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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

http://smallworldinitiative.org/colorado-state-university/john-kelley/fort-collins-colorado-near-csu-campus http://www.smccd.edu/accounts/case/graphics/serialdil.gif

https://www.mtholyoke.edu/courses/cwoodard/biol210/GelElectrophoresisFigure.jpg

Pick isolate/isolates with largest zones of inhibition most zones of inhibition produced: Isolate #3 chosen due to number and size of zones of inhibition vs. ESKAPE Relatives.

http://www.chemicool.com/img1/graphics/extractn.gif

Actual Gel Electrophoresis of Isolate #3

Used to differentiate between Gram Positive

bacteria and Gram Negative bacteria based on cell wall.

(Purple = positive, Pink = negative)

Tests for fluorescin production by creating an optimal environment to enhance fluorescin production if the strain

is capable

http://biosistemika.com/wp-content/uploads/2015/03/real-time-pcr-4.jpg

opu/normal/395.jpg

hKp://www.agrisol.co.nz/assets/worms.jpg ad.wikimedia.org/wikipedia/commons/0/08/Thermal_hot_spring.jpg

Nucleotide LAST Search

ESKAPE Pathogens - six most antibiotic-resistant emerging pathogens due to their ever-changing pathogenesis, transmission, and resistnce

Bacillus subtillis Stahpylococcus epidermis Escherichia coli Acinetobacter baylyi Enterobacter aerogenes Pseudomonas putida (Enterococcus faecium) (Staphylococcous aureus) (Klebsiella sp.) (Acinetobacter baumannii) (Enterobacter sp.) (Pseudomonas aeruginosa)

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  Co

unt  

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  Ran

ge    

Cell  Co

unt  

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%