QC for Small Brewers Made Simple

1. QC for (Small) Brewers Made Simple Chris White MBAA District Mid-South December 2008

2. Why Test??? Small breweries, single brewer Selling beer on site Already too late when making beer? Reusing yeast? Find the source of the problem � think like a microbiologist

5. 5 Pub Brewies Small Brew Pub- < 1000 bbls of beer produced per year. >90% sold on-site, mostly from serving tanks. All draft, no bottles. Large Brew Pub (a)- Up to 3,000 bbls of beer produced per year. Limited distribution to local area, >50% sold on-site. Bottles beer for distribution

6. 6 Small Regional Breweries < 3,000 bbls of beer produced per year. All off-site distribution Primarily kegs with limited bottle distribution

7. 7 Mid-size Regional 3,000 to 30,000 bbls of beer produced per year Distributes to local and surrounding areas roughly within 800 miles Bottles and kegs

8. 8 Large Regional 30,000 to 100,000 bbls of beer per year Multi-State distribution through numerous Distributors

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10. How we do it for Cheap? Sensory, sample panel Forced Wort Test Microscope

11. Forced Wort Test Sterilize sample cock with isopropanol and flame Obtain wort sample cleanly into sterilized container-tube, bag, bottle.

12. Forced Wort Test Incubate in a warm area for several days. Optimal temperature is 30�C.

13. Forced Wort Test Results Clear = Beer is clean Cloudy wort or wort with bubbles = bad news Can analyze or miniferment with the contaminant How long does your wort stay clear?

14. If You Have More Money� Basic Testing Supplies Selective Media Incubator Anaerobic Chamber Pressure Cooker Microscope

15. Microscope

16. Hemacytometer

17. Hemacytometer- Full Grid This photo shows the 25 squares seen with the microscope at 10X power. Check for uniformity of cells. If okay, you can use the quick, 5 square method count.

18. Establishing a Counting Protocol Use the same counting protocol for all 5 squares- Cells touching or lying on the top and right triple boundary lines are not counted, whereas cells touching or lying on the bottom or left triple boundary lines are counted. Yeast buds emerging from the mother cell are counted as separate cells if the bud is at least one-half the size of the mother.

19. Other notes Yeast cells are easily seen at 40X power. You may also notice trub in your viewing field that may stain. Trub can be seen in this square at top center and mid center.

20. Viability protocol Dead cells will stain dark blue Cells that are clear or pale blue in color are considered alive. Some budding cells will stain dark blue, but they are not dead! Buds are busy with growing metabolism and not extruding the dye.

21. Square #1 Keeping with the counting protocol, you should have counted: Total cells: 69 Dead cells: 1

22. Calculating Your Cell Count Take the total # of cells you counted in the 5 squares. In this case= 302 Multiply cells 5 squares by 5 to generate the number of cells in 25 squares? 302 x 5 = 1,510 Determine your dilution factor of your sample. In this case= 1:100 Volume in Hemacytometer chamber is 1/10,000 ml Yeast cells/ml= Total cells in 25 squares x dilution factor x (1 x 104) 1,510 x 100 x (1 x104) = 1.51 x109 or 1.51 billion cells/ml

23. Calculating Your Yeast Viability # of live cells � total number of cells x 100% = viability percentage. In this example: 7 dead cells total 295 � 302 x 100% = 97.7% viable

24. What do you do with this? 1.51 billion cells/ml, 98% viability Calculate volume for pitching Confirm pitching rate- what is your rate? Track fermentation progress Bottle conditioning beer Yeast health Troubleshooting Become a better brewer!

25. Starting a Lab!  15ml tubes for dilutions & HLP testing Sterile serological pipets with bulb or transfer pipets Pyrex glass flasks or bottles Media Microwave Sterile petri plates Portable propane flame Plate spreader Incubator Sink

26. Micro testing basics Aseptic technique Use a flame & isopropanol when possible Used sterilized equipment Avoid air drafts and foot traffic

27. Setting Up a Testing Regime Keep it simple or you won�t do it Every brew: Wort Test & test pitching yeast Every week: Brewery tanks Post CIP: Swab test Sterilization time dependent on microbial load, nature of contaminants, size of containers, thickness of walls, nature of contents. Example, agar in media doubles the time versus the same volume of water. Sterilization time dependent on microbial load, nature of contaminants, size of containers, thickness of walls, nature of contents. Example, agar in media doubles the time versus the same volume of water.

28. Media Options Purchase ready made plates or pour your own Best bet for anaerobic organisms- HLP

29. Media Options Best bet for aerobic organisms and wild yeast- WLN, WLD.

30. Testing Pitching Yeast Requires dilutions Test for aerobic bacteria, anaerobic bacteria, & wild yeast Results in 1 � 5 days

31. Plating your samples Wipe down counter with isopropanol Isopropanol on hands/gloves Label plates with sample type and date Work near flame and transfer 0.1ml � 0.25ml to plate. For HLP tube transfer 0.5ml to 1ml sample. Spread sample evenly on plate Let sample dry on plate for ~20 minutes Once dry?turn plate upside down and incubate in warm area.

32. Incubating samples Aerobic= with air. Primarily looking for bacteria such as Acetic Acid bacteria and also contamination by wild yeast. Anaerobic bacteria= without air. Primarily reveals Lactic acid bacteria.

33. What if something grows?

34. What will you do?