From: arf@ddsw1.mcs.com (Jack Schmidling)
Subject: Re: Dragging up More Dregs.Culturing
Organization: ddsw1.MCS.COM Contributor, Chicago, IL
Date: Sun, 23 Aug 1992 03:31:38 GMT
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I think the following should answer most of your questions....

 
 
                  JACK SCHMIDLING ON YEAST CULTURE
 
 
                          Objective
 
 The objective of culturing yeast 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 to the naked eye.
 
 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.  This is done using a calibrated X-Y stage on the 
 microscope and making careful notes.  Fortunately, however, I do not 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 1.020 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.
 
 It should be noted that a pressure cooker is the preferred method of 
 sterilization but for our purposes, one could probably get by with steaming 
 in any pot with a lid and a half inch of water.  Set the dishes or slants in 
 or on a cup or some other support to keep them out of the water.
 
 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 when 
 vertical.  They are also stuffed with cotton before going into the pc.  You 
 can also use tubes with plastic screwcaps and avoid the cotton.
 
 It should be noted that gelatine melts around 75 F so its use in summer is 
 precarious.   The better alternative to gelatine is agar agar.  This is 
 available at oriental food stores in stick form.  Half a stick (about 4 
 inches) in a cup of wort will get you through the hottest weather.
 
 
 
                      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.  If starting with dry yeast, desolve one granule of 
 yeast in a test tube with about one inch of sterile water.  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 and as controls.
 
 
                       Pure Culture
 
 The next step is to visually inspect the surface of the petri dish under low 
 magnification (hand lens or naked eye will do) 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 poke this into 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 25% or more 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 pint 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