Why Is Cell Viability Important?

Introduction

• Dr. Ulrich M. Tillich

• Co-CEO & CTO at Oculyze

• Co-founded the company in 2016

• Masters in Bioinformatics / Biosystem Technology

• PhD in Molecular Biology with focus on laboratory automation 1. Yeast • What is yeast? • Why cell concentration is important C O N T E N T S • Why cell viability is important

2. Yeast analysis • When should you Analyze • Using the hemocytometer • Using the Oculyze BB2 • Other Methods – Overview

3. Further reading 1. Yeast • What is yeast? • Why cell concentration is important C O N T E N T S • Why cell viability is important

2. Yeast analysis • When should you Analyze • Using the hemocytometer • Using the Oculyze BB2 • Other Methods – Overview

3. Further reading 1. Yeast • What is yeast? • Why cell concentration is important C O N T E N T S • Why cell viability is important

2. Yeast analysis • When should you Analyze • Using the hemocytometer • Using the Oculyze BB2 • Other Methods – Overview

3. Further reading • Member of the fungus family What is yeast? • Capable of living in the absence of oxygen

→ Turn sugar into CO2 and alcohol • One of the best studied microorganisms 1. Yeast • What is yeast? • Why cell concentration is important C O N T E N T S • Why cell viability is important

2. Yeast analysis • When should you Analyze • Using the hemocytometer • Using the Oculyze BB2 • Other Methods – Overview

3. Further reading

• Avoid Under-Attenuation → + Low ABV +Sweet Why is cell conzentration • Avoid Unwanted Flavors: important? • High → + ethyl butyrate (tropical), ethyl hexanoate (fruity apple) • Low → + isoamyl acetate (banana), acetaldehyde (fresh pumpkin / green apple) & diacetyl (butter) • Slow → + i.e. Ethyl mercaptan (rotten drain gas),

H2S (rotten egg)

• Brewing Efficiency → + tank turn-around

• Pitching → + Fusels +hangovers

• Flavor Consistency (!) 1. Yeast • What is yeast? • Why cell concentration is important C O N T E N T S • Why cell viability is important

2. Yeast analysis • When should you Analyze • Using the hemocytometer • Using the Oculyze BB2 • Other Methods – Overview

3. Further reading • Viability: Yeast being either alive or dead; i.e. percentage of live Viability cells within population vs. Vitality • Vitality: Well being of the yeast. Is it alive and well or barely holding on? → Very difficult to measure! → Correlates strongly with viability

VIABLE YEAST CELL NON-VIABLE YEAST CELL

+ dehydrogenase - dehydrogenase activity activity POSSIBLE STAINS / DYES

Methylene Blue (MB) Trypan Blue Methylene Violet • Standard dye for years • More uncommon, but popular • Modern stain, very low toxicity • Low toxicity among some brewers • Easier identification than MB • Very widely used • Toxic! • Better at lower viabilities than MB • Not accurate below 90% viability • Avoid Under-Attenuation: low viability same as low yeast count • LowABV • Sweet • Avoid unwanted flavors (Autolysis flavor) • Avoid stalled fermentations • Brewing efficiency (Tank Turn-Around) • Flavor consistency (!) Why is cell viability • Characteristics of healthy yeast: important? • Cellular growth and proliferation • Strong resting metabolism • Strong membrane integrity

• REPITCHING = cost saving RE-PITCHING COST REDUCTION

• Re-pitching your yeast has several advantages when done right. • Taste often improves after the first use but more importantly you can reduce your cost for dry yeast. • Checking your yeast for concentration and viability before re-pitching can save you between 8 $ and 15 $ per barrel.

