From the HBD Archive
From: "Brett Lindenbach" <Brett_Lindenbach@qms1.life.uiuc.edu>
Subject: Re- Homebrew Digest Request
Date: 1992-04-30 17:31:49 GMT

Mail*Link(r) SMTP Re: Homebrew Digest Request



Microbiology for the Home: A Primer on Yeast Culturing

by: Brett Lindenbach
(brett_lindenbach.microbiology@qms1.life.uiuc.edu)

After quaffing a good yeasty beer I thought to myself, "Why
throw away the yeast that's stuck to the bottom of the bottle,
especially when I have to pay four bucks for a Wyeast culture?" And
so, with this do-it myself attitude, and some training in microbiology,
I set out to construct my own library of yeast strains. Let me tell you
how I did it.

A WORD ON SANITATION First of all, to have success in
manipulating microorganisms, you must have an appreciation of
sterile technique. It is one thing to pitch an active culture into
fresh
wort, and quite another to revive a yeast that has been sitting
happily in alcoholic dormancy for months: the chance for
contamination are at least ten-fold. A good thing to bear in mind at
all times is that microbes are everywhere: on your hands, in the air,
your countertop, you name it. I am amazed at my roommate's brewing
technique (he's an engineer). He will sanitize something by swishing
it around in our bucket-o-bleach water, and promptly set it in the
kitchen sink! So, when dealing with sanitary/sterile things it is
important to work quickly, but to not get sloppy. Soak things in
bleach water at least 5 minutes. Wipe down the area you in which
you plan to work with a bleach based sanitizing solution.
Contamination can be common until you are well practiced in sterile
technique. If you have access to an autoclave, by all means, learn
how to use it. If not, the next best methods are boiling all
ingredients, which does not guard against spores but will suit most of
a homebrewer's needs, and sanitizing all equipment with bleach
solution. Also, it would not be a bad idea to check out a book on
microbiological techniques (1,2) from your library for this all
important concept

MAKING MEDIA The next thing to do is to prepare some media to
grow and keep the yeast on. I decided to use agar plates for storing
my yeast. The advantages of this method are that it is easy; single
colonies can be isolated, thus allowing you to "purify" yeast away
from contaminating organisms; and that cultures can be kept for
months in a refrigerator with a properly stored plate. To do this
requires getting some pre-sterilized disposable plastic petri dishes
(Fischer Scientific 711 Forbes Avenue Pittsburgh, PA 15219 ). The
best buy is cat#08-757-14G, p.683 ($50/ case of 500). Also needed is
some agar. The best is from Difco Laboratories ( P O Box 331058
Detroit, MI 48232 1-800-521-0851) and it is called Bacto-Agar. Start
by buying 1/2#, which should run about $37. Also, we will need
some DME, and a source of hop oil (any flavor). A good pot to use is
one that has a handle, a tight fitting lid, and preferably a lip for
pouring on the side. It should be big enough to avoid boil-overs, yet
small enough for handling with one hand. Mine is 12 quarts. Start by
boiling up 3 cups of water. Throw in a good amount of DME (up to 1
cup) or pure maltose, (if you can get it), stir to dissolve, and
continue boiling for 15 min. Keep the lid on loosely, to allow steam to
escape
and "steam sanitize" the lid. Add 18 IBU's of hop oil. Use oil. We do
not want to have to strain this mixture. The hops, as any casual
reader of Papazian might know, is to act as a microbicide, thus
helping to select for our hop-tolerant yeast. Then slowly add 1/2
teaspoon of agar, stirring constantly to avoid boiling over. Watch it
carefully, and continue boiling for another 20 minutes. When done,
remove from heat and put the lid on tightly. Allow to cool. Do not use
a wort chiller, or similar device. Agar melts around the boiling point,

and solidifies at around 50 degrees Celsius (your body temp is 37 C).
While you are waiting, crack open a sleeve of plates. Take out 10 and
put them on your freshly scrubbed-down counter in two piles of 5,
with the lids on top. Do not take the lids off. When the pot is still
very warm, but within handling temp., quickly flame the lip you plan
to pour out of by passing it over your stove burner (for people with
electric ranges, see below under "other equipment"). Now, with one
hand tilt the lid, and the stack of plates above it, off the bottom
plate.
Pour the media in to fully cover the bottom of the plate, but only go
about 1/2-3/4 of the way up the sides. Try not to mar the surface
with bubbles. Replace the lid and stack of plates, and proceed to the
next highest plate, etc. Let the plates sit undisturbed for 45 minutes
to an hour. You will know the agar has solidified when the media
color lightens to a buff, and the media stays when tilted. When you
are sure the agar has solidified, turn the plates upside down, and
store them that way to avoid dehydration, in a cool area, like a
cupboard, away from air currents. If your plastic sleeve wrapper is
empty, slide it back on the stack of plates before flipping, and seal
with a twist tie. These can be stored for weeks at room temp., and
longer if you wrap them and refrigerate.

