Craft Beer Yeast Handling (3/3) - Pilot Batch Propagation
When brewing, the equipment is typically cleaned and sanitized. When preparing a fresh sample for yeast growth, a sterile environment is needed to ensure that unwanted micro-organisms do not propagate alongside. Sterile materials can be achieved with a wet environment for 15m at 121°C or in a dry environment for 2h at 160°C (White and Zainasheff, 2010).
Figure 9- Flow Diagram of a Proposed Laboratory Propagation of Brewing Yeast (Bolton & Quain, 2008)
- Clean and sanitize an area of the workspace. Light the Bunsen burner or alcohol lamp. Use sterile microbiological techniques:
- Only use equipment that has been adequately heat-sterilized.
- Work in proximity (<10cm) to the open flame.
- Flame all openings of any glass or metal containers.
- Do not flame the opening of any disposable containers. (White and Zainasheff, 2010)
- Prepare a 20mL sterile solution of 1.040SG Wort. Use a 50mL test tube. This may be autoclaved
- Identify the yeast colonies on the plate for harvesting.
- Using a flamed inoculation loop:
- Open the plate.
- Touch the flamed inoculation loop to the agar to cool.
- Collect a single entire colony.
- Close the plate.
- Open the container of wort and deposit the colony into the medium.
- Seal the wort container (WC), place upright. Store at ambient temperatures for 24-48 hours.
- Manually agitate the wort, gather a sample and perform a diluted sample cell count using methylene blue (White and Zainasheff, 2010).
- Prepare a 200mL sterile solution of 1.040SG Wort. Use a 500mL Erlenmeyer flask. This may be autoclaved (Bolton & Quain, 2012).
- When the secondary WC is at ambient temperature:
- Agitate the primary WC and vent.
- Empty the primary WC into the secondary (White and Zainasheff, 2010).
- Place the secondary WC on a stir plate with a sterile stir bar. Agitate for 72 hours. Biological activity should be evident within 12-24 hours (Bolton and Quain, 2012).
- Prepare a 3L sterile solution of 1.040SG Wort. Use a 5-10L Erlenmeyer flask. Autoclave the wort for sterility. Repeat Step 9 for the tertiary WC combined with aeration (Bolton and Quain, 2012).
- Agitate the WC, gather a sample, and perform a diluted sample cell count using methylene blue.
- A quaternary WC may be required if not enough cells are present:
- Chill and decant the spent wort from the tertiary WC keeping the condensed yeast. Yeast may be kept cold for up to two weeks (White and Zainasheff, 2010). Allow the temperature to rise to ambient.
- If there is not ample yeast, prepare a sterile solution of 1.040SG Wort. Use a 5-10L Erlenmeyer flask. This may be autoclaved. Repeat Step 9 for the quaternary WC for 24-48h (Palmer, 2017).
- When the final wort propagation is at ambient temperature, add to the pilot batch of wort.
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