1. Use 14 grams of dry yeast (usually 2 packets) per 4 gallons of brew. Rigorously sterilize everything used in the hydration procedure. This should include boiling and cooling the water for rehydration, so that chlorine is boiled off and the water is sanitized.
2. Add the dry yeast to 1/2 cup of water at 90F (32C). Leave for 14 mins.
3. Combine the hydrated yeast with 1-2 gallons of wort that is as close to the wort to be fermented as possible. You can take samples from the main wort at the end of the mash/sparge and rapidly boil and cool it.
4. Aerate the starter as much as possible under sanitary conditions.
5. Don't forget to properly oxygenate the main wort once it is chilled. (Shaking hot wort is dangerous, but even worse it can cause oxidation and give your beer funny flavors.)
6. Pitch the starter into the main wort once the latter has been chilled to the recommended fermentation temperature (64-68F or 18-20C). Yeast with good viability will result in minimal lags. (The longest experienced in test brews using the new Red Star Ale Yeast was 2 hrs.)

1. Sanitize everything in sight.
2. Aerate your wort to insure rapid initial yeast growth--your best defense against secondary infection.

PART 2: Propogation of Yeast Strains

or

How to have your very own yeast ranch!

I am deeply indepted to George Fix for both giving me these chapters and letting me alter and condense them for the homebrewer. His support was an essential impetus for getting this FAQ off the ground.

General Comments

There is no single item as important as the selection of a yeast strain, or if appropriate strains, to be used in commercial brewing. The same applies to homebrewing. Sensory characteristics taste and smell will normally determine the type of yeast that is appropriate to any particular beer formulation. This section contains the necessary procedures for achieving self-sufficiency in pitching yeast. The part treated in this section is often called the Hansen pure culture system. The heart of this system is the so-called yeast slant. It is a test tube containing a solidified media sloped at an angle. Often Petri dishes are used, but the media is level, and hence the term slant is not always appropriate. In any case, yeast cells are streaked on the surface of the solid media. When refrigerated, these slants will keep at least 3-4 months before they have to be recultured. Yeast are taken from the slants, and built up so there is enough to pitch a full batch. The system also contains procedures for doing the exact opposite, i.e., adding yeast to slants for storage and future use.

Equipment

The equipment needs for operating a pure culture system with slants are rather modest. The following are the major items.

Refrigerator

This is needed for slant and media storage.

Autoclave or pressure cooker

This will be needed to sterilize equipment and media for yeast work. A pressure cooker will do, but it should have a pressure gauge attached so that the conditions during sterilization can be controlled.

Media

The preferred media for slants is malt extract and agar. These can be obtained from any scientific outlet. Food grade agar is also available from some oriental markets. The flaked form is easier to work with.

Misc.

A number of minor items will also be needed. These include inoculation loops, glassware, petri dishes, and test tubes.

Propagation of Yeast

This process consists of transferring some of the yeast on slants to a small flask or jar containing wort, then building this up until there is enough to pitch a full brew. The most delicate steps are the initial ones. Experience has shown that the best results are obtained by using full strength hopped wort for propagating yeast. The ideal situation is when the wort used in propagation is identical to the wort that will be used in brewing.

Practical experience has also shown that it is best to pitch yeast freshly harvested from slants at the maximum acceptable rate. Anticipating the results in the next section, this for lager yeast amounts to pitching 1 volume of yeast solids for each 240 volumes of wort. Thus, we need 4gal/240 = 0.02gal*128oz/gal = 2.4oz of yeast solids for a 4 gallon batch. Using the estimation that yeast solids are 1/10 the total volume of a yeast culture after the krauesen dies down (i.e. just entering lag phase), that means that one needs about 24oz or a little more than 3 cups culture. For ale yeast all of these numbers are reduced by a factor of two, so (3/2) to 2 cups of an ale yeast culture would be sufficient.

In the procedure described below new wort is added just after the end of the period of high krauesen, and in particular after the foam starts to recede. The reason for this is to keep the yeast in the aerobic exponential growth mode. This will insure a steady buildup of yeast cells, and thereby minimize the number of wort charges that are required. The importance of taking great care when adding fresh wort can not be overemphasized. To avoid infections not only is it necessary to properly sanitize equipment, but it also important to sterilize necks of vessels and jars by flame or 200 proof alcohol solutions. The easiest way to flame a jar at home is with a lighter (esp. the ones for pipe- smokers!). Be extremely careful, and don't use both alcohol and a lighter.

