Useful Information and Resources

Useful Information and Resources Information Yeast: The Practical Guide to Beer Fermentation. By Chris White and Jamil Zainasheff, this book has detailed explanations of most of the QC procedures that small breweries should be performing. Master Brewers Association of the Americas Membership benefits include admission to your region s meetings, discounts on reading material and webinars, as well as a network of support of brewery professionals. American Society of Brewing Chemists Membership benefits include access to the ASBC Methods of Analysis, which list the industry standard methods for hundreds of analyses of beer and raw materials. It also includes access to excellent ASBC webinars and discounts on products. Laboratory Equipment and Materials Cynmar Corporation www.cynmar.com Offers a huge assortment of laboratory equipment at very competitive rates and has excellent customer service. American Instrument Exchange www.americaninstrument.com This company sells a very large assortment of used laboratory equipment at significantly reduced prices from new equipment. Local Colleges Often times local colleges sell old equipment when they upgrade. Nasco Whirl-Pak bags www.enasco.com/whirlpak Sterile sample bags sold in various sizes that are incredibly useful and very affordable. Siebel Institute of Society Siebel sells dry microbiological media for use in the brewing industry. Hsu s Lactobacillus/ Pedicoccus Media is a particularly useful buy.

LAB Sterile Sampling The purpose of this SOP is to ensure proper technique for taking a sterile sample from cellar tanks and bottles. Author: Nathan Sanborn Version: 1.0 Effective Date: 10/07/2013 MATERIALS! Sterile 15ml graduated tubes with caps! Spray bottle of 70% minimum isopropyl alcohol! Butane lighter or other flame source! Fine tip sharpie marker or similar! Sanitized sample cock, gasket, and tri clamp (tank only)! Squeeze bottle of 70% minimum isopropyl alcohol (tank only)! 5 gallon bucket with an inch or two of water (tank only)! Sterile 5 or 10 ml serological pipettes (bottle only)! Pipette bulb (bottle only)! Small tray or dish with ¼ inch of water (bottle only)! Clean metal bottle opener (bottle only) SAMPLING PROCEDURE, CELLAR TANKS 1. Ensure the sample port valve seat is well cleaned. 2. Spray sample port valve, sample cock tri-clamp face, and gasket with isopropyl alcohol. 3. Attach sample cock to sample port valve. 4. Place 5 gallon bucket with an inch of water below sample cock nozzle. 5. Label sterile graduated tube with the brand, batch ID, tank sampled, sample date, and brewer s initials. ex: DM150-FV2-091513-NAS. 6. Rinse interior and exterior of sample cock nozzle thoroughly with isopropyl alcohol from the squeeze bottle. Ensure that the squeeze bottle dispenser tip goes all the way up into the nozzle of the sample cock. 7. Apply flame to the nozzle allowing the alcohol to burn away. 8. Repeat steps 5 and 6. 9. While the flame is still burning, open the sample cock and allow beer to flow freely into the bucket for 2-3 seconds before taking the sample. 10. Remove the cap on the sample tube. 11. Place the opening of the sample tube into the freely flowing stream of beer close to the nozzle but ensuring that you do not touch the nozzle with the sample tube. Fill tube at least half-way but do not overfill. 12. Quickly cap the tube. 13. If the samples will not be used directly, store them under refrigeration. 14. Thoroughly clean sample cock and sample port valve seat. 1 of 2

