11/20/2019 A Practical Guide to Yeast Management | MoreBeer

A Practical Guide to Yeast Management

09/25/2018
by Fal Allen (Brewing Techniques)

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.

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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.

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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.

Yeast Tracking Sheet

1. Date yeast was pitched
2. Quantity of yeast pitched (weight and/or volume)
3. Fermentor from which it was harvested
4. Batch number (brew number) of the beer from which it was
harvested
5. Acid washed? (Yes/No)
6. pH, if acid
washed
7. Length of acid wash
8. Cell count of the brew, and time the cell count was taken
9. Viability of the yeast at the time of cell count

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10. Time at which the trub was removed from the fermentor
before harvest
11. Visual observations (i.e., powdery, liquidy, etc.)
12. Taste observations (off-flavors, etc.)

Figure 1: The 12 items on the Pike Place Brewery yeast tracking sheet. Although the yeast tracking
information can be kept on a separate sheet, it safer and handier if recorded as part of the brew log.

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).

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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 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.
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. You should see a grid with 25 large squares, each of
which contains 16 smaller squares. 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.
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. 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.
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.

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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.

Laboratory Equipment Used at Pike Place Brewery

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All of the equipment in List I can be purchased at a relatively low cost, much less than the cost of one
dumped batch of beer. The most expensive item on the list will be the microscope. An entire article can
be written on selecting the right microscope for your lab and how to find it. (Look for such an article in
an upcoming issue of BrewingTechniques.—Ed.) The microscope should have at least two objective
lenses, a 10x lens and a 40x lens (a 100x lens is nice if you can afford one). A 10x eyepiece and a 40x
objective lens will provide 400x magnification, which is sufficient for most yeast work. I bought a 16x
eyepiece, which together with my 40x objective lens gives me 640x magnification. I find 640x to be
perfect for most work, and the combination of 16x eyepiece and 40x objective lens is an inexpensive
trick. If you want to see bacteria close-up you will need a 100X oil immersion lens. The objective
lenses on your microscope can be changed out like the eyepiece, so you can buy a more powerful
lens later to replace a less powerful one.
You can find used microscopes at some suppliers and at university sales. I bought our microscope
new and on sale from Nurenburg Instruments (Portland, Oregon). It is a good make and I was able to
get it for less than $ 275. It would have been even less expensive, but I added fine focus and an
electric light source.
List 2 is a more advanced set of equipment with prices to match.
List 1 List 2

Hand-held counter*
Hand torch†
Test tubes (24) (either glass
Hot plate
reusable or sterile disposable)
Triple-beam scale
Test tube rack
Ethanol in spray bottle
One-hole rubber stoppers (2)
Incubator box‡ Gram stain kit
Pipettes and pipette pump
Autoclave or pressure Carbon dioxide tester
Innoculation loop (Zahm & Nagel, Buffalo,
cooker§
Petri dishes (6–10) New York)
Safety glasses
Erlenmeyer flasks (6) Membrane filtration
Gloves setup#
pH paper or pH meter
QC data sheets Oxygen tester (Zahm &
Microscope Nagel, Buffalo, New York)
Hsu’s Lactobacillus–
Hemocytometer (Bright line, 1 Pediococcus medium (HLP)
mm deep, Reichert, Buffalo, New mediumІІ
York)
Lin’s Wild Yeast (LWY)
Cover slips mediumІІ
Methylene blue or
rhodamine B stain
*Used during cell counting to help keep an accurate tally.
†The kind used in plumbing to solder fittings.
‡You can build this yourself by closing in a cupboard or building a wooden box. A light bulb can serve
as a heat source. The temperature can be controlled by changing the wattage of the bulb or by
placing a thermostat for an AC unit in the box and using a hair dryer to raise the temperature.
§I found that a medium-large pressure cooker works great and can be purchased at a swap meet or
the like for about $ 20.
ІІJ.E. Siebel Sons Co., Chicago, Illinois; U.S. distributor: Crosby and Baker, Westport, Massachusetts.
#Autoclavable or disposable.

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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.

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Rules and Guidlines

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.
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.

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