The first four steps described below are done under the cleanest conditions possible using 1000 ml. starter jars. At the end of the fourth step there will invariably be more than enough yeast in each starter jar to pitch a 24 liter brew (about 6gal); i.e., there will be at least 1/10 liter of yeast solids as can be checked by visual inspection. These numbers are based on the requirements of lager yeast. As will be seen below there will be no harm in producing too much yeast in this procedure since at the end only the correct amount will be added to the fermenter.

1. Preparations:

   1. Carefully inspect all the slants that are to be propagated. Those which have unusual growth patterns and/or discoloration should be discarded. The ideal is thin white yeast layer on top of the solid media.
   2. Autoclave the starter jars and the rubber stoppers for the airlocks for 4 mins. at 14 psi. Alternatively, use your favorite chemical sanitizing agent.
   3. Add 240 ml. (about 8 oz) of wort to each starter jar. Wipe their necks with a 200 proof alcohol solution. After this add the airlocks.
   4. Pasteurize the wort by adding the starter jars to a water bath at 60 C (140 F), and hold this temperature for 20 mins. Cool to 18 C (74 F).
   5. In a clean room with no air movement (turn off fans and air conditioning for at least 14 min to give the dust a chance to settle), place starter jars, yeast slants, inoculation loops, and a 200 proof alcohol solution in a clean, quiet spot (i.e. lock the door after first insuring that Fido, Fluffy, and Junior are on the other side of it :-) !).

2. Inoculation:

   For each jar, start by sterilizing its neck. Then sterilize ("flame") the inoculation loop. Open a slant, quench the loop in clean agar ("sizzle") and use the loop to remove some yeast. Remove the airlock and then add the yeast to the starter jar. Replace the airlock, and then start work on the next jar.

3. Initial Buildup:

   1. Place the starter jars in a location where 68F (18C can be held). Resuspend the yeast twice daily by vigorously swirling the jars. 1L Erlenmeyer flasks are excellent for this purpose because they permit vigorous swirling without getting the wort up by the neck and opening. Also good--and more fun to prepare, Hic :-) --are 1.4L ex-wine bottles. The wine bottles are also cheaper, even with the wine. But be careful heating them--I have not used them, and I forget who recommended them as an alternative!
   2. A widely used practice is to discard any starter that is not active within 48 hours. Certainly if some of the starters are active within this period, then the inactive ones should be discarded. In any case, any starter not active within 72 hours should definitely be discarded even if this means they are all discarded.

4. Second Wort Charge

   1. When the foam has receded prepare 240ml. of fresh sterile and aerated wort for each starter.
   2. The new wort is to be added to each starter, and this should be done as cleanly as possible.
   3. Before pouring the wort into the starters, it is very important to swab the necks of the starter jar and the wort jar with a 200 proof alcohol solution to prevent contamination or flame them with a lighter.
   4. It is also desirable to reduce the temperature to a point closer to the temperature that will be used in production if that is lower than 18 C. The temperature should be reduced slowly, e.g. few degrees a day. Large shifts in temperature (>10 deg F or >4 deg C) can cause marked slowing of yeast growth.
   5. The starters should be swirled at the start and then again after 12 hours. New activity should be seen before 24 hrs. Those which are not active within 36-48 hours should be discarded.
   6. Increase the volume of wort until you have sufficient volume to pitch.

5. Pitching the Yeast

   1. At this time you should have a jar with about 400ml (a little more than 2 cups) of yeast for a 4gal ale batch. I would suggest pitching just after the krausen (foam) dies down, the logic being that the yeast have amassed glycogen reserves and are at their healthiest. Some other sources recommend pitching at high krausen, reasoning that the yeast are in the exponential growth phase. Whatever you do, avoid overdilution and keep accurate notes. The total volume will vary with batch size, yeast type, and your personal experience/whim. Remember to keep yeast notes along with your beer notes so that you can learn from experience!
   2. Clean the outside of the jar with 200 proof alcohol or weak bleach and allow to dry.
   3. Pour the yeast slurry carefully into the primary.

