Yeast stocks have been kept and maintained for a long time as a part of brewing beer, and making wine, cheese, sausage, bread, etc. Different strains of yeast or molds have been collected and cultivated as different types of beer and wine have been created. Maintaining these stocks has been an integral part of brewing, and a natural extension of the process, just as growing grain, hops or grapes has been. The need for yeast, or at least something from the environment has been known to brewers and vintners for millennia, but knowledge of yeast, and isolation of various types has been very recent. Brewers or vintners who maintain yeast cultures on a “farm” allows the to create a library of yeast strains from which they can create a variety of products. For really adventurous home brewers, many unique beverages, or one consistent product can be created through culturing yeast at home.
Yeasts are single-celled organisms known as eucaryotes. They are related to bacteria, or procaryotes, and it is thought that procaryotes evolved into eucaryotes. Like bacteria, yeast reproduce by division, making copies of themselves. Theoretically, one yeast cell can create millions of exact copies of itself, (in reality, this isn’t quite the case), meaning that the desired qualities of one yeast cell can be multiplied by millions, and create a large amount of beer or wine. This also means that through special techniques, a cell or a small group of cells with special qualities can be isolated, picked up, and put on medium to grow. Mixtures of yeast with unique qualities can be used to create truly unique beverages, or a beverage with exceptional characteristics.
Yeast cells need nutrients to live and divide. Nutrients can come from several sources, and be present in several forms, but they all must include CHNOPS. CHNOPS stands for Carbon, Hydrogen, Nitrogen, Oxygen, Phosphorus and Sulfur, and represents the major chemical elements needed to make a cell. Trace elements such as magnesium, calcium, iron, copper, sodium, potassium cobalt and zinc are also needed, and can be supplied through tap or mineralized water or yeast extract. A reducing substance is also needed, and is usually provided in the form of a sugar. All of these nutrients, including vitamins and proteins are mixed and available in a matrix, which can be liquid broth or gelatinous agar (a protein extracted from seaweed). The matrix, also called medium, not only hold nutrients, but it also provides support for the yeast cells and a place for them to grow.
Yeast cells can be in one of four phases of growth: rest, lag, log and stationary. Most of a cell’s life is spent at rest, not growing or dividing. When cells are transferred to fresh media, wort or must, nutrients become available, and cells begin to take them up. These cells enter a lag phase, where they are biochemically active but not dividing. As soon as the cells have enough nutrients to divide, they enter log phase growth. The cells divide, and the population of yeast cells doubles every 20 minutes or so, creating a population explosion. During log phase the nutrient uptake is enormous, and nutrients are quickly depleted. Colonies of cells on agar become visible, and wort or must becomes turbid. When most of the nutrients have been depleted (especially oxygen), the cells stop dividing and enter the stationary phase. They then begin anaerobic fermentation until they run out of nurients and die. If yeast cells are to be taken out of the carboy for culturing, they should be removed during log phase growth, or soon after the stationary phase begins (when CO2 bubbling begins to slow down). These cells will enter a brief lag phase on new medium, and go through log to stationary phase. New stocks need to be re-inoculated on fresh medium before the cells die off, or once every seven to ten days.
During stationary phase, yeast cells continue to break down sugars or other reducible organic molecules as they did during log phase, and harness the energy from liberated electrons. Like many other aerobic organisms, they use oxygen to accept those electrons and form water (H2O). In the absence of oxygen, they ferment anaerobically, and use other molecules to accept these electrons to generate other compounds, including ethanol. Thus, it is important to first aerate the must or wort to allow log phase growth, and then fit an air lock on the carboy to create anaerobic conditions. For culturing, it is important to maintain an aerobic atmosphere so that the cells can use the available nutrients most efficient. Ethanol is ultimately poisonous to yeast cells, and it is not desirable in stock cultures.
- List of supplies
- Work Area Set Up
- Agar Prep / Pouring / Sterilization
List of Supplies
You will need the following to make, pour, sterilize and store nutrient agar:
- heat resistant plastic or glass tubes measuring at least 16mm X 120mm
- caps to fit tubes
- pan for boiling
- measuring cup
- nutrient agar
- tube rack to hold tubes
- petri dishes
- Sharpie (c) marking pen
- alcohol burner (do not use candles – they generate soot)
- household cleaner
- 10% bleach solution (1 oz bleach in a healthy cup of water, or 25 ml bleach in 225 ml water)
- pressure cooker (optional)
The best location for preparing medium and working with stocks is the kitchen. Basements are not recommended because they are often damp and moldy. Garages are also not good for the same reason, as well as being drafty. A clean bathroom with a large sink area will also work. The location needs to be well lit, clean, easily cleanable, and free from combustible material. It also needs to have a flat, non-pourous surface that is at least 2 1/2 feet wide, and as deep as you can reach comfortably.
