Attachment One-SOP Enumeration Of Recombinant Raccoon Poxvirus In Viral Serials And Sylvatic Plague Vaccine .pdf

Attachment One - SOP: Enumeration of Recombinant Raccoon Poxvirus in Viral Serials and Sylvatic

Plague Vaccine Baits (SPVB) Using a Vero Cell-Based Bioassay

Reagents and materials:

1. Vero cell line (provided by Dr. Tonie Rocke, USGS)

2. HyClone™ Dulbecco's Low Glucose Modified Eagles Medium (DMEM), Fisher Scientific, product number: SH3002102

3. HyClone™ Fetal Bovine Serum (U.S.), Characterized (FBS), Fisher Scientific, product number:

SH3007103

4. Gibco™ Trypsin-EDTA (0.25%), Phenol Red, Fisher Scientific, product number: 25-200-056

5. Falcon 50 mL Conical Centrifuge Tubes, Fisher Scientific, product number: 14-959-49A

6. Fisherbrand™ Petri Dishes with Clear Lid, Fisher Scientific, product number: FB0875712

7. Eppendorf™ LoBind Microcentrifuge Tubes: Protein, Fisher Scientific, product number: E925000090

8. Corning® 75 cm² U-shape Cell Culture Flask, Canted Neck, Millipore Sigma, product number:

CLS430641U

9. Corning® 96 Well TC-Treated Microplates, Millipore Sigma, product number: CLS3599-100EA

10. Crystal Violet, Millipore Sigma, product number: C0775-100G

11. Trypan Blue solution, Millipore Sigma, product number: T8154-100ML

12. Ethyl alcohol, pure, Millipore Sigma, product number: 459844-1L

13. Antibiotic solution (See Appendix A for composition and preparation)

14. Phosphate buffered saline, pH 7.3 (See Appendix B for composition and preparation)

15. Sterile Pipettes and pipette tips (use barrier tips) (contractor’s discretion)

Equipment:

1. Biosafety cabinet

2. Dewer, and liquid nitrogen

3. Pipettors

4. Pipet-Aid (or similar)

5. Vortexer

6. CO2 incubator, and medical grade CO2

7. Sterile scalpels (for SPVB only)

8. Sterile scissors (for SPVB only)

9. Sterile forceps (for SPVB only)

10. Inverted microscope

11. Compound microscope

12. Hemacytometer

Draft

Protocol (part A): Propagation of Vero cells from seed stocks:

1. ___ Initiate Vero cell cultures in accordance with the methods described by Ammerman et al. (1) for

“Basic Protocol 1” with the following considerations:

a. Vero cell seed stocks are to be stored in the vapor phase of liquid nitrogen.

b. Antibiotic solution should be added to all media.

c. The antibiotic solution, FBS, DMEM are sterile, thus no sterile filtration is required.

d. Pass Vero cells 5 times from the seed stock before use in the bioassay.

e. In ‘step 1’ of the Ammerman protocol, use two vials of Vero cell seed stocks to initiate the culture.

f. In ‘step 5’ of the Ammerman protocol, use a 75 cm flask, with 20 mL of media+FBS+antibiotics.

Protocol (part B): Maintenance of Vero cells:

2. ___ Maintain Vero cell cultures in accordance with the methods described by Ammerman et al. (1) for “Basic Protocol 2” with the following considerations:

a. In ‘step 3’ of the Ammerman protocol, incubate with trypsin for 1 min until the cells begin to lose transparency. Draw the remaining trypsin using a sterile pipette, and incubate the flask for 2 to 3 min.

b. In ‘step 4’ of the Ammerman protocol, add 10 mL of media+FBS+antibiotics to wash cells.

c. Disregard ‘steps 7 & 8’ of the Ammerman protocol.

d. In ‘step 9’ of the Ammerman protocol, cells are diluted approximately 1:10 to 1:20 for splits.

e. Pass cells approximately once per week, re-initiate the cultures from seed stocks after

50 passages for use in the bioassay.

Protocol (part D): Viral enumeration of serials via Vero cell bioassay:

3. ___ Pre-warm reagents and media to room temperature (approximately 23°C) prior to use. Perform all manipulations below in a biosafety cabinet using appropriate aseptic technique.

4. ___ Begin with Vero cells split 3-4 days prior to use, which are growing in a 75 cm2 flask.

