Starting an EffectiveQC Program on a Budget

jmello@bootlegbiology.com

ibrannon@bootlegbiology.com

: @bootlegbiology

: /bootlegbiology

• Bootleg Biology, LLC

✓ Est. 2013

✓ Operating in Nashville, TN (Welcome!)

• Jeff Mello

✓ Founder

✓ Chief Yeast Wrangler

• Isaac Brannon

✓ Lab Manager

✓ Yeast Whisperer

Contact

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WHO ARE WE?

WHAT IS BOOTLEG BIOLOGY?

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▪ Pioneered the Local Yeast Project to catalogue wild yeast from every U.S zip code & country

▪ Our mission is to expand the universe of unique brewing microbes and teach brewers basic science skills

▪ Creator of the Backyard Yeast Wrangling Tool Kit

▪ Offering Commercial-Sized Culture Pitches:

Funk Weapon 1-3, Sour Weapon L & P, Sour Solera,Mad Fermentationist Blend, Saison Parfait, S. Arlingtonesis, Chardonnay, Custom Blends

▪ Services include: House Culture Banking & Analytical labtesting including IBU/SRM, Contamination, Cell Counts

OUTLINE

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1. Ask Yourself a Question (anyone but me)

2. What Equipment Do You Really Need?

3. Cell Counting & Viability

4. Contamination Checks

5. Cleaning & Proper Sampling Techniques

6. Neglected Tests

7. Yeast Storage

8. In-House vs Outside Testing

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WHO

Who’s on the QC Team? EVERYONE!

WHAT

What are we testing? What do we want to learn?

• Don’t just test what you’re told to test

• Define upper/lower thresholds (tolerance) for each beer/test

WHERE

Where is the information recorded?

WHEN

When are we testing?

• Yeast: Day of harvest/Before pitching

• Wort/Beer: Before transferring to next vessel or packaging

WHY

Why are we testing?

• No zombie testing! Know why you’re testing. If no corrective action will be taken, don’t do it!

ASK YOURSELF A QUESTION…OR 6

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What is your budget? Consider…

• What is the batch cost of dumping your largest fermenter?

• Costs of outsourcing testing

• Time and in-house expertise/training

Where to find?

• Start first at Amazon, eBay, DoveBid, Labx, University surplus before buying new equipment

• Try before you buy – get competing demos/quotes from vendors

WHAT EQUIPMENT DO YOU REALLY

NEED?

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✓ Clean Work Space: Office/Closed Room/Laminar Flow Hood

✓ Flasks - 100ml, 1L, 2L, 5L: $75-$150

✓ Stir plate/stir bar/stopper: $65-$120

✓ Micro pippettor/tips (1000 microliter/1ml): $60-$100

✓ Microscope: $250

✓ Viability Stain: $10-$40

✓ Hemocytometer: $30-$100

✓ Test Tubes (15 ml), Rack: <$20

✓ Germicidal UV Bulb: $20

✓ Burner/Torch/Click Lighter

✓ Alcohol/Ethanol

✓ Nitrile Gloves

✓ Loop

✓ Incubator/Anaerobic Chamber

➢ Convert a keg!

✓ Agar Media: $50-$200/500g

✓ Sterile Petri Dishes: $40-$60/case

✓ Autoclave/Pressure Cooker: $200-$1000 (direct fire -> electric)

MATERIAL LIST: YOUR BUDGET LAB

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Goal: Avoid over or under pitching

✓ Prevent under attenuation

✓ Prevent long lag times

• Holds up tank space, gives contaminants chance to thrive

✓ Prevent off flavors

✓ Prevent reduced yeast viability in subsequent pitches

• Don’t pitch yeast with less than 80% viability

• Verify proper pitch with fill count

CELL COUNTING AND VIABILITY

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Expensive Method:

▪ Cellometer, NucleoCounter ($10k+)

▪ Pros: Mostly automated, very accurate, easy to operate

▪ Cons: $$$, must adjust based on cell size/clumpy yeast

Inexpensive Method:

▪ Hemocytometer (+ Microscope, Slides, Stains, Tubes)

