, you get early-flocculating yeast that has not completely attenuated the beer. If you crop too late, or from the top, you get nonflocculant yeast that stays in suspension too long and clouds your beer, making later clarification more difficult. When you repitch yeast that has not been harvested from the center, these negative characteristics that you have inadvertently selected will be accentuated.

Reordering/reculturing yeast: It is also important to reorder your yeast (or reculture it from stock) at least every 30th pitching or every six months, depending on the strain of yeast you use and the frequency of your brewing. The need to reorder/reculture sooner than this depends on the performance of your yeast. Evaluate the yeast in terms of attenuation, flocculation, the number of times the yeast has been acid washed, viability, trub content, and contamination level.

CELL COUNTS

Most small-scale brewers pitch their yeast by weight or volume. The standard rule of thumb is 1 lb of yeast slurry/bbl. Measuring by weight or volume, however, can be very inaccurate. If your slurry is thin and liquidy, you may have relatively few cells per milliliter compared with a thick powder slurry of the same weight or volume. There is no way to tell at this point what percentage of the slurry is yeast and what percentage is trub. The only way you can get an accurate idea of how much yeast you pitch is to take a cell count.

Cell counts can be taken at any time during the life of your beer. They can be taken from the yeast slurry to determine the volume of yeast to pitch. They can be taken directly after pitching to check the pitching rate. They can be taken during chilling to determine the best time to filter or fine the beer. The following procedure assumes that you have taken a sample from the fermentor right after casting back (or knockout) is completed and the yeast has been pitched.

Equipment: The following equipment is needed for performing cell counts: microscope, hemocytometer and hemocytometer cover slip, pipette and pipette pump, flask, and a hand-held counter (for tallying).

Procedure: Using a clean, dry flask, take a small sample from the fermentor. Swirl the sample around to help break up any clumps of yeast and to make sure it is properly mixed. The swirling also helps to remove any gas in solution. The hemocytometer and the cover slip should be clean and dry. Place the cover slip over the counting areas. Next, affix the pipette into the pipette pump. Pull out ~2 mL of beer into the pipette, and then purge out 2-3 drops to clear the tip of any differentiation that may occur. Immediately place the tip of the pipette on the V-shaped groove (see Figure 2) and gently fill the counting area under the cover slip without disturbing the cover slip; only a drop or so is needed. If you desire, you can turn the hemocytometer around and fill the other side the same way. The counting chamber must be completely filled, but not overfilled. The sample should not run out into the canals or bulge at the edges (Figure 2).

The sample on the hemocytometer is now ready to be viewed under the microscope. Without spilling any of the sample, carefully place the hemocytometer on the microscope stage. Using a 10X objective lens, frame up one of the counting chambers (Figure 2). You should see a grid with 25 large squares, each of which contains 16 smaller squares (Figure 3). You can count all of the cells within the 25 squares, or, when there are large numbers of cells, you can count the cells in the four corner squares and in the center square and multiply the total by 5 (Figure 3).

To count the cells within an area, switch over to the 10X objective lens with the 40X objective lens, frame up the counting area of one of the 25 large squares, and take a count. At this point, I like to replace the 10X eyepiece with a 16X eyepiece; it increases magnification and makes counting easier. Use your hand-held counter to keep an accurate tally of the cells.

To eliminate the chance of counting a square twice or a cell twice, it is important to use a standardized counting procedure. Always count in one direction (left to right, top to bottom, for example). Cells touching the top or right-hand boundaries are not counted. Cells touching the bottom or left-hand boundaries are counted (Figure 4). Cells that are budding are counted as one cell, unless the daughter cell is equal to or greater than one-half the size of the mother cell, in which case the daughter cell is counted as a separate cell (Figure 5).

If there are more than ~50 cells per large square (that is, per square that contains 16 smaller squares) dilute the sample to be counted. The dilution factor must be used to calculate the final cell count. Remember that 1 mL of sample mixed with 9 mL of distilled water or saline solution gives you a 10X dilution factor. It is important to dilute and count samples of yeast slurry as soon as possible after sampling to prevent inaccurate counts due to cell multiplication. Because there can be a high degree of inaccuracy inherent in this procedure due to human error, I always do a second count to confirm my first tally.

Calculation: Use the following calculations to arrive at a final cell count: the number of cells counted (multiply by 5 if you counted only 5 boxes) X dilution factor (if any) X (1 X 104) = the final count, in cells per milliliter.

For example, if you counted a total of 300 cells in your 5 boxes (the four corners and the center box), you would calculate the total cell count as follows:

(300 X 5) X (1 X 104) = 15,000,000 cells/mL

Or, if you diluted 1 mL of slurry in 9 mL of distilled water and then counted 30 cells in the 5 boxes, the calculation would be as follows:

(30 X 5) X 10 X (1 X 104) = 15,000,000 cells/mL

Remember, the proper pitching rate is 1,000,000 cells/mL/° P (degree of Plato of wort). Therefore, for a 14 °P wort, you should be counting ~280 cells in each large square: (280 X 5) X (1 X 104) = 14,000,000 cells/mL.