SAMPLING PROCEDURE, BOTTLES 1. Label sterile graduated tube with the brand, batch ID, number of days since packaging, sample date, and brewer s initials. ex: DM150-PACK+5-091513- NAS. 2. Prepare serological pipette by opening the sterile package at the bulb end just enough to expose one half inch of the pipette. Place pipette bulb on the end of the pipette. Leave the pipette in the package until ready to sample. 3. Holding the bottle neck over the tray of water, spray the top of the neck and the cap area with isopropyl alcohol. 4. Flame the neck allowing the alcohol to burn away. 5. Loosen the cap on the sample tube, but leave it sitting loosly on the tube. 6. Spray the bottle opener with isopropyl alcohol. 7. Carefully open the bottle. 8. Spray the mouth of the bottle with isopropyl alcohol, being careful to minimize overspray onto the surrounding lab bench or equipment. 9. Flame the mouth of the bottle, allowing the alcohol to burn away. 10. Immediately draw a sample using the serological pipette. Be careful to ensure that you do not touch either the bottle or the sample tube when transferring the sample.. Use a new pipette for every new sample. 11. Immediately cap the sample tube. 12. Dispose of the pipette. 13. If the samples will not be used directly, store them under refrigeration 14. Clean up. 2 of 2

Wort Sterility Test April 8, 2015 Purpose Ensure the integrity of your heat exchanger sanitation procedure. Procedure Obtain a sterile sample of wort as it exits the heat exchanger. Allow the sample to sit at room temperature, or if possible, at 80 degrees. Visually inspect the sample after 2 and 4 days for signs of fermentation.

Diacetyl Force Test SOP February 4, 2015 Haley Campbell Purpose Determine if a beer is ready to be crashed through the presence or absence of VDK aromas. Procedure - Obtain a sample of ~200 ml of beer in a 1000 ml Erlenmeyer flask. Place a submersible thermometer in the sample and cover. - Heat the sample to 150 F, remove from heat, and allow to stand for 10 minutes. - Place the sample in an ice bath and cool to ~65 F (or close to the temperature of the beer in the tank). - Pour about 50 ml of sample into a glass and take another 50 ml sample directly from the tank. Compare the aromas of the two samples. - If VDKs are present in both samples: beer needs much more contact time with the yeast. - If VDKs are present in the heated sample only: beer needs more contact time with the yeast. Perform this test again at the end of the day, tomorrow, etc. until VDK aromas are not present. - If neither sample displays VDK aromas: beer is ready to be crashed.

Cell Counting SOP February 3, 2015 Haley Campbell Purpose Determine the amount of yeast in a slurry to calculate pitching amount. Procedure - Obtain a well- mixed sample of slurry. It must be representative of what is in the brink. - Perform a serial dilution of the slurry at a 1:200 concentration. To do this: - Weigh out 1 gram of slurry and dilute with distilled water to a final weight of 10 g. Shake well for ~2 minutes. - Weigh out 1 gram of this and dilute with distilled water to a final weight of 10 g. Shake well for ~2 minutes. - Weight out 1 gram of this and add 3 drops of methylene blue. Dilute with distilled water to a final weight of 2 g. Shake well for ~1 minute. - Place the cover slip over the hemocytometer counting area so that the outside edges overlap. Draw up the final solution into a pipette and carefully fill the counting chamber by touching the tip of the pipette to the edge of the counting chamber/cover slip. Make sure to not overfill the chamber. - Find the 5 X 5 grid under the microscope that contains 25 squares bounded by triple lines. Count the cells in the outside squares of the grid. Also count the cells in the center square. Make note of how many of the cells are blue, which means they are dead. - Add the 5 numbers together. Determine which generation of yeast you are counting and multiply by the corresponding viability percentage according to the chart behind the microscope. - Multiply this number by 5 to account for the total cells in the grid. - Multiply this number by 200 to account for the serial dilution. - The volume of the counting chamber is 1/10,000 th of a ml. Multiply this number by 10,000 to determine the number of viable cells/ gram of slurry. - Consult the Total Cells Needed for chart hanging behind the microscope to determine how many cells are needed for the entire batch, or determine the number empirically yourself. (This is explained at the end of this SOP).

