← Soul of Beer
Open the protocol in the app →

Same lesson, with the figures in motion.

From a Single Colony (CFU) — Slant/Plate to Pitch-Ready Culture (20 L)

Builds a full culture from a single colony (1 CFU) — one isolated colony on a Petri plate or slant, roughly 1–10 million cells. Because that's ~10–100× fewer than a streaked slant, we add one small wake-up step up front; after that it's the same path as the slant protocol. Target: ~450–500 billion cells for a strong 20-litre (5 US gal) wort (>1.075 SG). Adapt the quantities to your own equipment.

Materials

  • Erlenmeyer flask, 1000 ml
  • Erlenmeyer flask, 5000 ml
  • Sanitised container with lid (e.g. glass jar) — to hold the culture between steps
  • Hot plate
  • Magnetic stirrer + stir bar
  • Light dry malt extract (DME / spray malt)
  • Yeast nutrient
  • Aluminium foil
  • Tap water
  • Kitchen scale (ideally 0.1 g resolution for the nutrient)
  • Thermometer
  • Sanitiser (e.g. StarSan) for surfaces, flask necks and foil
  • Inoculation loop (sterile disposable, or metal that you flame)
  • Spirit lamp / burner (to flame the loop)
  • Sterile empty test tube with cap (for the wake-up step)
  • Petri plate or slant with a well-isolated colony

Before you start — rules throughout

  1. Sanitise all surfaces, flask necks and foil before every step.
  2. Drop the stir bar into the flask before boiling, so it's boiled clean at the same time.
  3. Always cool the wort to ~20 °C (check with the thermometer) before adding yeast — wort that's too hot damages it.
  4. Run each liquid step on the stirrer until the krausen has risen and fallen (~24–48 h) before moving on.
Boiling wort causes burns. Never cover airtight — steam must escape, or the pressure can shatter the glass.

Step 1 — Pick the colony (~15 ml, test tube)

  1. Weigh ~1.5 g DME into a test tube and top up with water to 15 ml (≈ 1.036 SG). Cap loosely, boil, then cool to ~20 °C.
  2. Flame the inoculation loop until it glows, let it cool ~5 s, then pick ONE well-isolated colony from the plate/slant.
  3. Swirl the loop in the wort to release the cells. Cap loosely.
  4. Keep at ~20 °C and swirl by hand a few times a day for 1–2 days until cloudy. (This tiny step is done statically — 15 ml is too shallow for the stir bar to couple.) (~1–10 million → ~0.1–0.3 billion cells.)
Flame the loop to glowing and let it cool ~5 s before touching the colony — too hot kills the cells you're picking. Pick just ONE isolated colony to keep the culture pure.

Step 2 — Wake up (~150 ml)

  1. Weigh 15 g DME into the 1000 ml flask and top up with water to the 150 ml mark (≈ 1.036 SG). A little boils off, and that's fine. Drop in the stir bar and cover loosely with sanitised foil.
  2. Bring to a boil and boil for 15–20 min.
  3. Cool in a cold water bath to ~20 °C and set on the stirrer.
  4. Pour the whole test-tube culture (step 1) into the flask and let it run 24–48 h. (~3–5 billion cells.)

Step 3 — Build up (~500 ml)

  1. Pour the step-2 culture into the sanitised container (lid on) and clean the 1000 ml flask.
  2. Weigh 50 g DME into the flask and top up with water to 500 ml (≈ 1.036 SG). Drop in the stir bar and cover with foil.
  3. Boil 15–20 min, cool to ~20 °C and set on the stirrer.
  4. Pour the whole step-2 culture into the flask and let it run 24–48 h. (~40–50 billion cells.)
In a hurry? You can skip this step and go straight from 150 ml to the final culture — but you won't reach the cell target. 150 ml holds ~4 billion cells, and the final step would then have to multiply them 40–50×, where the other steps go ~10×.

Step 4 — Final culture (~3 L / 3 qt)

  1. Weigh 300 g DME into the clean 5000 ml flask, top up with water to 3000 ml and add 0.3 g yeast nutrient (≈ 1.036 SG). Drop in the stir bar and cover with foil.
  2. Boil 15–20 min, cool to ~20 °C and set on the stirrer.
  3. Pour the whole step-3 culture (from the 1000 ml flask) straight into the 5000 ml flask and let it ferment out 24–48 h. (~450–500 billion cells.)
No holding container needed here — the 5000 ml flask is free, so you make the wort straight in it and pour the previous culture right over. One contamination step fewer.

Before pitching

  1. Cold-crash the final culture in the fridge for 12–24 h so the yeast settles to the bottom.
  2. Decant most of the spent wort and pitch the slurry.
  3. Do not guess the final count — measure it → The cell counts above are estimates. With a haemocytometer, the Measured mode tells you exactly how many mL of this slurry to pitch.
  4. Allow ~5–7 days total (~2 days per step + cold crash).
Pitching the whole culture means ~3 L (3 qt) of old starter beer in your 20 L (5 US gal) batch (~15 %) — decant and you avoid it, keeping the beer clean.

Assumptions & cell counts

  1. Starting point: a single colony holds roughly ~1–10 million viable cells (varies with colony size and age).
  2. Wort strength ~1.036 SG in every step (100 g DME/L).
  3. ~10–30× growth per step, final density ~100–150 billion/L (realistic for a stir plate).
  4. The pitch calculator reckons a starter from a pack at a cautious 100 billion/L – a pack's viability is uncertain. This protocol's higher figure comes from a fresh culture built up step by step.
  5. After step 1: ~0.1–0.3 bn · step 2: ~3–5 bn · step 3: ~40–50 bn · step 4: ~450–500 bn cells.
  6. The ~450–500 bn target suits a lager or strong ale (>1.075 SG); a normal ale needs less.
Next: Cell Counting (mi­cro­scope) →
See also: Sterile Tech­nique & Sani­ta­tion →
The Kitchen Lab