Work Area Set Up
When making up agar and pouring it, the tubes and/or petri dishes (aka plates) need to be on a level surface near the pan of agar. Agar liquefies near 100 deg C and solidifies near 37 deg C (body temperature), so it needs to be poured quickly when about 45 deg C. The rack will be used to hold tubes, and the caps should be kept in a clean container if they have been sterilized. The pencil will be used to make agar slants in the tubes, and the marking pen will be used to date the tubes after they have been poured.
When maintaining stocks or making new ones, the set-up is different. The alcohol burner should be placed nearest to the dominant hand (i.e. right side for a right-handed person, left side for a left-handed person), with nothing behind it or in front of it. The inoculating loop should be toward the center of the work area, with the rack of tubes further away from the burner. The old tubes and new tubes should be separated in the rack to prevent confusion and contamination. The marking pen will be used to write the date on the tubes as they are inoculated. Before brewing equipment is set up, the workspace should be wiped down first with a household cleaner, then with a solution of 10% bleach. After work is finished, it should be wiped down again with the bleach solution.
Agar Prep / Pouring / Sterilization
There are two ways of preparing nutrient agar, and ending up with sterile usable slants and plates: the easy way, and the hard way. The easy way requires a pressure cooker to re-melt and sterilize pre-poured tubes and plates at the same time, while the hard way requires pre-sterilized tubes and equipment, and very carefully handling during pouring to maintain sterility.
The easy way:
- Wash hands.
- Melt the agar and nutrients in a pan of boiling water according to directions, usually 15g agar in 1000ml water (1.5% w/v agar solution), plus nutrient powder. Alternatively, wort or must can be substituted for the water.
- Remove the pan from heat, pour solution into tubes, filling them halfway, capping the tubes. If sterilizable glass petri dishes (plates) are used, fill the bottom dish (the smaller dish) halfway and replace the lid (the larger dish).
- Allow tubes and/or plates to cool to a comfortable temperature, and load them into the pressure cooker. The plates can be stacked, but the tubes must be upright.
- Add water to the pressure cooker, seal, and apply heat. Maintain a temperature of 121 deg C (250 deg F) for 15 minutes.
- Remove from heat, cool, and release seal when safe, but before temperature drops below 45 deg C.
- Remove the plates, and place them on a cool surface where they will not be disturbed while the agar solidifies. If the agar is still runny when cool, try a slightly higher concentration of 2.0, 2.5 or 3.0 % w/v agar, until a gelled surface forms. When they are cool, turn them upside down, seal them with plastic wrap around the edges, and store in a refrigerator. Keep one plate out at room temperature for two or three days as a batch quality control plate, and look for bacterial or yeast colonies that indicate contamination.
- Remove the tubes, and place them on a level surface, the caps resting on a pencil. The agar solution should almost reach the top of the tube, but not spill out into the cap. Allow to cool undisturbed, and then store upright in the refrigerator. If the slant is won’t be used for several months, it should be wrapped with plastic wrap to prevent the agar from drying out and cracking. Leave out a tube at room temperature as a batch quality control.
The hard way:
- Wash hands
- Sterilize tubes and caps and/or sterilizable glass plates by boiling for 15 minutes or baking in a 350 deg F oven for 30 mins. Sterilize any other equipment that will be used, such as funnels, measuring cups, etc. Baking them on a cookie sheet will help keep them together, and provide a sterile surface until they are ready to be used. Be very sure that plastic items are not baked, unless they are specifically marked as bakeware. Boiling is preferable to dry heat sterilization, but handling things in boiling water is trickier than baking.
- Dry wet tubes upside down in a sterile rack, or recap dry tubes and allow to cool. Dry wet plates upside down and put together as soon as they are dry (even if they are still hot). Recap wet tubes as soon as they are dry.
- Melt the nutrient agar in boiling water, and boil for 15 minutes. See (2) above for details on preparation.
- Allow agar solution to cool, and arrange tubes, plates, and funnel for quick pouring.