When you have finished counting, thoroughly clean both the hemocytometer and the cover slip. Be careful not to scratch the surfaces. Dry them with a soft lens tissue and store them in a safe place. A hemocytometer costs about $80, and the cover slips cost a little over $50 for a package of 12.

YEAST VIABILITY:
STAINING WITH METHYLENE BLUE OR RHODAMINE B

To ensure that your pitching rate is correct, you must check the viability of the yeast you are pitching. I like to check the viability at the same time I take a cell count. I use the lower counting chamber on my hemocytometer to count cells and the upper chamber to check viability. The grid pattern of the counting chamber makes counting viable and nonviable cells easier.

The theory behind the use of staining techniques is that nonviable yeast cells tend to take up the stain more readily than do viable cells. Viable cells contain reducing compounds (nicotinamide adenine dinucleotide, reduced form [NADH] and nicotinamide adenine dinucleotide phosphate, reduced form [NADPH]) and will be able to metabolize the stain; dead cells lack these compounds and retain the stain. It should be noted that some small buds will also retain the stain even though they are viable; therefore, do not count these cells as dead. Staining with methylene blue measures the ability of cells to reduce the stain, not the ability of cells to reproduce. It must be stressed that methylene blue staining gives an indication of the percentage of viability rather than an absolute number value.

Staining for viability is a less reliable method than using a slide culture, with which you can count the number of cells that bud and produce daughter cells. In addition, as the percentage of viable cells decreases, so does the accuracy of the staining test. Staining with methylene blue is not considered to be an accurate test when the viability is less than 80%.

The fact that the accuracy of a staining test is statistically unreliable after viability drops below 80% should be of no concern to brewers because such a low-viability yeast should be pitched only when there is no other option. The more nonviable cells you pitch, the more likely it is that those dead cells will create a yeasty off-flavor in your beer. Dead cells can also host bacteria.

Equipment: The following equipment is needed for the staining process: microscope, slide, cover slip, pipette and pipette pump, flask, and stain.

Procedure: Methylene blue is available in many forms: powder, liquid, premixed, technical grade, and nontechnical grade. I opt for the less expensive dry powder form and mix my own stain. Mix 1 g of powder in 1 L of water.

Take a sample of beer or yeast slurry in a flask and, if necessary, dilute it with distilled water until you have a sample that contains ~100-200 cells in the microscope viewing field. Mix the sample thoroughly. Then pipette out about 2-4 mL and place it in another flask. Put 10-20 drops of methylene blue or rhodamine B stain into the flask (the exact amount depends on the strength of your stain solution) until the sample is dark blue (methylene blue) or pinkish (rhodamine B). Do not put so much stain into the sample that you cannot see the cells with the microscope. Mix well and let stand 3-5 min.

Place a drop on the slide and cover with the cover slip. Try not to trap any bubbles under your cover slip, and avoid having excess liquid surrounding the edges. Examine the sample under the microscope at ~400-600X magnification. Count 100 cells, keeping track of how many are dead. With methylene blue stain, dead cells are blue; with rhodamine, dead cells are pink. Report the percentage of dead cells, including broken and shriveled cells. If the sample you are checking is your yeast slurry, now is a good time to try to get a feel for the percentage of the slurry that is yeast and the percentage that is trub. This should be fairly obvious when you view it under a microscope. I like to see <20% trub. Yeast cells should be round or oval in shape. Non-Saccharomyces wild yeast cells are often bipolar and elongated.

ACID WASHING

Acid washing should never be considered a cure for bacterial infection. It should be used only for preventive maintenance or as an emergency measure. Some brewers regularly acid wash their yeast as a preventive measure, sometimes as often as every pitching. Other brewers acid wash only when an infection shows up. In this case, they will continue to acid wash until the brewery can obtain a fresh pitching culture; at that point the old, infected culture is discarded.

The frequency with which you should acid wash depends on your brewing process, your brewing cycle, and the nature of your yeast. Some yeast seems to thrive on being washed. The yeast we use at Pike Place Brewery is more active after it has been washed. Other yeasts are severely damaged by washing.

The theory behind acid washing is that it lowers the pH of the yeast slurry to the point at which bacteria and weak yeast cells are killed off, but does no harm to healthy cells. One drawback to acid washing is that it often fails to kill all of the bacteria. It sometimes merely reduces their number, and the culture remains infected.

Remember, acid washing can be very hard on some strains of culture yeast. We have also found that repeated acid washings can reduce a culture's long-term viability.