Use the examples below to get an idea what this means for your brewery:

Calculate your yeast cost Yeast cost/ barrel (US) Output barrels per Yeast cost p.a. year

Use yeast for 1 generation $8,00 1000 $8.000

Use yeast for 2 generations $4,00 1000 $4.000

Use yeast for 3 generations $2,67 1000 $2667

Use yeast for 4 generations $2,00 1000 $2.000

Interactive calculator available at: https://www.oculyze.de/en/products/bb/better-brewing-yeast-counter-yeast-viability/new-user/ 1. Yeast • What is yeast? • Why cell concentration is important C O N T E N T S • Why cell viability is important

2. Yeast analysis • When should you Analyze • Using the hemocytometer • Using the Oculyze BB2 • Other Methods – Overview

3. Further reading 1. Yeast • What is yeast? • Why cell concentration is important C O N T E N T S • Why cell viability is important

2. Yeast analysis • When should you Analyze • Using the hemocytometer • Using the Oculyze BB2 • Other Methods – Overview

3. Further reading When should you measure your yeast? WHEN TO COUNT

• Minimum When is cell count • Before pitching (after careful rehydration) important? • After harvesting the yeast at the end of fermentation • Before re-pitching (after harvesting and storage period)

• Recommended • Make one measurement each day during fermentation phase • Track yeast health through generations → This will allow you to detect problems in Alsuhaim et al. va, E. 2012, 'Effects of non-thermal microwave exposures fermentation long before your gravity on the proliferation rate of saccharomyces cerevisiae yeast', in World Congress on Medical Physics and Biomedical Engineering, IFMBE meter or taste buds detect them Proceedings 39, M. Long (ed.), Springer, Beijing, China, pp. 48-51 1. Yeast • What is yeast? • Why cell concentration is important C O N T E N T S • Why cell viability is important

2. Yeast analysis • When should you Analyze • Using the hemocytometer • Using the Oculyze BB2 • Other Methods – Overview

3. Further reading • For almost any method a dilution will be needed Dilution • This will not be significantly different, no matter how you are counting • While technically simple this is still a potential source of error!

For a step by step video on dilution please check: https://www.youtube.com/watch?v=HCKPAqZC4hk THE HEMOCYTOMETER EXPLAINED THE HEMOCYTOMETER EXPLAINED THE HEMOCYTOMETER EXPLAINED THE HEMOCYTOMETER EXPLAINED

Counting Grid THE HEMOCYTOMETER EXPLAINED THE HEMOCYTOMETER EXPLAINED THE HEMOCYTOMETER EXPLAINED

Actual counting area (large square) consisting of various small squares (25 here) THE HEMOCYTOMETER EXPLAINED

Potentially minor differences in the countig grid

Improved Neubauer / Thoma (new) Neubauer / Thoma (old) THE HEMOCYTOMETER EXPLAINED

Typically 5 (small) squares are counted

Improved Neubauer / Thoma (new) Neubauer / Thoma (old) PREPARING THE HEMOCYTOMETER FOR USE

What you need

PIPETTE AND TIPS ETHANOL

+ OPTIONAL: STAINING DYE HEMOCYTOMETER & COVER SLIP (DILUTED) YEAST PREPARING THE HEMOCYTOMETER FOR USE

Put a drop of ethanol on your finger tip PREPARING THE HEMOCYTOMETER FOR USE

Moisten the support structures on both sides

(DILUTED) YEAST PREPARING THE HEMOCYTOMETER FOR USE

Slide on the cover slip carefully from the side

(DILUTED) YEAST PREPARING THE HEMOCYTOMETER FOR USE

After short drying make sure Newton's rings are visible PREPARING THE HEMOCYTOMETER FOR USE

The empty space between the cover slip and the grid creates the chamber of defined height for counting PREPARING THE HEMOCYTOMETER FOR USE

The empty space between the cover slip and the grid creates the chamber of defined height for counting LOADING THE SAMPLE

Carefully and slowly load the sample from the side using capillary force; make sure to not overload the chamber COUNTING UNDER THE MICROSCOPE

Load the prepared hemocytometer into the microscope, allow the cells to settle and focus on the cells/grid COUNTING UNDER THE MICROSCOPE

