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The Budget Microbiologist

By Gus Cabuso

When I decided to add to my add to my list of home brewing skills by culturing my own yeast, it didn't take long to realize that it might not be as easy as I anticipated. I live in a small town in the mountain west that does not enjoy the availability of many of the items that are readily available in larger cities. There was also a shortage of information on exactly how to go about beginning a yeast ranch. What follows are the results of my efforts in gathering materials and information. This information was compiled from a variety of print and virtual sources and I make no promises as to your success with it, nor do I take credit for creating it.

My total investment was in the neighborhood of thirty dollars, (not counting my tuition last semester, during which I took two Microbiology classes.) That was adequate to provide everything I needed to begin and maintain a ready and renewable supply of yeast with a minimum of repeat purchase requirements.

A Word on Sanitation

This is probably as good a time as any to talk about sanitation. You will notice periodic references to "sterile" items in this writing. I will not go into a great deal of detail about the processes of sterilization unless there is a special technique involved. Anyone who may find this information of use to them already knows the importance of sanitation during the brewing process and I won't waste time or space telling you what you already know. Suffice it to say that working with microorganism cultures requires an attention to sanitation that is far above that required for home brewing. In brewing you can (you shouldn't, but you can) get a little careless and still come out okay. The margin for error in working with cultures is far less forgiving.

Any time you are working with these cultures you must ensure that the area you are working in is as clean as possible. Dust, grease, heavy breathing, pet hair, and anything else that provides a vehicle for contaminants should be avoided. Don't expose your media to air any more than is necessary to inoculate it or remove a sample. If the medium becomes contaminated, or is in any way suspect, throw it away and begin again. This is too cheap and easy to use a potentially contaminated source, it defeats the whole purpose. Remember, in micro, the operative word is PURE. Treat anything that comes in contact with your brew or anything that goes into it, with the care that all brewers know is essential, MAKE SURE ITS SANITARY.

Getting Started

Many methods are utilized for this process by different brewers, that require various types of equipment ranging from the most simple, (beer bottles and a cigarette lighter), to the most complex, (requiring microscopes and grid filters.) Mine is a melange of several of these and my practical experience. Once again, the only credit I take for this is in compilation. This information came from many sources, perhaps including some of you now reading this and from various books including one by the patron saint of homebrew, Charlie Papazian.

The purchases that were required began with a package of petri plates, 20 for $5, and a dozen or so plastic test tubes with caps, 40 cents each. These were purchased mail order from Williams Brewing out of San Leandro, California. The advantage to using these items is that they are already sterile, and packaged to remain that way until used. In my view, its two less things to have to sanitize. I did a fair amount of research to find a source and this company is the only one that had reasonable prices and allowed purchases of anything less than a gross.

I also purchased from them a 500 ml Erlenmeyer flask. It cost about $5 but a glass milk bottle or something similar will do just as well. I like the flask because it is graduated, which simplifies my life. Foam stoppers and rubber corks are readily available for this item as well and it is easy to fit with an airlock for use as a starter vessel. Agar Agar is supposed to be available from most Asian grocers, but if you live in an area where there are none, it can be mail ordered as well. Six ounces cost me less than three dollars and six ounces is plenty. Some people use Knox which works okay, but Agar gels at a much higher temperature. I wouldn't want to try to work with Knox here in August.

The food source for the yeast will be light dry malt extract. Some recommend adding a yeast nutrient to your agar, you can if you like but I haven't found it necessary. Some kind of inoculating loop is also needed. There are again many ways to go with this one. You can pay upwards of $100 for a platinum one, purchase a cheapie for 4 or 5 dollars, or make your own with a paper clip and an exacto knife handle.

So much for the stuff I paid for. I begin my starters in baby food jars. They are free (if you know someone with a baby), and easy to sterilize. Go for juice or fruit jars because they are less likely to retain odors. Discard any that do have odors or discolored lid liners. The other items we all have; a brewpot, fridge, plastic baggies, etc.

The Medium

I begin with my brewpot with about 1 or 2 inches of water in it and a teaspoon of vinegar, (it keeps the glass from clouding.) I place the baby food jars and the lids in it with my flask. In the flask is 250 ml of water, three teaspoons of Agar powder, and five teaspoons of dry malt extract, dissolved. Cover, bring to a boil, and continue boiling for at least a half hour. If you have access to one, the preferred method is to use a pressure cooker instead of a brewpot and steam them at 15 psi. for the same amount of time.

My work area is sanitized with a bleach solution, and I am working in a draft free, clean room. The heat is turned off beneath the pot and I begin by filling the baby food jars. I pour about one half inch of liquid agar into each one and place the lids on without tightening them. I place them upright in the bottom of the pot with what remains of the steaming water. I keep the pot covered as much as possible during these steps. Next I remove the petri dishes from their package and immediately reseal it. I stack the dishes on my sanitized surface and beginning with the one on the bottom, lift the lid (and all of the plates above it), just enough to allow me to complete the next step.

(Try to avoid removing the lid completely.) I pour enough agar into the plate to cover the bottom and replace the lid. The longer the flask is out of the pot, the more opportunity exists for it to become contaminated so this should be done as quickly as possible without making a mess. This step is repeated until all of the plates are filled. The plates are then placed in the pot, on top of the food jars.

The last step is to fill the tubes. They are filled halfway, and placed in a bowl inside the pot so that they are on a slant somewhat less than 45 degrees. The caps are not tightened just yet because the cooling agar may cause a low pressure inside that will crack them. The reason for this approach is a fantasy that I have that the steam that remains in the pot will help to keep any transient microbiota from contaminating the medium while it is being prepared and until cooling takes place. The pot is then covered and let the "blank slants", plates, and food jars are allowed to come to room temperature.