Preparation of New Slants

Two steps are needed in the preparation of new slants. The first consists of adding the proper media to test tubes or petri dishes. Once prepared the slants will store well far a very long time when refrigerated, so many can be prepared at one time. The second step consists of inoculating the slants with yeast. For the homebrewer who cannot afford several refrigerators: Please be advised that your refrigerator is a haven for bacteria, mold, and wild yeast. Anyone wishing to store sterile slants in their refrigerator is advised to

• Wipe down the slants before storage with ethanol or your favorite sanitizing solution.
• Seal the slants with parafilm or electrical tape.
• Keep the slants in a ziplock bag.
• Wipe down the bag with ethanol or your favorite sanitizing solution before opening.

The media consists of dry malt extract and agar. As a general rule 4 tablespoons of malt extract and 1 tablespoon of agar per cup of water will yield 16-18 slants.

1. Bring the water to a boil, and then stir in the malt extract. Boil for 10 mins.
2. Remove from heat, and then start stirring in the agar. This will take some effort, but this usually indicates that a good solidification will ultimately be achieved. If your slants "sweat" too much, you may want to increase the amount of agar you use. Although commercial/scientific agar will vary little, I cannot answer for "food grade" supplies. Gelatin is easier to dissolve, but it sometimes does not give a good solidification.
3. When the agar is dissolved, the malt/agar solution should be added to the test tubes, filling each to approximately a third of their volume. Add the screw cap, but do not fully tighten.
4. Autoclave the tubes at 14 psi for 14-20 mins.
5. Allow the tubes to cool. Don't tighten the caps until they are cool or they may implode! Although this sounds fun, in reality, flying glass shards and hot agar blobs are a nasty combo. They can be left overnight in the autoclave/ pressure-cooker so that they can cool in a sterile environment. Tighten the caps on the tubes, and place the tubes at a 30 degree angle. Allow them to solidify at room temperature. Solidification should become apparent within a few hours. Tubes which are not solid after 24 hrs. should be discarded.
6. Refrigerate until needed, heeding storage precautions above.

Note: Plastic petri dishes can not be autoclaved, and so alternate procedures are needed for them. You may use the above techniques with pyrex petri dishes if you so desire. A common practice is to autoclave the malt/ agar solution in small jars or flasks. The agar solution is then poured into the petri dishes. Let the agar cool until the jars are just slightly too hot to handle bare handed--about 40 deg C; the media will start to set around 40 degrees. If the agar is too hot it will warp plastic plates. Swirl it gently to mix but avoid bubbles. A few bubbles around the edges are unimportant, but sometime the whole surface of the plate is bubbles. You can pop the bubbles with the flame of a lighter! Or use a hot inocculation loop. Do not use your finger or blow on the plates. Let the poured plates dry 2 or 3 days in a clean quiet room before bagging. Condensation is normal, but you have to deal with it. Once the plates have cooled, turn them over (agar side on top) and always incubate them and store them in this position. That way the water vapor wafts into the agar and keeps it humid (slightly) and any condensation that does form drops to the lid and can be shaken off. Wipe them down, seal them, and bag them, but leave them at room temperature for 1 week. The bad bugs, if they are there, will be visually apparent at the end of that period and the contaminated plates can be discarded. While Petri dishes are more trouble than test tubes, they do offer the distinct advantage of having more surface area and being easier to store. After the trial period the dishes should be refrigerated.

Another Note: If you find mold (not wild yeast) contamination to be a persistent problem, Pierre Jelenc suggests:

From 0.4 to 1% sodium propionate in the medium will suppress practically all molds, without affecting the growth or viability of yeasts. The propionate can be either added before autoclaving, in which case the medium will turn cloudy, or as a sterile solution just before pouring the plates, in which case the medium will stay clear. There is no growth difference in either case. While not reinheitgebotmaessig, propionate is FDA-approved to prevent molds on foodstuffs.