View at 100X (10X Objective) COUNTING UNDER THE MICROSCOPE

View at 200X in phase contrast (20X Objective) COUNTING UNDER THE MICROSCOPE

View at 400X (40X Objective) -> single small square with 16 areas COUNTING UNDER THE MICROSCOPE COUNTING UNDER THE MICROSCOPE

General counting rules: • Count each area separatly, "snaking" through the entire small square COUNTING UNDER THE MICROSCOPE

General counting rules: • Count each area separatly, "snaking" through the entire small square • Cells whithin the outer edge of the square are not counted COUNTING UNDER THE MICROSCOPE

General counting rules: • Count each area separatly, "snaking" through the entire small square • Cells whithin the outer edge of the square are not counted • Within each area count cells which are touching left or top grid COUNTING UNDER THE MICROSCOPE

Yeast counting rules: • Daughter Cells are only counted if their area is ≥ 50% than the mother cell COUNTING UNDER THE MICROSCOPE

Yeast counting rules: • Daughter Cells are only counted if their area is ≥ 50% than the mother cell

Stained yeast rules: • Stained cells are counted as dead COUNTING UNDER THE MICROSCOPE

Yeast counting rules: • Daughter Cells are only counted if their area is ≥ 50% than the mother cell

Stained yeast rules: • Stained cells are counted as dead • Budding cells are excluded from viability calculations CALCULATING THE CONCENTRATION - EXAMPLE

BASIC FORMULA Cells Counted Concentration [cells/ml]= Volume of area counted [ml]

EXAMPLE CALCULATION

Volume of counting area (V) = length (L) x w i d t h (W) x height (H) Volume of counting area (V) = 1mm x 1mm x 0.1 mm Volume of counting area (V) = 0.1 mm3 = 1 x 10- 4 ml (or 0.1 µL)

1 Concentration [cells/ml]= Cel l s Count e d * = Cel l s Cou nt e d * 10000 10− 4

But we also have to correct since we are not counting all the squares:

s q u a r e s in c o u n t i n g a r e a 25 Concentration [cells/ml]= C e l l s C o u n t e d * 1 0 0 0 0 * = C e l l s C o u n t e d * 10000 * c o u n t e d s q u a r e s 5 Concentration [cells/ml] = Cel l s Cou n t ed * 50000 PRACTICE COUNT

We will do a practice count using only 2 squares (normally you should to AT LEAST 5) PRACTICE COUNT - CALCULATION

BASIC FORMULA Cells Counted Concentration [cells/ml]= Volume of area counted [ml]

ASSUMPTIONS • Chamber counting area volume 1 x 10- 4 m l (as in the example before) • 1:100 dilution

CALCULATION s qu ar e s in c oun t i ng ar ea Concentration [cells/ml]= Cel l s Count e d * 10000 * * Dilution factor c ou nt e d s qua r es Concentration [cells/ml] = Cel l s Cou n t ed * 125000 *100 = Cel l s Cou n t ed * 12500000

Write your result in million cells / ml into the chat! PRACTICE COUNT - CALCULATION

BASIC FORMULA Cells Counted Concentration [cells/ml]= Volume of area counted [ml]

ASSUMPTIONS • Chamber counting area volume 1 x 1 0 - 4 m l (as in the example before) • 1:100 dilution

Concentration [cells/ml] = (52+ 56) * 12500000 = 141750000000 = 1417,5 mil. cell / ml PRACTICE COUNT

Which cells to exclude from the count REMEMBER: Alternative Our example Methods calculation did not consider viability. Doing viability counts accurately requires a bit more practice! HEMOCYTOMETER MAINTENANCE

Disasemble the chamber be pulling the cover slip of sideways (don‘t lift it off, as it might break!) HEMOCYTOMETER MAINTENANCE

Clean both parts with distilled water and then dry before storage GENERAL MANUAL COUNTING TIPS

