From the HBD Archive
From: arf@ddsw1.mcs.com (Jack Schmidling)
Subject: yeast culture
Date: 1992-03-23 18:49:00 GMT


To: Homebrew Digest
Fm: Jack Schmidling

Several readers have asked, both here and on usenet, for info on yeast
culturing. No one has responded thus far so, although I know only enough to
be dangerous, I will share what I do know.

For openers, I called Yeast Culture Kit as suggested by someone and got
nothing but a price list in the mail. I poured through my library and dusted
off some glassware and made a few starts to get the feel.

Objective

The objective is to isolate a single cell from a beer or culture that has the
characteristics desired and encourage this cell to reproduce enough offspring
to start a new batch of beer.

This is easier said than done but with reasonable care, luck and modest
investment, can be accomplished by the serious home brewer.

General Program

The general program is to dilute the original culture and spread it over the
surface of a growth medium in a petri dish so that individual cells are far
enough apart to allow them to grow into visible colonies without touching
each other.

A sample from one of these typical colonies is transferred to a test tube
containing a growth medium. When this colony is actively growing, it is
considered a pure culture and can be refrigerated for later use or started by
covering with beer wort. When this starter is actively fermenting, it is
poured into a larger amount of wort which, when active, is pitched into the
beer.

Basic Assumptions

The procedure makes a number of assumptions which are correct, often enough
to allow it to work well enough, to satisfy most requirements.

The first assumption is that one can select the desired strain by looking at
colonies on a petri dish. This is more or less true because the overwhelming
majority will be the same, i.e. the dominant strain. Bacteria, molds and
many wild yeasts are obvious and recognizable.

The second assumption is that, while still very small, all round colonies are
the progeny of single cells.

The third assumption is that all such colonies, at least in the center are
mono clonal or at least mono-cultures and otherwise sterile.

To do the job right, one would have to study the original diluted culture
under high magnification and do a presort at that level. This is revealing
and fun. It also gives an indication of any bacterial contamination in the
culture but the rub is marking individual cells and finding them later when
they grow into colonies. I currently have no way of doing that. However,
neither do I believe that it is really necessary for the home brewer,
although a must for the lab selling selected strains.

Details

There are many growth media available for the purpose and no doubt someone
can recommend a source or recipe for the ideal but for my experiments, I
mixed two packets (16 gr) of Knox gelatin with one cup of wort. After
heating and disolving, this is poured into petri dishes and test tubes and
sterilized in a pressure cooker for 15 min at 15 lb.

The petri dishes are turned upside down after solidifying and cultured this
way to prevent water of condensation from falling on the medium. The test
tubes are cooled on a slant to allow the water to settle on the bottom. They
are also stuffed with cotton before going into the pc.

Isolating Cells

The first step is to inoculate the petri dish with as diluted a mixture as
possible. The books are full of procedures for doing this but I find the
simplest is just as good. Take a copper wire or thin glass rod and heat
several inches in a flame to sterilize. Dip this, when cool, into a working
beer or yeast culture. Gently drag this across the gelatin in the petri
dish, trying not to break the surface. Next, draw the wire across this line
at several points, to further dilute the sample. Turn the dish over onto the
cover and "incubate" at room temp for several days. Do this on several
dishes just for insurance. As gelatin melts just above room temp, any
attempts to rush the process by increasing the temp will prove disasterous.
If in a hurry, substitute agar for the gelatin and incubate at 80F.

Pure Culture

The next step is to visually inspect the surface of the petri dish under low
magnification to pick out a "typical" colony that appears to have come from a
single cell. All colonies should be rejected that are any shape other than
perfectly round and differ in any way from the majority.

Flame your wire again and after cooling, remove a small sample from the
center of the selected colony and rub this on the surface of the medium in a
"slant" test tube. You can do this to several slants, with the same sample,
to assure all slants are the same or flame the wire and take a new sample
from a different colony. You can make as many slants as you will need for
several months and throw away the petri culture.

You now incubate the slants until most of the surface is covered with the
pure colony and then refrigerate them till needed.

Starting

When needed for use, cover the slant with sterile wort and pitch when ready,
i.e fermenting. For best results, this starter should be used to pitch about
a quart of wort, a day or so before brew day.

This process can be used on anything from a packet of Red Star to a bottle of
your favorite beer and will produce a pure culture. There is no guarantee
however, that the strain will remain the same for ever because of natural
mutation. As it is my experience that the most common and objectionable
contaminents of dry yeast are bacteria and mold, this process will guarantee
at least, to eliminate these most serious problems.

I was intrigued by the recent posting on the quality of beer made from Red
Star that was re-cultured. I was also "impressed" by the number of contest
winners who use Wyeast and now rise to the challenge of winning the "World's
Greatest Brewer" trophy using re-cultured Red Star instead of just joining
the Wyeast bandwaggon.

js




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