▪ Pros: Pretty accurate, easy to perform

▪ Cons: Time intensive, easy to perform incorrectly

CELL COUNTING AND VIABILITY

COUNTING W/HEMOCYTOMETER

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Stains show viability of cells

▪ Cells that take up dye have permeable cell membrane and are considered dead

Two most useful:

▪ Methylene Blue (MB)

• Most common for counting brewing yeast

• Inexpensive, user friendly

• Can give false positives/negatives, especially with Brett

▪ Trypan Blue (TB)

• Most accurate

• Use gloves

• Toxic to yeast cells, decreases cell viability if count not done shortly after staining

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COUNTING W/HEMOCYTOMETERSampling:

▪ Count same day as harvest/pitching

▪ Homogenous sample, same density as brink

TB Dilutions:

▪ 1:110 (1x10x11)

• 1ml sample->9ml dH2O, Repeat, 1ml TB->Final Dilution

▪ Clumpy yeast can be diluted w/ 0.5% (0.1M) sulfuric acid instead of water

Proper Loading:

• Hemo cover slip first

• Fill indentation using capillary action

• If troughs are full, overfilled

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▪ Count both sides, average two (should be within 10%)

▪ No fewer than 75 total cells, no more than 48 in one square

▪ Don’t count cells that touch left/bottom sides, be consistent

▪ Budding Yeast/Daughter cells:

• Stained doesn’t mean dead

• Count if daughter >50% the size of mother

▪ Slow/More Accurate: Count entire field

• X counted cells * 10,000 * X dilution factor = X million cells/ml

▪ Faster/Less Accurate: Count 5 squares

• X counted cells * 50,000 * X dilution factor = X million cells/ml

COUNTING W/HEMOCYTOMETER

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Cell stained blue = dead

Daughter cell < 50% of Mother, do not count

Cell outside left border,

do not count

Daughter cell > 50% of Mother, count

THERE’S AN APP FOR THAT

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▪ Count is only as accurate as your total volume

▪ Counts based on weight are possible, but less accurate. Thrown off by hop matter/trub

▪ Many apps available for iPhone/Android to do “tap” count, and perform calculation (hemocytap)

CONTAMINATION CHECKS

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Goal: Know what’s in your beer and make informed decisions

✓ Prevent contaminated beer from being released

✓ Ensure packaged beer is shelf stable

✓ Determine keg vs can/bottle

✓ Prevent future issues

▪ Avoid repitching yeast from contaminated batches

▪ Adjust CIP process

CONTAMINATION CHECKS

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Identification Methods

PCR: Kits/Invisible Sentinel/PiKA

✓ Pro: Know in hours

• Con: $$$, viability unknown, limited microbes

PIKA FastOrange (pH indicator broth):

✓ Pro: Less work than making plates

• Con: More $ than making your own plates, same TAT as agar plates, limited microbes

Old School Plating (Gold Standard):

✓ Pro: Cheap, enumeration, viability

• Con: Making/preparing plates time intensive, 3-7 day TAT

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Agar Plate Media

WLN/WLD:

• Fungi (some bacteria), differential

MRS:

• LAB (w/Cyclo, Brett only yeast that grows)

HLP:

• Great starting media

• Doesn’t require anaerobic chamber, more $

CONTAMINATION CHECKS

CONTAMINATION CHECKS

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Plating: 3 Simple Methods

1. Streak

Single streak of sample with loop

2. Spread

0.2ml sample on to agar plate, place cell spreader/hockey stick in alcohol, flame, spread evenly w/ hockey stick

3. Pour

Pre-make agar in tubes, 1ml sample into empty dish, keep in liquid form warm (110-120F), pour into dish w/ sample, lightly swirl

CONTAMINATION CHECKS

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Differentiation

▪ Yeast species can’t be identified under microscope (Sequencing required)

▪ Genus can often be guessed based on morphology (Sacch vs Brett, Lacto vs Pedio)

▪ Cycloheximide: Prevents growth of yeast (except Brett), handle carefully!!!

