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Date: 26 Mar 1995 15:50:21 -0500
From: "Daniel S McConnell" <Daniel.S.McConnell@med.umich.edu>
Subject: YeastLab QA procedures
In response to Donovan (Rick) Bodishbaugh who asked about the QA/QC procedures (among other things) of commercial yeast vendors, let me take a stab at documenting the methods for YeastLab/Yeast Culture Kit. The original thread was about culture ID and QA so this is the issue I would like to address. This is going to be long.
This may lead to the most useful discourse that I have seen on the net yet. I personally feel that an educated customer is a good thing and encourage those with laboratory skills to use them. I try to teach my students to maintain a healthy skepticism, ask questions and test hypothesis. Sometimes carefully collected and documented data will prove the dogma wrong. I think this applies to your hobby as well as your life. The world is no longer flat.
At great risk of sounding like an infomercial..........I own The Yeast Culture Kit Company (YCKC) which sells cultures on slants, culture kits, glassware, media and culturing supplies (all the media discussed below are available in small aliquots, enabling you to duplicate these techniques). YCKC also produces Yeast Lab liquid Saccharomyces, Brettanomyces and Pediococcus cultures for G.W. Kent. I perform all the maintenance and QA. Stop by the lab at GWKent (3667 Morgan Rd) if you are ever in Ann Arbor.
QA procedures should be tailored to an individual lab and as such what may apply to YL/YCKC may not apply to others. The most important thing is to know thine enemy. Know your house contaminant and find a method that is particularly sensitive in locating it. With that said, here are my procedures.
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For Saccharomyces Sp, I am basically looking for bacterial/wild yeast contamination. Plates and tests are as follows.
1) Wallerstein Laboratories Media (WL) plate
A good general media that will support the growth of yeast and
bacteria. It contains an indicator dye (bromocresol green) that is
taken up differentially by different yeast strains. Mixed cultures
will therefore sometimes demonstrate mixed colony morphology
(different shades of green and white).
2) Wallerstein Laboratories Differential Media (WLD) plate
Same formula as WL but with added cycloheximide (actidione) (CHX)
to suppress the growth of CHX sensitive yeasts which include
Saccharomyces Sp. For Saccharomyces, if anything grows on this
plate, the culture is considered contaminated.
Sometimes I will substitute Yeast Malt Extract Agar with added CHX (YMA/CHX) because a) the color is different (reduces the chance of confusion) and b) I can control and reduce the concentration of CHX.
3) Hsu's Lactic Acid Pediococcus Media (HLP)
Specially formulated to select for lactic acid bacteria. A quick
and easy, semi-quantitative test for the presence and enumeration of
lactobacillus and Pediococcus. I pipette 0.1 mL of culture to the
tube which allows the estimation of the number of lactobacillus or
Pediococcus/mL. Cultures that show the presence of these bacteria
are considered contaminated.
4) Lin's Wild Yeast Media (LWYM)
Specially formulated with crystal violet which inhibits brewery
yeast to enable the detection of wild yeast. Note: some normal yeast
will grow on this media (certain Belgians and Weizens). Depending
on the culture that is screened, growth is considered contamination.
An alternative is Lin's Cupric Sulfate Medium (LCSM) but my personal
preference is LWYM due in part to its longer shelf-life.
5) Gram strain
Quick and easy but not very useful. Only gross contamination will
be apparent in a gram stain, but this procedure takes 5 min. opposed
to a few days for the plated media to grow out.
6) Cell count and Viability
I dilute the culture in a buffer that contains Methylene Blue
and count stained cells (considered non-viable) and non-stained cells
(viable) on a hemacytometer. One can then calculate the number of
cells/mL in the culture. Greater than 95% viable cells is considered
acceptable.
7) A reserved vial is held at 26C for 2 weeks than recultured to WL and WLD. Low level contaminants may be seen by this method.
8) 2 reserved vials are held at 4C until used by myself or given to the local brewers (who generally use about 20% of these freebies). I get very fast feedback on culture performance. These tests are done on every batch that goes out. Secondary master cultures are stored and retrieved from -80C annually. Slant submasters are restreaked every 4 months.
9) Tetrazolium Chloride (TTL) overlay plates are done when submastering in addition to the above routine plates and HLP. TTL detects respiratory deficient mutants.
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For Brettanomyces Sp, I am basically looking for non-Brett yeast and bacterial contamination.
1) De Man Rogosa Sharpe Media (MRS)
A buffered, highly nutritive media. Lactobacillus and Pediococcus
contaminants grow well on MRS.
2) Yeast Malt Extract Agar with CaCO3 and CHX (YMA/CaCO3/CHX)
Brettanomyces will grow in the presence of up to 100 ppm CHX.
Saccharomyces Sp are suppressed at less than 10 ppm. In addition,
Brettanomyces will produce acid which results in a dissolution of the
insoluble CaCO3 showing a zone of clearing around the colony. For
Brettanomyces, growth on CHX and acid production in the absence of
bacterial contamination are important tests.
3) Wallerstein Laboratories Differential Media (WLD) plate
Same formula as WL but with added cycloheximide (CHX) to suppress
the growth of CHX sensitive yeast which include Saccharomyces Sp.
For Saccharomyces, if anything grows on this plate, the culture is
considered contaminated. Brettanomyces and bacteria will grow just
fine on this media.
4) Lin's Wild Yeast Media (LWYM)
Specially formulated media with crystal violet which inhibits
brewery yeast to enable the detection of wild yeast. Brettanomyces
will grow just fine on this media.
5) Galactose Fermentation test
Most Brettanomyces are unable to ferment galactose, thus this
provides a good test for contamination by Saccharomycs Sp. Growth in
Phenol Red Galactose Broth usually indicates a contamination problem
most often by non-Brettanomyces yeast.
6) Corn Meal Agar (CMA)
Corn meal stimulates sporulation while inhibiting vegetative
growth of fungi. Brettanomyces produce pseudomycellia on this media.
This is an important diagnostic test for Brettanomyces.
7) Gram Strains
Only gross contamination will be apparent in a gram stain, but
this procedure takes 5 min. opposed to a few days for the plated
media to grow out. A second gram stain on colonies from the CMA
plate is needed to observe the pseudomycellia.
8) Cell count and Viability
I dilute the culture in a buffer that contains Methylene Blue
and count stained cells (considered non-viable) and non-stained cells
(viable) on a hemacytometer. Greater than 95% viable cells is
considered acceptable.
Retrieve from -80C every batch. Brettanomyces are difficult to maintain on slants without frequent subculturing. Since I make a batch of these every 4 months or so, I have found it easier to simply keep my submasters frozen until needed.
These are done on every batch that goes out. I check a gram stain and an MRS plate at the 1L stage for potential problems. The next batch will probably have a Phenol Red Raffinose fermentation added (another sugar that Saccharomyces will and Brettanomyces will not ferment).
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Feel free to comment on these procedures. I am confident that my QA is adequate (OK, maybe a little overanal), but would welcome someone pointing out a fatal flaw. QA is something of a moving target, what is good enough today may not be tomorrow. One must be always be on guard against complacence.
And yes, in case you are wondering.........I LIKE to do this stuff.
DanMcC