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JBK Pipetting_Cellometer Final Poster
- 1. RESEARCH POSTER PRESENTATIONDESIGN © 2012 www.PosterPresentations.com The Problem In brewing, the yeast pitch rate refers to the amount of yeast that is added to cooled wort in millions of cells per milliliter. Repeatable fermentations are not possible without accurate and precise pitch rates, and healthy fermentation is vital to successful re-pitching of yeast for generations. Thus, pitch rate has a large effect on the final flavor and aroma (seen below). Currently, there is no widespread methodology for determining cell counts using new technology, therefore capabilities of these measurement methods are unknown. OBJECTIVE The Goal The goal is to be able to get a repeatable volume of yeast (within 500,000 cells) when pipetting yeast slurry of all thicknesses, using the reverse pipetting method and analyzing cell count on the Cellometer. Why Reverse Pipetting? The reverse pipetting technique is used to pipette solution with high viscosity or a tendency to foam, both which are current problems faced when pipetting yeast slurry. INTRODUCTION How to Reverse Pipette Materials per Person • 1 P1000 pipette and 12 wide orifice tips • 1 P20 pipette and 24 20uL tips • 12 eppendorf tubes and 12 50mL conical tubes w/ caps • 12 cellometer slides • 1X PBS diluent • KimWipes, Eppendorf tubes, and 50ml Centrifuge tubes • Analytical balance, stir plate, and vortex Design 5 technicians of varying pipetting experience used the reverse pipetting method to pipette 4 yeast slurries of differing thicknesses in random order 3 different times for each slurry. Yeast slurries were stir plated before trails, and pipettes were cleaned with a KimWipe before dispensing yeast slurry from tip. Procedure 1. Reverse Pipette 1mL (using P1000 pipette with wide tip) of yeast slurry into the pre-weighed diluent (PBS) in a 50ml centrifuge tube without mixing by rinsing tip. Eject tip and remaining yeast slurry. 2. Record weight of added yeast slurry and label this dilution #1. Place tube on vortex for 5-10 seconds for mixing. 3. Reverse Pipette 20uL (using P20 pipette) of dilution #1 into pre-weighed diluent in eppendorf tube. Eject tip and remaining yeast slurry. 4. Label this dilution #2. Place tube on vortex for 5-10 seconds for mixing. 5. Load 20uL (using P20 pipette) of dilution #2 onto Cellometer slide using Reverse Pipetting method. Eject tip and give slide to Lab Supervisor for Cellometer analyzing. The Experiment CONCLUSIONS/RECOMMENDATIONS RESULTS My Experimental Trials • Done to gather information to form experiment design and procedure by analyzing % error of average weight recorded during trials. • This data shows reverse pipetting of thin and thick yeast slurry to be more accurate and precise at the 1000ul level, but was more precise at 20ul while showing similar % Error (accuracy). Multiple Operator Trials REFERENCES • http://www.artel-usa.com/resources-library/in-the-lab-pipetes/ • http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3962157/ • https://research.aston.ac.uk/portal/files/13572921/Antifoams.pdf • http://www.pros.co.nz/PDF/MSDS/Antifoam%20FermCap%20S%20MSDS%2025-04-07.pdf • http://www.braukaiser.com/blog/blog/2013/03/25/stir-speed-and-yeast-growth/ • http://www.thelabrat.com/protocols/ProperPipettingMethod.shtml • http://www.bio.davidson.edu/molecular/protocols/pipette.html • http://www.pipettecalibration.net/pipette-calibration-files/guide-to-pipetting-2.pdf • https://www.eppendorf.com/uploads/media/USERGUIDE_20_GB_Final.pdf ACKNOWLEDGEMENTS This work was supported by New Belgium Brewing Company and the Fermentation Laboratory at Colorado State University. Jeff Callaway Jeff Biegert Katie Fromuth Kimberly Cox-York J. Brendan Kelley Accuracy and Precision of Reverse Pipetting Yeast Slurry to load onto Cellometer Under-pitching effects • Excess levels of diacetyl • Increase in higher/fusel alcohol formation • Increase in ester formation • Increase in volatile sulfur compounds • High terminal gravities • Stuck fermentations • Increased risk of infection Over-pitching effects • Little to no ester production • Quick fermentations • Lacking mouthfeel • Autolysis of yeast and resulting off-flavors Overall, there is still a considerable amount of noise observed throughout the pipetting process and a method for pipetting a repeatable volume of yeast (within 500,000 cells) when pipetting yeast slurry of all thicknesses using the reverse pipetting method was not achieved. Operator experience and repeatability were found to be the biggest areas of concern, as seen in the Multiple Operator results, though in my experimental trials I found that with enough practice repeatability is achieved. Thus, I recommend that extensive training is implemented to ensure skill and repeatability before an operator is qualified to perform the procedure. It is also recommended that operators are tested monthly to quarterly for technique and quality assurance purposes. 0 20000 40000 60000 80000 Repeatability Part-‐to-‐Part Reproducibility Measurement System Variance Components 0 200 400 600 800 1000 1200 1400 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 Part Average Cell Count Part Number (Order from thinnest to thickest = 3, 1, 2, 4) Averages Chart Operator 1 Operator 2 Operator 3 Operator 4 Operator 5 UCL = 1193.942 Center = 806.336 LCL = 418.731 0 200 400 600 800 1000 1200 1400 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 Part Range Cell Count Part Number (Order from thinnest to thickest = 3, 1, 2, 4) Range Chart Operator 1 Operator 2 Operator 3 • Analysis of the variance components of our measurement system displays Operator’s ability to repeat procedure is where error is predominantly coming from. • The averages chart supports data revealed above, showing the operators had difficulty repeating the procedure accurately and precisely. • The range chart shows the standard deviation of operators. This data displayed Operators 1, 3, and 4 were the most skilled, in terms of standard deviation, while 2 and 5 were not. Data was supported by skill levels Operators listed before experimentation took place. Operator Experience 1 Expert 2 Novice 3 Intermediate 4 Intermediate 5 Novice Thin Slurry (Forward) 1000 uL 1000 uL (wide Rp) 20 uL Average .92419 .917325 .0197 Percent Error -‐7.581% -‐8.62675% -‐1.5% Thick Slurry (Reverse) 1000 uL 1000 uL (wide Rp) 20 uL Average 1.005545 1.012365 .020245 Percent Error .5545% 1.2365% 1.225% Thin Slurry (Reverse) 1000 uL 1000 uL (wide Rp) 20 uL Average .1.0265 1.00741 .0201 Percent Error 2.65% .741% 1.5% Thick Slurry (Forward) 1000 uL 1000 uL (wide Rp) 20 uL Average .954395 .89855 .01981 Percent Error -‐4.5605% -‐10.145% -‐.95%