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Why Is Cell Viability Important?

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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 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 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 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 • Exclude cells which are touching the bottom or right grid 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 • Exclude cells which are touching the bottom or right grid 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 • Exclude cells which are touching the bottom or right grid Yeast counting rules: • Daughter Cells are only counted if their area is ≥ 50% than the mother cell COUNTING UNDER THE MICROSCOPE General counting rules: • Count each area separatly, "snaking" through the entire small square • Cells whithin
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