5. ___ Wash cells gently by adding 5 mL PBS, and then pipet off all liquid.

6. ___ Add 4 mL trypsin solution to the flask of washed Vero cells and then incubate at room temperature (approximately 23°C) for 1 min. Following incubation gently pipet off trypsin solution.

7. ___ Incubate the flask at 37o with 5% CO2 for approximately 3 min for the Vero cell sheet to slough off the flask.

8. ___ Add 10 mL DMEM (supplemented with 10% FBS, and antibiotic solution) and pound the Vero cells down. Shake gently and pipet up and down to disperse the cells. Pipette the Vero cell suspension into a 50 mL centrifuge tube.

9. ___ Add 100 µL of the Vero cell suspensions (or dilutions of Vero cell suspensions in

DMEM+FBS+antibiotics), to 900 µL of the Trypan Blue solution and count the Vero cells using a hemacytometer. Dilute the Vero cells to a final concentration of 1 × 105 cells/mL using

DMEM+FBS+antibiotics as the diluent in 50 mL conical tubes.

a. Note for two 96-well tissue culture plates, a total of 30 mL of the diluted Vero cell suspension should be prepared.

10. ___ Pipette the diluted Vero cell suspension into sterile 100 mm Petri dishes. Using a multichannel pipet, dispense 100 µL of the diluted cell suspension into each well of a 96-well tissue culture plate.

11. ___ Incubate the 96-well tissue culture plates at 37oC with 5% CO2 overnight (up to 18 hr). Following incubation, Vero cells should be approximately 75% confluent.

12. ___ The day of infection, prepare 10-fold serial dilutions (vortexing is appropriate) of viral serials in

1.5 mL conical tubes. The diluent should consist of DMEM+FBS+antibiotics and typsin solution (300 µL/mL). See Protocol (part E) for preparation of SPVB. Typically dilutions ranging from 10-2 through

10-9 will be tested. Two plates will be used for each sample (ie. 20 technical replicates of each dilution).

13. ___ Add viral preparations (100 µL) to the appropriate wells of the 96-well tissue culture plates containing Vero cells. Typically, rows 1 and 12 will be utilized for positive and negative controls.

Each dilution of a single sample will have 10 technical replicates / 96 well plate (rows 2 through 11).

14. ___ Incubate the infected 96-well tissue culture plates for 3 days at 37oC with 5% CO2.

15. ___ To view cytopathic effect, forcibly shake out liquid from each well. Perform this step in a biosafety cabinet, expelling the liquid from tissue culture plates into a basin containing 10% bleach.

Next, add 100 µL of the Crystal Violet solution to each well. Allow plate to stain for at least 20 min at room temperature (approximately 23°C). Shake out Crystal Violet and rinse gently with distilled water by holding the plate at an angle to the stream. Pound the plate dry on an adsorbent pad.

16. ___ Record the position / dilution of any cytopathic effect (CPE). Here, any CPE will be considered a

‘positive’ well. Determine TCID50/mL and pfu/mL using the Reed & Muench method (see Appendix

C). See image below for typical CPE:

Protocol (part E): Viral enumeration of SPVB via Vero cell bioassay:

17. ___ Perform all manipulations below in a biosafety cabinet using appropriate aseptic technique.

18. ___ Record the weight of the bait.

19. ___ Place the bait into a sterile 100 mm × 15 mm Petri dish and cut into small slivers using a sterile scalpel and forceps.

20. ___ Transfer the bait slivers to a 50 mL conical tube, and proceed to cut the bait into even smaller pieces using sterile scissors.

21. ___ Add 10 mL of DMEM+FBS+antibiotics to the bait slivers and vortex at maximum speed for a total of 1 min (two 30 sec intervals).

22. ___ Incubate the baits at 37oC with 5% CO2 for 1.5 hr. Vortex the baits twice, for 30 seconds, after

30 min and 1 hr of incubation.

23. ___ Proceed to dilute the bait as described in ‘Protocol (part D), step 12.’ A wide bore tip may be helpful for preparing these dilutions. Typically, dilutions of the 10 mL suspension of 10-2 to 10-6 should be tested.

24. ___ Proceed to ‘step 13’ as defined in ‘Protocol (part D)’ to enumerate the recombinant raccoon poxvirus.

Notes:

Manipulation of all samples and media should be performed using proper aseptic technique.