- Divide the total cells needed by the number of cells/ gram counted to determine the total number of grams needed for the pitch. Divide this number by 1,000 to convert to kilograms. Here is an example with actual numbers. I counted the following numbers of cells in a serial dilution of a X generation slurry. I want to know the volume of slurry I need to pitch into a batch of Daymark, which requires 39,800,000,000,000 cells Total 26 25 29 32 30 Dead 3 2 3 4 3 26+25+29+32+30= 142 Account for the viability by generation: 142 x.92 = 130.64 Account for the total squares in the grid: 130.64 x 5 = 653.2 Account for the serial dilution: 653.2 x 200 = 130,640 Account for the volume of the counting chamber. 130,640 x 10,000 = 1,306,400,000 viable cells/ gram 39,800,000,000,000 cells = 30,465 grams 1,306,400,000 cells/ gram 30, 465 grams = 30.5 kilograms 1,000 grams/ kg So, pitch 30.5 well-mixed kilograms of slurry!

Pitching amount is determine by the following equation: number of viable cells = 750,000 cells / ml of wort / P. For higher gravity beers, the rate can increase to 850,000 or even 1,000,000 cells/ ml of wort / P. There are 117,348 ml in a barrel. Consult a chart to convert specific gravity to P.

LAB HLP Media Tests The purpose of this SOP is to ensure proper technique for preparing Hsu s Lactobacillus/Pediococcus media, inoculating same, and reading and recording results. Author: Nathan Sanborn Version: 1.0 Effective Date: 10/07/2013 MATERIALS! HLP powdered media! 15 ml sterile sample tubes with caps! Fine tip sharpie marker or similar! Test tube rack! Aluminum foil or clean foam plug! 500 ml flask! Hot plate, trivet, and oven mitt/glove! Plastic spoon and weighing tray or paper! Electronic balance! 1ml sterile serological pipettes! pipette bulb! Sterile collected samples! Positive control sample consisting of approx 1tsp of crushed malt in 2oz of 80-100 degree F water PREPARING THE MEDIA 1. Measure 100ml clean water into a flask. 2. Weigh 7grams of powdered media onto a weighing tray or paper. Be careful to avoid breathing the dust. 3. Slowly add the media to the flask while gently swirling the water. 4. Close the mouth of the flask either with a square of aluminum foil or a clean foam plug. 5. Place the flask on the hotplate. Turn to high heating. Continue to swirl the liquid intermittently during heating as necessary to help any clumps dissolve. 6. While the media is heating, prepare media tubes by labeling a tube with the sample code from each sample that will be tested. If another brewer drew the sterile sample, append your own initials to the sample code. [Ex: DM150-FV2-091513-NAS-SS] Also prepare one tube labeled POSITIVE CONTROL and the date, and a second tube labeled NEGATIVE CONTROL and the date. Loosen the cap on each sample tube and allow to rest atop the tube keeping the mouth covered. 7. Bring the media to a boil and boil for 2.5 minutes. 8. Remove the flask to a trivet and allow to cool for about 5 minutes. 1 of 3

