Same lesson, with the figures in motion.
Maintaining a Yeast Library
A yeast library in the fridge isn't an archive — it's a culture that has to be renewed. Every three months each strain is streaked out afresh: slant → plate → new slant. That gives you a fresh, controlled culture and, at the same time, a chance to check that the strain is still clean and healthy.
This protocol describes a single batched session — every strain at once. It is more hours at the bench that evening, but afterwards you don't have to think about the library at all for three months, and your hands learn the routine while you work.
Materials
- One ready-poured agar plate per strain + a couple of spares
- Slant tubes — you reuse the ones you have; they are emptied and re-poured ONLY once the plates have passed
- Inoculation loop (metal)
- Spirit burner or gas burner
- Test tube rack
- Labels + permanent marker
- Your log (paper, spreadsheet or app) with strain, generation and date
- Sanitiser for the bench
The rhythm — one session every three months
- Run every strain in the same session rather than rolling dates per strain. One date to keep track of, one occasion to get the burner out — and three months of quiet afterwards.
- Three months is the cadence for slants in the fridge: the yeast goes on living slowly in the cold, and beyond ~3 months genetic drift can't be ruled out.
- The session runs in two sittings: slant → plate now, and colony → new slant once the colonies have grown (2–5 days later). Book both at once.
- Reckon on about 10 minutes per strain in the first sitting and rather less in the second.
Write the next date into your log the same evening you run the session — three months ahead. It is the only note that has to be made while you remember.
Preparation
- Pour the plates in advance so they have set and dried a little. One plate per strain, plus a couple of spares for mishaps.
- Agar & Slants — cook the medium, pour plates and set slants →
- Don't prepare the slant tubes yet. The old slants must stand untouched until every plate has passed — they are your backup if something grows wrong — and they are the same tubes that will later be re-poured as the new slants.
- Label every plate before you start streaking — strain name, date and generation. Labelling afterwards is how you mix two strains up.
- Print out or bring up your strain list, with the generation for each strain. Tick them off as you go.
- Wipe the bench, close windows and doors, light the burner and work in the updraught around the flame.
Take the slants out of the fridge and let them come to room temperature while you label the plates. Condensation on a cold tube easily drips into the agar.
Open flame and alcohol on the same bench: never leave the alcohol bottle open next to the burner, and let the loop cool before it goes anywhere near the alcohol.
Sitting 1 — slant → plate (streaking)
- Flame the loop until it glows, then let it cool for 5–10 seconds. A hot loop kills the very thing you were about to pick up.
- Open the slant, put the loop in and touch the growth lightly. You need hardly anything — a visible film on the loop is enough.
- Streak phase 1: a tight zigzag back and forth across roughly a third of the plate. It is the only phase streaked directly from the slant.
- Flame the loop, cool it, and drag two or three times through the end of phase 1 and out into a new field = phase 2. Only what the loop carries along comes with it — that is the whole point. Miss that crossing and you carry no cells with you: the new field simply stays empty.
- Flame and cool one last time, drag through the end of phase 2 and streak phase 3 as a continuous zigzag with rounded turns back and forth across the rest of the plate. It is in those strokes that the single colonies should appear.
- The point of the pattern: every phase dilutes the material one more time. The figure shows the same plate twice — on the left the path of the loop, on the right how it looks after 3–5 days: a thick continuous mat in phase 1, thinner and broken growth in phase 2, and in phase 3 single colonies where every dot has grown from one single cell. Those dots are what the whole exercise is about.
- Turn the plate upside down (lid down) and incubate at room temperature for 2–5 days, until the colonies are distinct but not yet grown together.
Stop guessing when the loop has cooled: press the glowing loop into the agar near the edge, on a free spot you will not be streaking in (the dashed box in the figure). It hisses, and the loop is instantly down to the temperature of the agar — no waiting, no killed yeast.
Upside down: condensation forming in the lid then does not fall onto the agar surface and smear the colonies into one mess.
Lagers take longer than ales at room temperature. Give them an extra day rather than picking a colony too early.
Sitting 2 — picking the right colony
- Hold the plate up to the light. You are looking for a single colony with a nice round shape and a smooth edge — not one sitting up against a neighbour.
- The reason: two colonies growing into each other compete for nutrients and crowd one another. The one you pick from there has had a worse start than one that stood free.
- Typical brewing yeast looks like this: creamy white, round, glossy to matt, with an even edge. That is the look you want to recognise again in three months.
- Pick one of typical size for the plate, too. Skip any that are noticeably larger or smaller than the rest — an odd size can point to a mutation, or to that colony having grown under nutrient stress.
- Discard the whole plate if you see fuzz (mould), coloured colonies (pink, orange, black), slime, or if several different kinds of colony are growing. The plate cannot be saved.
- If you have a freezer bank: restart the strain from there. If you don't — streak out again from the same old slant, which is still in the fridge. The contamination may just as easily have come from the bench as from the slant, and a second attempt costs nothing.
- Nothing grew at all? Do the same: a fresh streak from the old slant. If it does not grow the second time either, the slant is dead — then it is the freezer bank or a new pack.
- This is why all the old slants stand untouched until every single plate has passed. They are your entire safety net during the session.
Photograph the plate with your phone the first time each strain looks good. Next quarter you have a reference to compare against — contamination is easiest to spot as a difference.
Sitting 2 — colony → new slant
- Only now, once every plate has passed, do you prepare the slant tubes. Empty the old tubes, clean them and pour fresh agar — they are the same tubes, and they were in use right up until the plates had been checked.
- Agar & Slants — cook the medium and set new slants →
- Label the tube before you streak: strain, date and generation +1.
- Flame the loop, let it cool, and pick the chosen colony — all of it, but only it.
- Streak in a zigzag up the sloped agar surface, from the bottom upwards, without cutting into the agar. Then screw the cap on loosely — never fully tight, or the yeast's respiration builds up pressure.
- Leave the tube at room temperature for 2–4 days until the growth is clearly visible. Never chill a freshly streaked slant straight away — it has to establish itself first.
- Once the growth is clearly visible: tighten the cap and put the tube in the fridge (1–4 °C). A loose cap is right while the yeast is growing and respiring — but in the fridge the tube stands for months, and there drying out is the real risk. Enter the date and generation in your log.
- So the order is: every plate passed → tubes emptied and re-poured → colony streaked onto the new slant. The old slant is there right up to the last step, and the session is finished only once the new one shows growth.
Never touch the inside of a tube, a lid or the loop, and do not put a lid down with the inside against the bench. The whole session stands or falls with that habit.
The log, generations & the way back to the freezer
- Note per strain: date, generation, source (which slant or which freezer vial it came from) and a line about how the plate looked.
- Every session is a passage. Slant → plate → slant forever stacks up generations, and drift creeps in without showing.
- Think master and working stock: the freezer is the master that stands still, the fridge is the working stock that wears. After roughly 4–8 passages — at a quarterly cadence that is every two to three years — you restart the working stock from a freezer vial instead of from the last slant.
- The same applies immediately, whatever the count says, if a strain starts fermenting sluggishly, tasting different or looking different on the plate.
- Glycerol Freezer Bank — the master you restart from →
During the session itself the old slant is your safety net — but only for that quarter. Without a freezer bank there is no way back once a strain has drifted over several generations: every new slant comes from the previous one. The freezer bank is the only point you can return to.