Our practice at Pike Place Brewery is to use acid washing as preventive maintenance. We acid wash every sixth to eighth repitching, and we reculture after 20-30 repitchings. If an infection shows up, we dump that yeast and take a slurry from another fermentor. If we had no choice but to pitch an infected yeast, we would acid wash every pitching until we could reorder or reculture fresh yeast. I also suggest having your yeast kept on file at a yeast bank. We keep ours at Wyeast Laboratories (Hood River, Oregon).

Equipment: The following equipment is needed for acid washing: a clean, sanitized bucket or yeast holding-tank; a sterilizable mixing paddle made of hard plastic or stainless steel, or a motorized, sterilizable mixing tool; pH paper or a pH meter with a range of pH 1-7; and 75% food-grade phosphoric acid.

Procedure: Mix the 75% food-grade phosphoric acid with sterile water, 9 parts water to 1 part acid (dilution factor of 10), which will produce a 7.5% solution. This solution will be used to acid wash your yeast.

Spray down the sterilized paddle and gloved hands with ethanol. While mixing continuously, slowly add a small amount of the 7.5% solution to the yeast slurry. I usually add about 0.25-0.5 cups of solution to 4 gal of yeast. Stir constantly until the solution is well mixed into the yeast slurry to ensure that it contains no pockets of high acidity and that it is thoroughly mixed. Pull out a sample and take a pH reading. If it is between 2.0 and 2.4, stop. If pH is above 2.4, continue to add small amounts of the acid solution and mix well. Continue to take pH readings until you reach pH 2.0-2.4. Leave the yeast slurry for 1-1.5 h and then pitch as usual.

If the pH drops too low, the yeast culture could be harmed. Some books recommend washing at 2.8-3.0 pH for 6-12 h. By exposing the yeast to higher pH washes (pH 2.5-3.0) for longer times, the effect on yeast viability is less dramatic, but at the same time some types of bacteria are not as effectively killed.

It should be noted that acid washing will not affect most types of wild yeast; it is effective only for controlling bacterial infections. It is also important to remember that when using acid washing as part of a maintenance program, you should wash all the yeast in your brewery. For example, wash the slurry from Fermentor 1 on Monday, the slurry from Fermentor 2 on Tuesday, and so forth.

RULES AND GUIDELINES

Finally, the following quality control procedures can be helpful in maintaining quality yeast cultures in your brewery.

  • Never let anything that touches your beer touch the floor. The floor is the greatest source of bacteria in a brewery.
  • Always sanitize a sample valve or butterfly valve before and after using or taking a sample.
  • Avoid working in the cellar when there is a large amount of dust being produced from construction, grain milling, or other activities. Dust vectors bacteria.
  • Clean and sanitize all hoses under pressure and replace the ends of the hosing at least every year. Bacteria can survive in the area between the rubber hose and the metal hose ends, or at any point where the hose has been damaged.
  • Check gaskets for defects and tears. Bacteria can survive in cervices and cracks in hoses, gaskets, and other places.
  • Before being sanitized, all equipment that contacts the beer should be cleaned of biological matter with a cleaner like sodium hydroxide (caustic) and should be cleaned of mineral deposits (beer stone). Bacteria can hide in beer stone deposits and can even survive the CIP process. To break down the mineral deposits, periodically use either chlorinated caustic or a fairly strong acid cleaner. We use both alternately on a periodic basis and always use a phosphoric acid-based sanitizer, which helps break down beer stone.
  • If you soak equipment in a tub of sanitizer, change the sanitizer regularly. Iodine sanitizers in particular need to be changed often because the iodine drops out of solution over time.
  • Work rapidly. A basic principle of working with yeast (during reculturing, pitching, or acid washing, for example) is to work rapidly to minimize the time that the yeast is exposed to nonsterile environment (i.e., air).
  • Have a spray bottle filled with a 70% solution of ethanol handy so that you can quickly spray down and sanitize any surface. Ethanol is a very effective and fast-acting sanitizer.
  • Never allow any free-standing water or beer in your brewery because they provide ideal environments for bacterial growth. At the end of each day, splash down the floors with sanitizer.

    Caveat Brewer

    Although I have not been formally trained in brewing microbiology, I have done extensive research on the subject, and the recommendations in this article are the result of that research and practical experience. I have read the course material from both of the major U.S. brewing schools (Siebel Institute [Chicago] and the University of California at Davis), the ASBC Guidelines, and other literature. I have also done a fair amount of experimentation at our brewery and have exchanged ideas with many other brewers.

    If you have any comments or suggestions please fax them to me at 206/622-6648 or write to me at Pike Place Brewery, 1418 Western Ave., Seattle, WA 98101. I will respond to your comments and suggestions in the upcoming installments in future issues of BrewingTechniques.

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