• Take a representative sample of your yeast • Keep careful track of your dilution! This is one of the most common causes of error • Never pipette the yeast solution onto the hemocytometer and then press the cover slip on top, this will make your measurement very inaccurate • It is advisable to count no fewer than 75 cells on the entire 1mm² ruled area and no more than about 48 cells in one of the 25 squares. • Get a manual clicker to reduce the chance of miscounts (or at least 2 if doing viability as well) • Best practice is to count the grids on both sides of the hemocytometer; both measurements should agree within 10%. • There is quite a bit of subjectivity involved when it comes to budding cells (human estimation of area is not great) and viability (what is counted as dead) → Make sure its always the same person in the brewery doing the counts! This will increase reproducibility a lot when counting manually. • Make sure to create a standard document to write down your counts, dilutions and calculations; if you can save the pictures. Don’t just write down the result or you will never be able to track an error! Why is taking the sample correctly important • Lets take a random container e.g. 20 hl (approx. 17 barrel) • Let‘s say your initial sample is 1 ml. → That is 1ml out of 2.000.000 ml or 1ml is 5*10-7 times smaller than your entire volume. → This shows why it is important to take a homogenous sample and always take it in the same way and the same time.

Why not dilute till I have only ~5 cells in the squares?

• If your 1 ml sample comes out of a fermenter you should probably do a 1:100 dilution. • If you take a sample from the dilution and put it in your counting chamber you are looking at 100th the concentration of your original sample. • This is still going to leave you with a lot of cells to count- so you might think: • Easier than counting a lot of cells is dilute again and adjust your formula… • If you dilute this again say 1:10 you are looking at 1000th of your original sample • Any variation (and due to unequal distribution there will always be some!) will get magnified 1000-Fold by the dilution alone 1. Yeast • What is yeast? • Why cell concentration is important C O N T E N T S • Why cell viability is important

2. Yeast analysis • When should you Analyze • Using the hemocytometer • Using the Oculyze BB2 • Other Methods – Overview

3. Further reading SMARTPHONE BASED CELL COUNTER OCULYZE BB2: ANALYSIS PROCESS PREPARING THE BB2 FOR USE

Connect phone and log into the Oculyze BB2 App LOADING THE SAMPLE

load the sample into one of the chamber ports COUNTING WITH THE OCULYZE BB2

Load the prepared chamber into the BB2 COUNTING WITH THE OCULYZE BB2

Focus the sample by using the wheel COUNTING WITH THE OCULYZE BB2

Using the app capture an image for analysis and move the slide to a new position (each image corresponds to 1 small square) COUNTING WITH THE OCULYZE BB2

After capturing the images you can enter the sample details and the sample will be automatically analyzed (5 images are default, but it can be increased to up to 20 in settings for better accuracy) COUNTING WITH THE OCULYZE BB2

The result will come back in a couple of seconds COUNTING WITH THE OCULYZE BB2

The result will come back in a couple of seconds.

You can also visually validate the counts COUNTING WITH THE OCULYZE BB2 COUNTING WITH THE OCULYZE BB2 ADDITIONAL FEATURES

A history of your analysis is automatically saved A pitch rate calculator is also included ADDITIONAL FEATURES

You can also access your results through our Web-App; admin accounts to monitor various devices are possible BB2 CHAMBER MAINTENANCE

Clean the chamber using a syringe of distilled water then dry using the provided bellow ALTERNATIVE HARDWARE: LAB-MICROSCOPE CAMERA ADD-ON Quickly upgrade existing microscopes: Use ocular or c-mount camera connected to smart phone to access Oculyze algorithms in cloud. We count for you, but you still need to do the rest correctly on your own

Advantages: • Keep your existing process and hardware in place • Even lower investment if you already own a microscope

Video showing this system in detail: https://www.youtube.com/watch?v=VVhEsTJnCqE THOROUGHLY TESTED

WE HAVE: • 20 years of combined experience in traditional image recognition know how • Some of the best AI/ deep learning hardware money can buy • You will gain automation which matches a human giving you an immediate improvement on reproducibility and future scaling and cost advantages

VALIDATION: • 3 years ago our software was as good as a person with 20 years of experience → We have steadily improved since then