Appendixes:

Appendix A – Composition and preparation of the antibiotic solution.

Appendix B – Composition and preparation of the phosphate buffered saline, pH 7.3.

Appendix C – Calculation of viral titer (TCID50/mL and pfu/mL).

Reference:

1. Ammerman, N. C., Beier-Sexton, M., and Azad, A. F. (2008) Growth and maintenance of Vero cell lines. Curr Protoc Microbiol Appendix 4, Appendix 4E

APPENDIX A

National Wildlife Health Center

United States Geological Survey

Antibiotic / Antimycotic Solution for Cell Culture Media

Product No.: A300 Page: 1 of 6

Effective Date: 12 May 2016 Revision: 3

C:\Users\apjcchandler\Desktop\SPV bait titration\Appendix A.docx

Biological Support Services Section

Media Lab Protocol

1.0 Purpose, Scope and Background

1.1 This protocol covers the production of the Antibiotic / Antimycotic Solution for use in cell culture media by the Biological Services Section at the National Wildlife Health Center. This solution is added to media to inhibit the growth of bacterial and fungal contaminants. It is prepared in 7.5mL aliquots with one aliquot being the correct amount to add to a 500mL bottle of media.

2.0 Safety

2.1 Wear gloves, eye protection and laboratory smock.

3.0 Materials and Recipe

Component Vendor Product

Number

Final concentration when added to 500 mls of cell culture media

For 20

Tubes

For 40

Tubes

Fungizone (Amphotericin B)

(250 g/ml) Sigma A2942 1.0 g/ml 40 mL 80 mL

Penicillin-Streptomycin 10,000 U/ml Penicillin G sodium

10,000 g/ml Streptomycin sulfate

Life

Technologies 15140-122

Penicillin 100 U/ml;

Streptomycin 100 g/ml 100 mL 200 mL

Gentamicin Reagent Solution 50 mg/ml gentamicin sulfate in distilled water

Life

Technologies 15750-060 0.026 mg/ml 5.25 mL 10.5 mL

Tube, sterile, 10 - 15mL any N/A N/A 20 40

4.0 Procedure

4.1 Prepare solution in Biological Safety Cabinet (BSC) using aseptic technique

4.2 Thaw Fungizone, Pen-Strep and Gentamicin in a 30ºC water bath or overnight in the refrigerator.

4.3 Combine all ingredients to a sterile flask containing a sterile stir bar and mix without heat.

4.4 Dispense 7.5 ml into each 15 ml centrifuge tube.

4.5 Seal tubes with a piece of parafilm.

4.6 Store at -20°C

4.7 Labeling

4.7.1 Label bottles and seal with a strip of parafilm.

5.0 Quality Control

5.1 Visual Inspection

5.1.1 Pass / Fail criteria: Media should be slightly turbid and yellow in color.

APPENDIX A

Antibiotic / Antimycotic Solution for Cell Culture Media

Product No.: A300 Page: 2 of 6

Effective Date: 12 May 2016 Revision: 3

C:\Users\apjcchandler\Desktop\SPV bait titration\Appendix A.docx

Biological Support Services Section

Media Lab Protocol

6.0 Labeling

6.1 Use Avery 5160 labels.

6.2 MSDS warning label

7.0 Storage Conditions

7.1 Store at <-10°C.

8.0 Shelf Life

8.1 6 Months. Estimate.

9.0 References

9.1 N/A

10.0 Revision History

A300

ABS for Cell Culture Antibiotic / Antimycotic Solution

Lot: Lot Number

Store at 4°C Exp:

WARNING

READ MSDS

Antibiotic / Antimycotic Solution for Cell Culture Media

Product No.: A300 Page: 3 of 6

Effective Date: 12 May 2016 Revision: 3

C:\Users\apjcchandler\Desktop\SPV bait titration\Appendix A.docx

Biological Support Services Section

Media Lab Protocol

11.0 Approval

DOCUMENT REVISION HISTORY

Edition

No.