9. Lift the cap off of its sample tube and carefully fill with 12ml of media. Set the cap back loosely onto the tube. Continue sequentially until all the tubes are filled. 10. Leave the cap off of the POSITIVE CONTROL tube and insert a thermometer to monitor the temperature of the media. 11. Allow the media to cool to between 110F and 105F. 12. Locate the lab record sheet for each sample. If this is the first HLP test for a beer, the sheet will still be on the clipboard at the fermentor. Otherwise it should be located in the lab logbook. Record the Sample Date, Sample Code, Sample Volume, and Sampling Brewer. INNOCULATING THE SAMPLES 1. Prepare serological pipette by opening the sterile package at the bulb end just enough to expose one half inch of the pipette. Place pipette bulb on the end of the pipette. Leave the pipette in the package until ready to sample. 2. Loosen the caps on the sample tubes. 3. Remove the pipette from the package and draw 1.0ml of sample from the sample tube. Transfer the sample to the correspondingly labeled media tube. 4. Immediately recap the inoculated media tube. 5. Dispose of the pipette. 6. Ensure the media tube cap is fully closed, then gently invert the tube TWO TIMES ONLY to mix the sample into the media. 7. Replace the tube into the rack. 8. Repeat for each sample. Innoculate the POSITIVE CONTROL sample first, then continue with the rest of the samples. Use a new sterile serological pipette for each sample. Work carefully but quickly. If the media cools too much before inoculation, you can not get a good even mix, as the media will begin to gel. Do not inoculate the NEGATIVE CONTROL tube with anything, but be sure to also invert two times to mimic the mixing procedure. 9. Place the test tube rack with all the media samples into the incubator at 85-86F. READING AND RECORDING RESULTS 1. Locate the lab recording sheet for the sample in question. They will be organized numerically by batch code in the lab logbook. 2. For each media date, ensure that the positive and negative control samples show positive and negative results. In case of either control showing an incorrect result, all samples from that media date must be discarded. Positive samples will often show a result within 24 hours but in any case should show a positive result by 48 hours. 3. Carefully observe the sample in the tube. Look first for any haze or fuzzy discoloration concentrated right at the surface of the media. This indicates an aerobic lactic acid producer. Indicate Y or N as appropriate in the Aerobes Y/N column. 4. Look for any lactobacillus colonies growing within the media. Lactobacillus colonies tend to be shaped like small white tornados or ghost-like shapes. 2 of 3

Count the total number of these colonies and record the number in the Lacto Count column. Do not be concerned with the size of the colonies, as the growth is limited by the amount of nutrient in the media. Fewer colonies will allow for larger growth size. 5. Look for any pediococcus colonies growing within the media. Pediococcus colonies tend to round, oval, or football shaped. Count the total number of these colonies and record the number in the Pedio Count column. Do not be concerned with the size of the colonies, as the growth is limited by the amount of nutrient in the media. Fewer colonies will allow for larger growth size. 6. Counts should be conducted daily for 4 days. After 4 days the sample can be discarded. 7. Report any positive results to the head brewer immediately. 3 of 3

Brew Date Brewer Gyle Batch Fermentor Brand ID Lbs Ingredient Wgh Chk Mill Lbs Ingredient Wgh Chk Mill Grain Lbs: Total Gal: Strike Gal: Min (lbs/55): Flow (gal/min): Strike Temp: Target Mash: MASH Mash In Sacch Rest Time Water ph Grain Bed Temp Notes Start End Base Strike Temp Meas Temp Panel Sample Note or Note Number Vorlauf Lauter Sparge Runoff Time Water Sight Glass Notes Start End Target Gal Actual Gal Temp Static Dynamic Note or Note Number LAUTER Time Kettle Gravity ph Notes Clock Timer BBLs Temp SG Meas Crx Temp SG Calc Meas Temp Note or Note Number Final Runnings Pre Boil Boil Add Clock Timer Qty Ingredient AA % Weighed By Notes KETTLE Whirlpool Time Volume Control Start End Kettle Vol Pump Knockout Time Control Notes Start End Pump O 2 Flow Time Temp Gravity ph Notes SG Meas Crx Temp SG Calc Meas Temp Final RISING TIDE BREWING COMPANY, LLC Portland, ME info@risingtidebrewing.com /reporting/tracking documents/brewtrack_v6.indd 020515 v6

Batch Code Fermentor Brand ID Gyles Date Time Yeast ID Notes Wort BBLs Pitching Temp Cell Count Viability Target Kilos Actual Kilos PITCH Process Anticipated Timeframe/Specs/Notes CELLAR SCHEDULE Date Time pv sv Crx SG Cells (x10 6 ) Viability ph Brewer Notes Combined Original Gravity GRAVITY 1.080 1.075 1.070 1.065 1.060 1.055 1.050 1.045 1.040 1.035 1.030 1.025 Forced Ferment Result FERMENT GRAVITY OVER TIME 1.020 1.015 1.010 1.005 1.000 0 12 24 36 48 60 72 84 96 108 120 132 144 156 168 180 192 204 216 228 240 252 264 276 288 300 312 0 1 2 3 4 5 6 7 8 9 10 11 12 13 TIME (Hours/Days) /reporting/tracking documents/cellartrack_v5indd 012915 v5 RISING TIDE BREWING COMPANY, LLC Portland, ME info@risingtidebrewing.com