Inoculation of Slants:

1. Collect a small portion of the yeast to be added to the slants. It goes without saying that one should strictly follow the standard sterilization procedures of all items used to collect this yeast. I usually open a Wyeast (or other brand) pouch, then streak a plate and make a starter at the same time, that way I know what I am getting. Feel free to culture from the dregs of your favorite unpastuerized brew or the roof of your favorite monastery.
2. With one hand sterilize the inoculation loop (flame or alcohol solution). With the other hand open the cap of a slant.
3. Dip the loop into the yeast solution, and remove a small amount.
4. Slowly insert the loop into the tube avoiding contact with either the sides or neck of the tube. Streak the yeast over the solid. Only a thin layer is wanted, and one should try to use as much of the surface area as possible.
5. Slowly remove the loop avoiding contact with tube walls or neck. Add the screw cap back on the tube and tighten.
6. When finished store the tubes at 24 C for one week. Visually inspect all tubes at this time both for yeast growth, and also for any irregularities (see below). Discard those which are not satisfactory. Growth for most Saccharomyces sp. should be evident within 3 days; Lambicophiles culturing Brettanomyces sp. are on their own (actually, these typically grow slower, about a week for the Brett--the species names have been withheld to protect the innocent). Brett may actually be better maintained in liquid culture, with an occasional streaking to check for gross contamination. Again, more rumor and hearsay.
7. Store the remainder at 2-8 C. After 3-4 mos. of storage, unused tubes should either be discarded or recultured; i.e., propagated by the procedures in Section III.2.c and then put on fresh slants. The best idea is to put production yeast on slants on a regular basis so that reculturing is not necessary.

Note: The larger surface area afforded by Petri dishes can be used to advantage in the above procedure. In particular, it useful to streak out yeast in parallel lines which make angles with each other. This allows for a better examination of growth patterns. Petri dishes should be sealed after the 1 week trial period with electrician's tape and refrigerated.

How can you tell contaminants (mold and bacteria) from yeast? J. Wyllie (The Coyote) slk6p@cc.usu.edu wrote in rec.crafts.brewing in answer to that question: (this has been slightly ammended)

*Things to look for: Colors: creamy off white. (red, yellow, etc likely to be contaminants) Textures/Shapes: Mostly roundish, like a demi-sphere. (Fuzzy=bad mold, flat=maybe bad). Light Transmittance: Hold the plate up to the light. Look for colonies which are transluscent- let light pass. If there are opaque ones (darker) consider them contaminants. You can still pick a pure colony off of a plate with a contaminant elsewhere on the plate (unless you have fuzzy fungal hyphae and spores all over)

PART 3: Yeast Washing for the Homebrewer

The following notes were taken from a demonstration given to the Oregon Brew Crew by Dave Logsdon of WYeast Labs, on September 12th. According to Dave, it was important for healthy yeast to be washed free of trub and hop residue so that it could be stored for future use. Dave said that the problem with simply storing the mixed contents from a carboy after fermentation was that the unwanted particulates would suffocate the yeast over a period of time. Most breweries, Dave stressed, use an acid wash; the sterile water wash is much more practical for homebrewers.

Objective:

To recover yeast from a finished batch of beer for repitching or storage for future brewing.

Materials:

• One primary fermenter after beer has been siphoned off or otherwise removed.
• Three sanitized 1-quart Mason jars with lids, half full of sterile or boiled water. They should be cooled down, then chilled to refrigerator temperature (ca. 38^F).

Procedures:

1. Sanitize the opening of the carboy (flame or wipe with chlorine or alcohol)
2. Pour the water from one of the quart jars into the carboy. Swirl the water to agitate the yeast, hop residue and trub from the bottom.
3. Pour contents from the carboy back into the empty jar and replace the cover.
4. Agitate the jar to allow separation of the components. Continue to agitate periodically until obvious separation is noticeable.
5. While the viable yeast remains in suspension, pour off this portion into the second jar. Be careful to leave as much of the hops and trub behind as possible.
6. Agitate the second container to again get as much separation of yeast from particulate matter as possible. Allow contents to rest (about 1/2 hour to 1 hour) then pour off any excess water--and floating hop particles--from the surface.
7. Pour off yeast fraction which suspends above the particulate into the third container. Store this container up to 1 month refrigerated. Pour off liquid and add wort 2 days before brewing or repitch into a new brew straight away.

