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Introduction Dr. Ulrich M. Tillich Co-CEO & CTO at Oculyze - PowerPoint PPT Presentation

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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

  2. 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

  3. 1. Yeast • What is yeast? Why cell concentration • is important • Why cell viability is C O N T E N T S important 2. Yeast analysis • When should you Analyze • Using the hemocytometer • Using the Oculyze BB2 • Other Methods – Overview 3. Further reading

  4. 1. Yeast • What is yeast? Why cell concentration • is important • Why cell viability is C O N T E N T S important 2. Yeast analysis • When should you Analyze • Using the hemocytometer • Using the Oculyze BB2 • Other Methods – Overview 3. Further reading

  5. 1. Yeast • What is yeast? Why cell concentration • is important • Why cell viability is C O N T E N T S important 2. Yeast analysis • When should you Analyze • Using the hemocytometer • Using the Oculyze BB2 • Other Methods – Overview 3. Further reading

  6. • Member of the fungus family • Capable of living in the What isyeast? absence of oxygen → Turn sugar into CO 2 and alcohol • One of the best studied microorganisms

  7. 1. Yeast • What is yeast? Why cell concentration • is important • Why cell viability is C O N T E N T S important 2. Yeast analysis • When should you Analyze • Using the hemocytometer • Using the Oculyze BB2 • Other Methods – Overview 3. Further reading

  10. • Avoid Under-Attenuation → + Low ABV +Sweet Why is cell Avoid Unwanted Flavors: • conzentration 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), H 2 S (rotten egg) • Brewing Efficiency → + tank turn-around Pitching • → + Fusels +hangovers • Flavor Consistency (!)

  11. 1. Yeast • What is yeast? Why cell concentration • is important • Why cell viability is C O N T E N T S important 2. Yeast analysis • When should you Analyze • Using the hemocytometer • Using the Oculyze BB2 • Other Methods – Overview 3. Further reading

  12. • Viability: Yeast being either alive or dead; i.e. percentage of live V iability 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

  13. POSSIBL POSS IBLE ST E STAINS / AINS / DYES 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

  14. • 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 •

  15. RE RE-PITCHING PITCHING COST COST REDUCTION REDUCTION • Re Re-pitching your yea east ha has se several ad advantages whe hen do done ri right. • Taste oft ften i improves aft after th the fir first us use bu but more importantly you can an red reduce your cost fo for dry dry yeast. • Che hecking your yeast fo for concentration an and viabil ility be before re re-pit itchin ing can an sa save you be between 8 8 $ $ an and 15 15 $ $ pe per ba barrel. l. Use se the the ex example les be belo low to to get et an an idea what thi this mea eans fo for your br 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 In teractive cal calculator culator availa available ble at: at: https://www.oculyze.de/en/products/bb/better-brewing-yeast-counter-yeast-viability/new-user/

  16. 1. Yeast • What is yeast? Why cell concentration • is important • Why cell viability is C O N T E N T S important 2. Yeast analysis • When should you Analyze • Using the hemocytometer • Using the Oculyze BB2 • Other Methods – Overview 3. Further reading

  17. 1. Yeast • What is yeast? Why cell concentration • is important • Why cell viability is C O N T E N T S important 2. Yeast analysis • When should you Analyze • Using the hemocytometer • Using the Oculyze BB2 • Other Methods – Overview 3. Further reading

  18. When should you measure your yeast?

  19. WHEN WH EN TO TO COUN 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 fermentation long before your gravity Alsuhaim et al. va, E. 2012, 'Effects of non-thermal microwave exposures on the proliferation rate of saccharomyces cerevisiae yeast', in World meter or taste buds detect them Congress on Medical Physics and Biomedical Engineering, IFMBE Proceedings 39, M. Long (ed.), Springer, Beijing, China, pp. 48-51

  20. 1. Yeast • What is yeast? Why cell concentration • is important • Why cell viability is C O N T E N T S important 2. Yeast analysis • When should you Analyze • Using the hemocytometer • Using the Oculyze BB2 • Other Methods – Overview 3. Further reading

  21. • For almost any method a dilution will be Dilution needed • 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

  22. THE HEMOCYTOMETE THE HEMOCYTOMETER R EXPLAINED EXPLAINED

  23. THE HEMOCYTOMETE THE HEMOCYTOMETER R EXPLAINED EXPLAINED

  24. THE HEMOCYTOMETE THE HEMOCYTOMETER R EXPLAINED EXPLAINED

  25. THE HEMOCYTOMETE THE HEMOCYTOMETER R EXPLAINED EXPLAINED Counting Grid

  26. THE HEMOCYTOMETE THE HEMOCYTOMETER R EXPLAINED EXPLAINED

  27. THE HEMOCYTOMETE THE HEMOCYTOMETER R EXPLAINED EXPLAINED

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

  29. THE HEMOCYTOMETE THE HEMOCYTOMETER R EXPLAINED EXPLAINED Potentially minor differences in the countig grid Improved Ne Neubauer / Thoma (new) Ne Neubauer / Thoma (old ld)

  30. THE HEMOCYTOMETE THE HEMOCYTOMETER R EXPLAINED EXPLAINED Typically 5 (small) squares are counted Improved Ne Neubauer / Thoma (new) Ne Neubauer / Thoma (old ld)

  31. PREPARIN PREPARING G THE HEMOCYTOMETE THE HEMOCYTOMETER R FOR FOR USE USE What you need PIPETTE AND TIPS ETHANOL + OPTIONAL: STAINING DYE HEMOCYTOMETER & COVER SLIP (DILUTED) YEAST

  32. PREPARIN PREPARING G THE HEMOCYTOMETE THE HEMOCYTOMETER R FOR FOR USE USE Put a drop of ethanol on your finger tip

  33. PREPARIN PREPARING G THE HEMOCYTOMETE THE HEMOCYTOMETER R FOR FOR USE USE Moisten the support structures on both sides (DILUTED) YEAST

  34. PREPARIN PREPARING G THE HEMOCYTOMETE THE HEMOCYTOMETER R FOR FOR USE USE Slide on the cover slip carefully from the side (DILUTED) YEAST

  35. PREPARIN PREPARING G THE HEMOCYTOMETE THE HEMOCYTOMETER R FOR FOR USE USE After short drying make sure Newton's rings are visible

  36. PREPARIN PREPARING G THE HEMOCYTOMETE THE HEMOCYTOMETER R FOR FOR USE USE After short drying make sure Newton's rings are visible

  37. PREPARIN PREPARING G THE HEMOCYTOMETE THE HEMOCYTOMETER R FOR FOR USE USE The empty space between the cover slip and the grid creates the chamber of defined height for counting

  38. PREPARIN PREPARING G THE HEMOCYTOMETE THE HEMOCYTOMETER R FOR FOR USE USE The empty space between the cover slip and the grid creates the chamber of defined height for counting

  39. LOAD LOADING ING THE SAMPLE THE SAMPLE Car Carefull lly an and slo slowly load the sample from the side using capillary force; make sure to no not t overload the chamber

  40. COUN COUNTING TING UND UNDER ER THE MICROSCOP THE MICROSCOPE Load the prepared hemocytometer into the microscope, allow the cells to settle and focus on the cells/grid

  41. COUN COUNTING TING UND UNDER ER THE MICROSCOP THE MICROSCOPE View at 100X (10X Objective)

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