- When agar has cooled to between 45-50 degrees, pour. Plates should be immediately covered, and tubes immediately capped and laid flat, the cap resting on a pencil. This requires quick action, as the agar will begin to solidify shortly after it is poured. The tube/plate sterilization can be timed with the agar preparation so that the tubes and plates are still warm (but not hot) when the agar is poured. This will provide more time for arranging tubes to make slants. The pan can also be kept on low heat if a large batch of agar is being poured.
- Date slants and plates, seal plates, and store both in a refrigerator. Keep one plate and/or tube out for batch quality control.
Sterile technique is vital to maintaining the purity of yeast cultures. Poor technique can cause contamination, and ruin hours of careful preparation. Sterile technique is not hard to perform, but requires some practice. Like any other skill, the more you practice, the better you will be.
The most important rule to remember is that bacterial contaminants are everywhere. Media, sterile containers and yeast colonies are all handled so that their exposure to potentially contaminating air or instruments is kept to a minimum. This means that dishes and tubes should be opened as few times as possible. Sterile surfaces are enclosed on the inside, and non-sterile surfaces are left exposed on the “dirty” outside of a plate or tube. Anything that touches a sterile surface must be sterile, and anything that isn’t sterile will contaminate a sterile surface. Hands should be washed before working with plates or tubes as well. Maintaining sterility, then, means keeping contaminants out, and sterile conditions in.
Petri dishes are easier to work with than tubes, and are a good place to start practicing. Inoculated plates are incubated upside-down, so that the condensation on the lid won’t drip down onto the agar and spread cells across the plate. When examining or transferring colonies, the plate is placed upside-down on a table top, and the bottom (containing the agar) is quickly and smoothly lifted off of the lid using the finger tips of the non-dominant hand. It is replaced on the lid using the same motion.
Colonies are transferred, or subcultured (also called “subbing”) to fresh agar using the following technique:
- The new plate is placed off to the side closest to the non-dominant hand, and the old plate placed in front of you.
- The dominant hand is used to pick up the inoculating loop by the base. The loop is flamed (disposable plastic loops aren’t flamed) by holding it as close to vertical as possible with the loop in the flame. The loop will be sterile when it glows orange.
- The loop is removed from the flame, and the old plate lifted up so that the colonies are in plain sight.
- The loop is stabbed into a clean spot on the agar to cool, and pulled through a pure colony. The glob of cells picked up is called the inoculum.
- The plate is replaced on its lid, and the new plate immediately lifted, and the inoculum smeared on the new plate as a small spot at the edge.
At this point, the plate can be replaced, and the loop flamed, and replaced in its holder. Alternatively, you can immediately proceed to the next step, which is to streak out the cells. Recall that one cell can divide to create new cells, forming a lump, or colony of cells. Streaking spreads out the cells deposited on the plate, separating yeast cells from each other and from contaminating cells that might be present, so that the colonies that form will be pure. Streaking is performed in the following manner:
- Using a sterilized loop, touch the inoculation point on the plate with the loop and move it lightly and rapidly back and forth, covering clean agar with each stroke. Do this for the first quarter of the plate, then stop.
- Rotate the plate one quarter turn.
- Move the loop to the top of the streaked area. Flick the loop back through the streaked area two or three times to pick up cells, and streak again from the top edge of the plate to half-way down, covering about a quarter of the area of the plate.
- Rotate the plate one quarter turn.
- Stab the loop in clean agar, flick it through the just-streaked area two or three times, and streak a small area of fresh agar. When done, there should be a rectangular patch of clear agar extending to the center of the plate, with the stab-marks at the bottom.
- Rotate the plate one quarter turn.
- Stab the loop, flick it through the just-streaked area, and streak the remaining fresh agar. Avoid touching the stab marks.
- Replace the plate on its lid. Sterilize the loop, and replace it in its holder.
There should be four areas of streaking that cover as much of the plate as possible. When streaking, the loop must not touch any other streak lines, except at the beginning of the action. This ensures that not too many cells are streaked out, and the colonies that grow are well isolated.
Tubes require a little more manipulation and manual dexterity, but with practice colonies can be subbed with ease.
Tube to tube transfer is performed in the following manner:
- The tube with colonies is picked up in the minor hand and held between the thumb and forefinger. The new tube is held between the fore and middle fingers.
- The loop is picked up in the major hand and flamed.
- Being careful to keep the loop from touching anything, the pinkie of the major hand is used to grasp the cap of the old tube and pull it off with a twisting motion. The pinkie is used to hold the cap.