It should be noted that in the actual demonstration, Dave eliminated the final step; the yeast in the second jar was essentially clean at this stage and seemingly fine for storage.

PART 4: Parallel Yeast Cultures

Rick Cavasin sent me (PW) the following method of "parallel" culturing liquid yeasts. This should work with most packaged liquid yeasts, not just Wyeast. The advantages here for the beginner are that (in additon to saving money) it minimizes the problems of strain drift and contamination that can plague yeast ranchers. As for the savings, it makes liquid yeast almost as cheap as dry yeast! Post follows:

Here's the (poor man's) method for stretching the Wyeast that I (Rick) have been using successfully. This method has worked for me with 4 different Wyeast ale strains (Whitbread, Irish, German, European). It's simple, and requires no special equipment. Also, it allows several brewers to swap yeasts with each brewer propagating one strain.

Briefly, my suggestion consists of converting the original Wyeast package into a number of 'copies' stored in beer bottles. ie. it is a parallel propagation rather than a serial propagation

1. Step 1: Prepare some starter wort (S.G. = 1.020), see Miller's book for recipe. Basically, you need about 1/2 gallon, but if you make more and can it in mason jars (using standard canning procedures), you will not have to prepare more at a later date. Note from PW--Most authorities now recommend using full strength (1.040), hopped wort for starters.
2. Step 2: Place 1/2 gallon or so of starter wort in a suitable container (1 gallon glass jug), pitch (inflated) Wyeast package at correct temp. and fit air lock. This is the 'master' starter.
3. Step 3: Allow to ferment to completion. When fermentation has ceased, agitate the 'beer' to suspend all sendiment, and very carefully bottle it.

You will now have about 6 bottles of very thin beer with a good deal of viable yeast sediment in each bottle. Use each bottle as you would use a package of Wyeast - ie. prepare a starter culture a couple days before brewing. This is facilitated by canning wort when you prepare the master starter. All you need to in that case is pop open a mason jar of wort, dump it into a sanitized bottle/jug of appropriate size, pop open one of your bottle cultures, add it, agitate vigorously, and fit an air lock. All yeast starters are of the same 'generation', ie. 'twice removed' from the original Wyeast package (as opposed to the usual 'once removed'). I've had the bottled cultures remain viable for more than 6 months. Observe proper sanitation and wort aeration procedures thoughout. Equipment: 1 gallon jug (for 'master' starter) 1.4 litre wine bottle (for subsequent starters) air lock 6 beer bottles, caps and capper Optional equipment: mason jars and canning pot. Cheers, Rick C.

PART 4: Send Yeast Through the Mail!

The following technique is reproduced as is from the pages of the HBD; it sounds like and interesting and useful method. Now, if someone could only figure out how to send yeast by e-mail!

From: drose@husc.harvard.edu

Subject: Mailing Strains

... I thought it worth mentioning that there is a cheaper alternative to agar slants for mailing strains, and it works just as well. We routinely send out laboratory strains on filter paper. Basically, you just put a drop of culture on a ~1 cm square piece of filter paper (probably any absorbent paper would do) and wrap the square in a piece of sterile foil. Then pop it into an envelope and send it off. When it gets to the other side, they drop the paper on a rich media plate, incubate for a day or so, and the yeast grow up. Then you streak for singles on another plate and you're set. I haven't rigorously determined the viability of cells dried on paper, but they are very stable. It works.

I can think of two possible disadvantages to this system. First, we use autoclaved paper and foil, and a surprising number of households STILL lack an autoclave. However, while commercial paper is probably not sterile, I imagine it is pretty close; the yeast are going to far outnumber anything else, and when you streak for singles you will get what you want. The foil you could always steam, but probably it would also be close enough to sterile for most people's purposes. The second drawback is that this method requires that you are set up to culture, and to streak for singles in particular. However, while this isn't absolutely necessary in the case of slants, it is certainly advisable. Anyway, just thought I'd throw it out there. Dave Rose Dept. of Cellular and Developmental Biology Harvard U.

This FAQ was HTMLized by Gary Rich and Paul Timmerman