Isolating Bugs (DRAFT)¶

Why isolate bugs¶

• Dregs make unbalanced cultures. Growing up bottle dregs usually gives a mixed culture dominated by strong acid producers and resistant bacteria. Building a mixed fermentation from individual strains gives some control over what ends up in the beer, and in what proportion.
• Cost. Buying three to five different commercial bugs for one batch is just too expensive.
• Choice. You get to pick up the bugs from bottles you really like.

The Holy Goat Shrinebuilder journey is a worked example of the whole process, and most of the photos below come from it.

The flow¶

Steps run top to bottom. Where a step fans out, the branches run in parallel (several plates, several tubes, several mini-beers). Dashed arrows are loops back when a round does not give clean results. Documentation (the box on the right) runs alongside every step.

flowchart TD
    S1["1 · Bottle selection and research"] --> S2["2 · Dregs under the microscope"]
    S2 --> S3["3 · Enrichment and selective media"]
    S3 --> P1["Plate: dilution 1"] & P2["Plate: dilution 2"] & P3["Plate: dilution 3 to 5"]
    subgraph step4 ["4 · Plating at several dilutions (3 to 5 plates)"]
        P1
        P2
        P3
    end
    P1 & P2 & P3 --> S5{"5 · Observe from day 3:<br/>target present and<br/>colonies separate?"}
    S5 -. "no: adjust dilution or media" .-> S3
    S5 -- yes --> S6["6 · Pick 3 to 5 lone colonies<br/>into sterile water"]
    S6 --> A1["Tube A"] & A2["Tube B"] & A3["Tube C to E"]
    subgraph step7 ["7 · Microscope check of each pick; discard mixed"]
        A1
        A2
        A3
    end
    A1 & A2 & A3 --> S8{"8 · Confirmation plating:<br/>plates clean after 2+ weeks?"}
    S8 -. "no, or first round: repeat" .-> S6
    S8 -- yes --> S9["9 · Pick 10 to 20 colonies<br/>into clear wort, traced"]
    S9 --> B1["Clear-wort tube 1"] & B2["Clear-wort tube 2"] & B3["Clear-wort tube 3 to 5+"]
    subgraph step9 ["Cultivate 2+ weeks"]
        B1
        B2
        B3
    end
    B1 & B2 & B3 --> S10{"10 · Full slide at 1000x:<br/>clean?"}
    S10 -. "no: correlate with plates, repeat" .-> S8
    S10 -- "yes: keep 2 to 3 tubes" --> S11["11 · Banking on slants, traced"]
    S11 --> S12["12 · Step up in sterile wort"]
    S12 --> M1["Mini-beer: temperature 1"] & M2["Mini-beer: temperature 2"]
    M1 & M2 --> S13["13 · Assess the mini-beers"]
    DOC[["Documentation, all along:<br/>colony and cell morphology,<br/>plate smells, dates, media,<br/>temperatures, mini-beer notes"]]

1 · Bottle selection and research¶

Start from a beer you really like, and find out as much as you can before opening it: read the label and the brewery's description, and ask the brewer. Knowing roughly what is in the bottle (which yeasts, whether there are bacteria, whether it was bottled with a separate yeast) tells you what to look for on the plates and what to expect under the microscope. For Shrinebuilder, the brewery's own description named a blend of Brettanomyces strains, and earlier messages with the brewery had filled in some of the process.

2 · Dregs under the microscope¶

A first look at the dregs, diluted straight from the bottle: which kinds of cells are there, roughly in what proportion, and how healthy they look. It is not easy: bottle dregs carry plenty of proteins and debris that get in the way. In the Shrinebuilder dregs there were Saccharomyces and Brett cells, rod-shaped bacteria singly and in pairs, and a lot of debris.

Shrinebuilder dregs · 1000x oil immersion, phase contrast, no stain

3 · Enrichment and selective media¶

Where needed and available, grow the dregs up on a medium that favours what you are after before plating. I don't plate straight from the dregs: I revitalise them in sterile wort first. A heavily hopped wort slows hop-sensitive bacteria while the yeast recovers; in the Shrinebuilder sample the lacto was hop intolerant, so it was held back while the yeast revitalised. Indicators in the medium are optional.

4 · Plating at several dilutions¶

Plate the enriched culture at three to five dilution levels. The bugs grow at different rates, and the ones we are usually after are the slowest, so bacteria (and sometimes fast-growing mould) can take over a crowded plate. The right dilution gives cells well separated, with still a few colonies of what you are after.

5 · Observe the plates and assess colonies¶

Look at the plates from day 3, then every day or every other day. How fast a colony appears is a first hint of what it is. Assess by colony morphology (size, shape, surface, edge, colour) and aim first to spot the undesirable or harmful bugs. If the organisms you want are not there, or the plate is too crowded to find lone colonies, adjust the dilution and/or the medium and start over.

On the first Shrinebuilder plate (wort agar, ~21 °C) the Brett colonies only started to appear after 2+ weeks: tiny smooth colonies were Brett, big smooth ones Saccharomyces, and the large volcano-shaped ones bacteria.

6 · Pick lone colonies¶

Look at the plates under a magnifying glass to find lone colonies, well clear of their neighbours. Pick three to five at a time, each into its own tube of sterile distilled water.

7 · Microscope check of each pick¶

Look at each tube under the microscope and discard anything mixed. The aim is to end up with three to five tubes, each holding a single strain.

8 · Confirmation plating, and a repeat round¶

Plate every tube that passed the microscope check, then discard the tubes. Repeat the round once more (recommended), especially if mixed colonies grew on the confirmation plates. Use the magnifying glass again and let the plates grow for 2+ weeks, as some organisms take that long to show visible colonies.

Both Shrinebuilder confirmation plates below were meant to hold only Brett; one got some Saccharomyces in, so another round followed.

Shiny and dull colonies of the same Brett · wort agar, ~21 °C

9 · Clear-wort tubes, traced¶

From the clean plates, pick 10 to 20 colonies into test tubes of clear wort with no inhibitors, aiming for five or more tubes. Label and trace each tube to the plate it came from. Cultivate the tubes for 2+ weeks.

Brett tubes in clear wort

10 · Full-slide check at 1000x¶

Examine each tube carefully under the microscope; scanning one full slide at 1000x is recommended. If undesirable bugs turn up, correlate them with what grew on the plates. Aim to end up with two or three clean tubes, then discard the plates.

Clean Brett tube · 1000x oil immersion, phase contrast, no stain

11 · Banking on slants¶

Prepare slants from the clean tubes and bank them, tracing each slant to its tube and culture. Record the bank in the bank inventory.

12 · Step up and brew mini-beers¶

Step the culture up in sterile wort until there is enough thick slurry to pitch into 200 mL of sterile wort, then ferment the mini-beers. Brewing the same wort at different temperatures, in parallel, shows how the organism responds. The Shrinebuilder Brett mini-beer was 200 mL in a 250 mL Erlenmeyer, 20 g DME, water under 50 mg/L alkalinity, ambient (~22 °C) for 4 weeks, repeated at 30 °C for 2 weeks.

13 · Assess the mini-beers¶

Assess each mini-beer for aroma, flavour, acidity, attenuation and clarity, and decide what the organism is for: bank it, test it further, or discard it. The Shrinebuilder Saccharomyces fermented super clean and super fast with no character, so, presumed bottling yeast, it was discarded.

Documentation, all along¶

Document every step as you go: colony morphology, yeast and bacterial cell morphology, plate smells, dates, media, dilutions, temperatures and days, and the mini-beer attributes. Each bottle gets a journey page; the organisms that come out of it get their own pages.