Batch Code Fermentor Brand ID Gyles Yeast Harvest Date Time pv Dump 1 Brink 1 Brink 2 Dump 2 Yeast Code Brewer Notes Post Harvest Checklist (Brewer s Initials) FV Head Pressure at Zero Blowoff Valve Open Dump Valve Cleaned and Capped Yeast Brink(s) Tagged w/yeast Code Yeast Brink Head Pressure at Zero HARVEST Preparation Checklist (Brewer s Initials) FV Manway Area Cleaned Blowoff Valve Closed CO2 Attached Date Time Pv Sv Tank additions Brewer Notes Post Addition Checklist (Brewer s Initials) TANK ADDITIONS 1 FV Manway Sealed Blowoff Valve Open Glycol Temp Set Preparation Checklist (Brewer s Initials) FV Manway Area Cleaned Blowoff Valve Closed CO2 Attached Date Time Pv Sv Tank additions Brewer Notes Post Addition Checklist (Brewer s Initials) TANK ADDITIONS 2 FV Manway Sealed Blowoff Valve Open Glycol Temp Set Preparation Checklist (Brewer s Initials) Ports Inspected and Cleaned Pump/Hose Sanitized Trub Dumped Date Time Pv Sv Tank additions Pump Speed Mix Time Brewer Notes FINING 1 Post Fining Checklist (Brewer s Initials) Glycol Temp Set to 32 F 3psi CO2 Head Pressure Set Valves Cleaned and Capped Preparation Checklist (Brewer s Initials) Ports Inspected and Cleaned Pump/Hose Sanitized Trub Dumped Date Time Pv Sv Tank additions Pump Speed Mix Time Brewer Notes FINING 1 Post Fining Checklist (Brewer s Initials) Glycol Temp Set to 32 F 3psi CO2 Head Pressure Set Valves Cleaned and Capped RISING TIDE BREWING COMPANY, LLC Portland, ME info@risingtidebrewing.com /reporting/tracking documents/cellartrack_v5indd 012915 v5

Package Date Transfer Date Anticipated Born Date Batch Code Fermentor Brand ID Gyles Transfer Start Transfer End BBLs Racked Package Temp Notes Brewer RACK Tank Manager Settings Date Start Time Tank Set Temp Supply Pressure Starting Head Pressure Set Volumes Flow Notes Brewer Pressure Zahm Date Time Tank Temp Tank Stone Pressure Temp Volumes Notes Brewer Initial Carb Settings / Target Vols FORCE CARBONATION Orders Cases 1/6 BBL 1/2 BBL Firkins Pins Notes Pallet Cases 1/6s 1/2s Firkins Pins Notes 1 2 3 4 5 PACKAGE 6 7 8 Totals Cases 1/6 BBL 1/2 BBL Firkins Pins Notes Grav at Packaging Temp CRX Grav Cells/ml Bottle Pressure Bottle Temp Bottle Vols Brewer Misc. Notes RISING TIDE BREWING COMPANY, LLC Portland, ME info@risingtidebrewing.com /reporting/tracking documents/cellartrack_v5indd 012915 v5

Fermentor Brand ID Gyles Brew Date Yeast ID Harvested From? Harvest Date Batch Code Test FV BBT Notes 2 Days HLP LCSM WLD Test FV BBT Notes LAB TESTS 4 Days HLP LCSM WLD Pitch rate and viability: Harvest amount and viability: Fermentation notes: YEAST NOTES Date Bottle/Keg Pack + PSI Temp Vols Airs Brewer Notes Package +0 CARBONATION TESTING /reporting/tracking documents/lab_track_2.indd 040414 v2.0