OTHER EQUIPMENT Other things you will need include a source of
flame, for sterilizing. A gas stove does the trick for me. Also good is
a
small alcohol lamp. Do not use an oil lamp. A disposable lighter works
in a pinch. We also need to construct a loop. This consists of a
straight
piece of wire, a little longer than a long neck bottle, with a handle
on
one end. The other end is twisted around into a circle, about 10 mm
dia., to form a loop. A good handle would be one of those twist-to-
clamp X-Acto knives, minus the blade. The more inert the loop
material, the better. A good bacteriological platinum loop is probably
out of the price range of most homebrewers. Try stainless or regular
steel, about .5mm dia. Fischer (see above) also sells pre-sterilized,
plastic, bad-for-the-environment loops for those so inclined. Also, a
source of Parafilm, a wax-like wrapping paper for the lab, will be
helpful in extending the life of your plates. Finally, find a good,
dark
spot in your house, preferably away from air currents, where the
temperature is consistently around 30-37 C. This will be our
makeshift incubator. I use this spot on our range above the pilot
light, and keep a bowl over my plates to shield from air, light, and
grease. Keep this area especially clean.

MAKING A PLATE Okay, so we've made it this far, let's start to
collect yeast. Take a bottle of beer with a good amount of yeast on
the bottom. Allow it to settle in your fridge overnight. Carefully pop
it open and slowly decant the brew. Set this aside. Be sure not to lose

too much yeast pouring. It is best to leave that last bit of beer/yeast

slurry in the bottle. Flame the lip of your bottle and the business end

of your loop. Insert the loop into the bottle. If the loop is still
hot,
touch it to the inner bottom of the bottle, so as to dissipate the
heat.
Scrape up some yeast sediment or swish the loop in the yeast slurry
to fill the loop. Taking care not to touch the loop to anything,
withdraw your sample. Grab a plate and remove the lid. Pick up the
plate and streak the loop back and forth across the plate. Do not
press too hard, or you will ruin the agar surface. To get nice isolated

colonies, confine your streaks to one region of the plate. Flame the
loop, and poke it into a spot of the agar to cool. Pull the loop
through
the streaked region twice and streak in a new region. Repeat this
dilution technique again. Replace the lid, and put this plate upside
down in your incubation zone. With practice comes speed, which is
important for avoiding contamination from airborne nasties. Within a
few days you should see signs of growth. Yeast colonies should be
round, white-to-brownish bumps on the surface, in the pattern you
have streaked. Hopefully your plate will not sport any contaminants,
which could look like almost anything. Contaminating wild yeasts are
hard to discern, but usually look slightly different than what you
have spread. Look carefully, but remember not to open up the plate.
To store your plates, cut a small strip of Parafilm (1 cm x 5 cm), if
you plan to use it. Holding it to the sides of your plate with a thumb,

pull it around the lid/bottom edges of the plate, taffy-like, to seal
the
sides. Sanitize a Rubbermaid container, big enough to hold plates, and
lid. Store the plates in your fridge.

CHOOSING BEERS Not all commercial beer has yeast in it. When
scouting for yeast in the liquor store, I hold the bottle up to the
light
and check for sediment. Also, beer yeast with high attenuation may
be hard to revive. They may have literally drowned in their own
alcoholic poop. Try as I might, I cannot seem to culture Old Peculier
Ale or Duvel's Belgian yeasts. Other yeasts will have simply run out
of food, and settled down for a nice nap in the bottom of our bottle.
These are the ones we want. So, choose a beer without too much
alcohol and a good amount of yeast. German lager yeasts come up
nicely, as do Weiss yeasts (note: many Weissbiers use two yeasts,
and you can see two discrete colony types). Ales can be had too: try
Chimay. Why not start out with one of your own? This is a good way
to keep a free culture of Wyeast on hand. Additionally, the re-use of
a yeast culture (assuming good maintenance) will make a yeast your
own. It will get used to your brewing methods by selecting for
variants that grow well in with your setup. This is how all the
different yeasts used in brewing came about in the thousands of
years B.G. (before genetics).

MAKING A STARTER Well, thats just great. We've got our yeast on
this little plate of agar. But let's not forget our reason for doing
all
this: to make better beer. So, we need to make a starter for these
critters, so we have something to pitch. Start by making the above
recipe, but leave out the agar. When cool, aliquot into very sanitary
beer bottles, about 1/4 volume and cap. Flame your loop and an open
bottle of starter wort. Tap the loop on agar surface to cool, and
scrape
up a single colony. Swish it around in the starter to get the yeast in
suspension. Cap the bottle with an airlock, and store in a nice warm
place. The yeast should be ready to pitch within a few days.

MAINTANING YEAST STOCKS The main reason a plate will go bad is
a contaminating organism may appear. If so, pick a clean yeast
colony with a sterile loop and streak onto a new plate. If you are safe

from contaminants, plates will go bad from dehydration. When a
stock plate shows signs of this, it is good to streak a fresh plate.

FURTHER READING
1. "Manual of Methods for General Bacteriology." Gerhardt, et al.
American Society for Microbiology, Washington, DC. 1981

2. "Microbiological Methods." C.H. Collins. Plenum Press, New York, NY

3. "Methods in Yeast Genetics" F. Sherman, et al. Cold Spring Harbor
Laboratory, Cold Spring Harbor, NY 1979

DISCLAIMER I have written this to introduce homebrewers to some
principles of microbiology. I make no claims about the use of this
information, nor my expertise in this area. Additionally, I am aware of
other methods of yeast culturing. I only describe what has worked for
me. Please distribute this document freely.








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