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 PRACTICESBacteria 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 SYSTEMManaging 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 COUNTSMost 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
(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:
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.
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.
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