▪ WLN great for differentiation due to Bromocresol Green (BG)

▪ BG is pH indicator: Acids turn blue -> green -> yellow

▪ Depending on how yeasts metabolize BG, colonies will take on different colors:

➢ Brewer’s yeast = dark green

➢ Wild Sacch/Phenolic yeast = white

➢ Brett = Rings of green/white

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Phenolic Sacch Brewer’s Yeast

Brett brux: White->Yellow->Green

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Spread plate: Beer Sample (001 + contaminant yeast)

Pour plate: Beer Sample (001 + contaminant yeast)

CONTAMINATION CHECKS

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Other Simple Differentiation Tests

Catalase Test (H2O2/Hydrogen peroxide):

• Catalase + (Fizz!) = wort spoilers (Yeast, certain bacteria)

• Catalase – (No Fizz) = beer spoilers (Lacto/Pedio)

Gram Stain/KOH Test (potassium hydroxide):

• Gram – (Stringy!) = Acetobacter, Megasphaera, Pectinatus

• Gram + (Not stringy) = Lactobacillus, Pediococcus

CONTAMINATION CHECKS

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Problem Areas

▪ Certain beers: Low IBU (<10-12), Low ABV, High Final Gravity

▪ Gaskets, fittings

▪ Yeast brinks/kegs

▪ Open pitching (vs In-line pitching)

▪ Sample ports

▪ Grain dust, spent mash

▪ Fans, floors, drains, open doors, people

CLEANING & PROPER SAMPLING

TECHNIQUES

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Cleaning verification

▪ ATP Luminometer - Handheld device (~$1500 + ~$200/box of swabs):

• Liquid swabs - CIP Rinse Water

• Dry swabs - Ports, connections, gaskets, hard to visually inspect areas

Proper Sampling

▪ Process:

• Alcohol (Ethanol/Isopropyl), Swab, Alcohol, Flame, Pour, Collect

▪ Wear gloves!

NEGLECTED TESTS

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Extract/Density:

▪ Hydrometer: Original & Final Gravity scales, Temp correction

▪ Digital: Expensive – DMA ($3-4K), Inexpensive – EasyDens ($500)

pH:

▪ First chance to catch issues with mash, contamination

Sensory: Are you drinking your own beer?

▪ Disinterested panel

▪ Gastrograph apps

Force Ferment:

▪ Over pitch small wort sample: Estimating Final Gravity

▪ Beer sample on stir plate: Determine likely gravity of contaminated beers

YEAST STORAGE

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▪ Death to Cornys

▪ SABCO/GW Kent Yeast Brinks ($300-$400)

▪ Keep cold

▪ Regularly relieve CO2 pressure

▪ No longer than two weeks of storage

▪ Hemogenize sample before doing counts (rolling, shaking)

IN-HOUSE VS. OUTSIDE TESTING

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IBU/SRM:

• Hop utilization, microbial stability, beer consistency

• Spectrophotometer ($1-3K), Centrifuge ($500-$1K)

• Send Out: $25-40/test

ABV:

• TTB compliance (0.3%), state laws, marketing materials, consistency

• Alcolyzer/GC ($30K-$60K)

• Send Out: $20-40/test

Other Testing (Diacetyl, DMS, Acetaldehyde):

• Gas Chromatograph ($$$!)

• Use in-house sensory panel to start

• Send Out: $90+/test

Consider testing frequency, quality & brewery priorities, DIY vs. Outside Costs (Time is $) – Start small, you don’t have to do it all at once!

Cell Counts: In-House - ~$1 + training/time vs. Send Out - $15-$20/count

Contamination Checks: In-House - ~$2 + training/time vs. Send Out: $20-$40/check

KEY TAKEAWAYS

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✓ Decide why you’re testing before starting your QC Program

✓ Cell Counts & Contamination Checks are inexpensive, user friendly and highly effective

✓ Don’t neglect common tests

✓ Verify your CIP process is working

✓ Don’t contaminate your beer or your sample

✓ Only you can determine what your budget and available time allows you to test

SPECIAL THANKS

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DON’T BE A STRANGER

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Bootleg’s new digs:

1033 3rd Ave S

Nashville, TN!

THANK YOU!Q&A time…