Report on the evaluation of the Oculyze yeast cell-counting system by the Research and Teaching Institute for Brewing in Berlin (VLB)

OCULYZE BB2 PRICING

Premium Package (not in Webshop) • Premium support • Admin account for WebApp • Payment terms 90 days via wire transfer • 7 day exchange device worldwide

$ $ $ 3.999 $

Extra Analysis Price: • Per Analysis: Starting at 1,80$ per Analysis (purchase of 1000) up to 1,99$ (100) • Flatrate: Starting at 150$ per month (yearly purchase) to 169$ (monthly purchase) 1. Yeast • What is yeast? • Why cell concentration is important C O N T E N T S • Why cell viability is important

2. Yeast analysis • When should you Analyze • Using the hemocytometer • Using the Oculyze BB2 • Other Methods – Overview

3. Further reading Overview of Methods to determine cell count MANY (YEAST) CELL COUNTERS IN THE MARKET

2008 2014 1953 Blackberry Iphone 5 UP TO DATE?

The process to update a cell counter is similar to updating a car navigation system (if updates are still available)

Often updates to analytical devices update the GUI (User Interface) but keep the same decade old analysis technology underneath (i.e. no good separation of clustering cells) BB2: DEVELOPED WITH BREWERS FOR BREWERS

• There are many one size fits none (all) cell counters on the market. Very few are targeted at brewers! • That‘s not because competitors are lazy- its just that it is less viable to do customs solutions for different types of (yeast) cells on old-school desktop based counters. • Our secret sauce is not the hardware but the software in the cloud. • We can and do keep on rolling out software updates to existing users around the world free of charge. microscope cell counter oculyze

Concentration Viability microscope cell counter oculyze

Concentration Viability

Contamination ( ) X ( ) microscope cell counter oculyze

Concentration Viability

Contamination ( ) X ( ) No h u m a n error X microscope cell counter oculyze

Concentration Viability

Contamination ( ) X ( ) No human error X

Mobile Device X X microscope cell counter oculyze

Concentration Viability

Contamination ( ) X ( ) No human error X

Mobile Device X X

Automatic X X docum entation microscope cell counter oculyze

Concentration Viability

Contamination ( ) X ( ) No human error X

Mobile Device X X

Automatic X X docum entation

Time: analysis & 20-30 min 10 min < 3 min docum entation microscope cell counter oculyze

Concentration Viability

Contamination ( ) X ( ) No human error X

Mobile Device X X

Automatic X X docum entation

Time: analysis & 20-30 min 10 min < 3 min docum entation

Hardware costs 300-15,000 $ 3,500-16,000 $ 999 $ - free microscope cell counter oculyze

Concentration Viability

Contamination ( ) X ( ) No human error X

Mobile Device X X

Automatic X X docum entation

Time: analysis & 20-30 min 10 min < 3 min docum entation

Hardware costs 300-15,000 $ 3,500-16,000 $ 999 $ - free

OUR FULL COMPETITOR ANALYSIS IS AVAILABLE AT https://www.oculyze.de/en/automated-cell-counter-comparison

1. Yeast • What is yeast? • Why cell concentration is important C O N T E N T S • Why cell viability is important

2. Yeast analysis • When should you Analyze • Using the hemocytometer • Using the Oculyze BB2 • Other Methods – Overview

3. Further reading Recommended further reading:

• Mary Pellettieri: “Quality Management: Essential Planning for Breweries” • Chris White: “Yeast: The Practical Guide to Beer Fermentation” • ASBC; Kelly Tretter and Rob Christiansen: „lab in a fish bowl presentation“ • https://www.oculyze.de/en/products/bb/training-en/ • VLB: Research and Teaching Institute for Brewing in Berlin (VLB) “The Yeast in the Brewery”

Contact information

Dr. Ulrich M. Tillich Co-founder, Co-CEO & CTO Tel: +49 1516 171-7961 E-Mail: [email protected]