After they are cool, the caps are tightened, and the plates are inverted so that the condensate can not drip onto the medium. They are stored in ziplock bags or a sanitized tupperware container in the fridge until they are needed. The baggie or container is essential to protect the medium from contamination from all of the microorganisms that live in your refrigerator.

This yields slightly less than 300 ml of liquid agar and will fill about five plates, three or four tubes, and three or four food jars. I leave one plate out of the fridge (in a ziplock), and check it in a day or so to make sure the medium was not contaminated.

Working with the Yeast

When working with yeasts and media, allow them to come to room temperature before manipulating. Take your slants or plates out of the fridge and allow them to warm up to room temp before transferring or inoculating. Drastic temperature changes may shock the yeast into inactivity, aborting or at the very least adding time to the processes.

The Streak Plate

There are several methods of doing a streak plate. I will describe the one that yields the best results for me. The reason for doing this is to isolate a pure colony, (that is a colony of yeast cells that is the progeny of a single cell), which will be used to grow a pure culture. I use an alcohol lamp to flame my loop for sterilization. Its one of those cheap, oil candles they sell during Christmas time, filled with ethyl (grain) alcohol instead of lantern oil. A gas range burner will work and some people simply use a cotton ball and alcohol or 100 proof vodka, which will work to a lesser extent.

The loop should be flamed until it glows red and then air cooled. Don't wave it around or blow on it, just be patient. After this, use it to obtain a bit of yeast from your starting yeast source. This can be a Wyeast pack, the sediment from a bottle of brew that contains a strain that you are fond of, or a bit of dry yeast dissolved in a tube of sterile water. Make sure the loop is sufficiently cool when you do this. If it sizzles when you touch it to the source, start over, you just killed it. Keep in mind that where ever you obtain your starting culture, make sure you inoculate your plate with it and not with contaminants from the package, bottle, counter top, or your hands. Use your standard homebrew sanitation technique, KEEP IT CLEAN.

One loopful of the source is plenty. Visualize the plate being divided in thirds,(if it helps divide it so by using a marker on the bottom of the plate and number the sections.) Lift the lid just enough to place the loop underneath. Streak it back and forth over one third being careful not to break the surface of the agar. Flame the loop when done. Streak the loop back and forth again, first going into the area you just inoculated twice, and then over the second third of the surface. Flame the loop again. Streak the loop through the second third of the plate surface twice and then over the last third of the plate. If this is done correctly each third will have less yeast in it than the one before it. Our hope is that at some point it will be so diluted that a single cell will come off of the loop and grow into a pure colony. Hopefully there will be several of these on the plate. I usually inoculate two or three plates to ensure I my choice of colonies and as a backup for an accidentally contaminated plate. "Incubate" the inoculated media at room temp, in a ziplock for 3 or 4 days. On top of or in a cupboard above the fridge is a choice spot.

This is where all of the expensive lab equipment comes in. However if all went well we should have a culture in which we can identify a pure colony without the benefit of it and with the naked eye. We are performing an inexact science here but we should be able to assume safely enough which one we want to choose. The yeast colonies will be whitish and should be perfectly round. The gas that has filled the bag will also have a pleasant yeasty smell. If the plate is contaminated with mold, (any dark or furry growth), or anything that doesn't look like what I have described, bag it, don't even bother. The dominant strain will be discernable by the fact that the majority of the colonies will be similar in appearance.

The Slant

Choose a healthy looking colony; good color, perfectly round, not touching any other colony, and remove it with your sterilized loop. Transfer it to one of your blank slants by smearing it over the surface of the agar in the tube. Take care not to touch the sides of the tube. If you do, the yeast will grow down the sides of the tube and accumulate in a gas bubble at the tip of the tube, beneath the agar, where you can't get to it. In a few of days you should see a good milky looking lawn of growth on the surface of the agar. The tube can now be stored in the fridge (in a ziplock) until needed.

The yeast in the slants can be used to make starters for a few months, after which time a new slant should be made. Use the old slant as your source and make another streak plate as described earlier and inoculate new slants from those colonies. This should only be done for four or five generations from the original sample because after that you will begin to lose quality due to natural mutation.

As you brew, you may use the sediment from your primary fermentation to make new plates and slants and replace your old ones with these as they expire.

The Starter

To prepare a starter, one of the food jars is inoculated with yeast from one of your slants. Transfer a loopful of yeast from the slant to the surface of the agar in the jar. When a substantial growth is present, (three days or so), it is ready to use. After the first 12 hours or so it might be a good idea to crack the seal on the jar (to receive the CO2 pressure head) and re-tighten. Prepare a 1040 wort by boiling 2 ounces of dry malt extract with 2 cups of water. The addition of one or two hop pellets will act as an added measure of protection against introduction of transient microbiota. Cool to below 85 degrees and add enough to the food jar to cover the yeast well. What wort is left over may be stored in the fridge in a sterile jar.

Within a day you should have active fermentation. Transfer this wort/yeast mixture to your sterile flask and add enough of the left over wort to equal about 300ml. Affix a foam stopper or airlock. You may use your sterile loop to scrape any yeast from the agar that is reluctant to make the move, but swirling the liquid around has always worked for me. If you use the loop and end up scraping up a bit of the gelatin, don't worry about it. If this step is completed a day or two before brew day, it should be ready to pitch when you need it. You will not notice a foamy head of kraeusen in the flask but that's okay. If you see a thickening layer of white sediment and small bubbles rising inside, its working.

After you have boiled and cooled your wort, pitch the contents of the flask to your fermenter and there you have it.

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