- The loop is inserted into the tube without touching the rim, and inoculum picked up.
- The loop is withdrawn, and the cap replaced using the pinkie. The pinkie is used to remove the cap of the new tube.
- The loop is inserted into the new tube as far as possible.
- The loop is pulled up the agar surface with a light back and forth motion so that the inoculum is spread over as much of the surface as possible.
- The cap is replaced using the pinkie, and the loop flamed and replaced in its holder.
When a new plate or tube is inoculated, it should be immediately labeled with the date and type of yeast. This is important, as the date will indicate when culture needs to be subbed, and the yeast information keeps the stocks organized. A chart or spreadsheet indicating the date of subbing will also help keep track of when cultures need to be subbed again (once every seven to ten days). After subbing, the tubes and plates should be kept at room temperature for a day, then placed in a refrigerator. Tubes should be kept upright, and plates sealed with plastic wrap around the rim and kept upside down.
Maintenance and Using Cultures
Maintaining cultures requires little time on a daily basis, and even subbing won’t take long, once a routine has been established. The temperature of the refrigerator used to store the cultures should be checked two or three times a day for two or three days, to be sure that it doesn’t dip below freezing. After the initial check, only periodic checks need to be made. As in brewing or preparing must, one day can be set aside for making, pouring, and sterilizing a batch slants or plates that will last for several months.
Normally, only one plate or slant per subspecies/packet of yeast needs to be maintained, so that the whole operation requires little room. Multiple slants or plates can be kept briefly if contamination is suspected, or if slants are being prepared for other brewers or vintners. For the best use of space, only slants need to be maintained. Plates will work too, but for the purposes of home culturing, they work better for isolating contaminants than for keeping stocks.
A unique problem exists when yeast stocks are maintained for extended periods of time. After four or five subcultures, yeast cells will begin to mutate to the point of losing their biochemical properties. If these cells are used, they will produce a poor or unusable product. Thus, the subcultures need to be restarted from the original source after the fourth or fifth subculture. If subcultures are made from lyophilized yeast packets, only one or two pellets need be cultured, and the pellets can be stored in a sealed sterile tube in the freezer. If they are made from liquid preparations, or directly from the carboy, the original sample must be in a broth and kept in a sterile tube, refrigerated, and sampled carefully.
Alternatively, the trub can be filtered through a sterilized mesh into a sterile container. Small portions of yeast can then be poured into sterile tubes, along with an equal volume of sterile 35% w/v glycerol (or 28 ml glycerol/100 ml water).The tubes are capped, and gently shaken to mix the cells. These tubes are then frozen below -13 deg C (8.5 deg F), and thawed when new stocks need to be started. Sampling from a container of lyophilized yeast can be a source of contamination, but freezing the pellets will at least slow down most contaminants. Sampling from a broth is trickier, as the broth will allow bacterial contaminants to grow if they are introduced, even at refrigerator temperatures. A way around this is to suspend yeast cells in a glycerol solution and freeze small amounts to be thawed as needed.
Contamination from other yeast or bacteria can does not mean that the stock must be thrown out. Monitoring for contamination involves careful examination of slants and/or plates. Yeast colonies or streaks will be off-yellow-white and slightly shiny, and isolated colonies are irregular in shape, with an undulating edge. The middle of the colony may have folds or heaps. Contaminating bacteria will form smaller colonies that will be smooth and rounded. The edge will be uniform, and the color may range from colorless to gray to white to cream (some bacteria are strongly colored, appearing as yellow/gold, pink, brick red, blue or green; these are rare contaminants). If contaminants show up, yeast colonies should be sampled, and streaked out on a plate. From that plate, pure, isolated yeast colonies should be sampled and streaked out. Incubation can be at room temperature for about two days. If any contaminants are seen, another plate should be streaked; otherwise a sample can be transferred to a slant, and stored.
The actual use of yeast from slants or plates in must or wort requires a little advance planning, as cells can’t be transferred directly to the carboy. For worts, a beer bottle filled halfway with prepared malt similar to the malt to be brewed is used to create a working culture that will provide enough cells for pitching. The bottle must be sterile, and fitted with an airlock. Yeast cells are transferred aseptically, and allowed to grow for a day or two. Once the airlock slows its bubbling, the yeast is ready to be pitched. The same procedure can be used for must, except that prepared grape juice is used instead of malt.