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.