Brief Description of Changes

Effective Date of

Document

0 Document Initiation 27 April 2006

1 Revised 27 November 2006

2 Updated to new format. Add COGS. 25 September 2012

*Corrected Description of: Penicillin-Streptomycin from 10,000 U/ml Penicillin G sodium and 1000 g/ml Streptomycin sulfate to: 10,000 U/ml Penicillin G sodium and 10,000 g/ml

Streptomycin sulfate. This concentration is on the label of Penicillin-Streptomycin Life

Technologies catalog# 15140-122

Corrected amount of Penicillin-Streptomycin being added to match the final concentration listed for 500 mls of cell culture media was changed from from 105 mls per 20 x 7.5ml tubes Antibiotic / Antimycotic Solution to 100 mls per 20 x 7.5ml tubes Antibiotic /

Antimycotic Solution

**Corrected the final concentration of Gentamicin Reagent Solution listed for 500 mls of cell culture media from 0.05mg/ml to 0.026mg/ml based the amount of Gentamicin that is being added and to Antibiotic / Antimycotic Solution.

Note:

*When looking at 10/27/2006 SOP Antibiotic mixture for CC Media Pen/Strep was also listed as 10,000 U/ml Penicillin G sodium and 1000 g/ml Streptomycin sulfate (Gibco BRL

#15140-122)

**When looking at 10/27/2006 SOP Antibiotic mixture for CC Media Gentamicin Reagent

Solution 50mg/ml (GIBCO BRL# 15750-060) final concentration in CC media is 0.05 mg/ml but that didn’t jive with the amount being added to Antibiotic / Antimycotic Solution.

(Mentioned correct concentration to Susan Smith and she said to leave it the way it is. Also, did nobody do the math in 2007? Susan remembers this being identified years ago, apparently nobody changed the paperwork to match correct concentration.)

12 May 2016

APPROVAL BLOCK

POSITION SIGNATURE DATE

BSS Group Leader

Antibiotic / Antimycotic Solution for Cell Culture Media

Product No.: A300 Page: 4 of 6

Effective Date: 12 May 2016 Revision: 3

C:\Users\apjcchandler\Desktop\SPV bait titration\Appendix A.docx

Biological Support Services Section

Media Lab Protocol

Costs of Goods Sold (COGS)

Component Manufacturer

Product

Number

Purchase

Size Cost

S/H

Cost

Total

Cost Cost / Unit

Fungizone

(Amphotericin B) Sigma A2942 100ml $87.60 $20* $99.90 $1.076 /mL

Penicillin-

Streptomycin

Gibco /

Invitrogen

15140-

100ml $17.58 $20* $37.58 $0.376 / mL

Gentamicin Reagent

Solution

Gibco /

Invitrogen

15750-

10mL $48.50 $20* $68.50 $6.850 / mL

Label, read MSDS Diversified

Biotech

TTSW-

2240-Y 2380/pk $60.00 $11.00 $71.00 $0.003 ea

Label, main Avery 5160 3000/pk $27.00 Unk $27.00 $0.009 ea

Tube, 15ml Corning 430052 50/pk $9.91 - $9.91 $0.199 ea

* estimate.

Cost for one unit 7.5ml Tube of media with labels

Component Cost / unit Amount Cost

Fungizone (Amphotericin B) $1.076 /mL 2.00 mL $2.152

Penicillin-Streptomycin $0.376 / mL 5.25 mL $1.9740

Gentamicin Reagent Solution $6.850 / mL 0.2625 mL $1.7982

Label, read MSDS $0.003 ea 1 $0.0030

Label, main $0.009 ea 1 $0.0090

Tube, 15ml $0.199 ea 1 $0.1990

Total Cost Per Unit: $5.9812

Price to Charge Per Unit: $6.00

Pricing updated 25 Sept 2012 by MDB

Antibiotic / Antimycotic Solution for Cell Culture Media

Product No.: A300 Page: 5 of 6

Effective Date: 12 May 2016 Revision: 3

C:\Users\apjcchandler\Desktop\SPV bait titration\Appendix A.docx

Biological Support Services Section

Media Lab Protocol

Batch Production Log

Production Lot Number Amount Produced Produced By Date Produced

Antibiotic / Antimycotic Solution for Cell Culture Media

Product No.: A300 Page: 6 of 6

Effective Date: 12 May 2016 Revision: 3

C:\Users\apjcchandler\Desktop\SPV bait titration\Appendix A.docx

Biological Support Services Section

Media Lab Protocol

Batch Production Record

Lot Number: Quantity: Produced By:

Production Date: Expiration Date:

Materials

Vendor Product # Lot Number Exp. Date Quantity Fungizone (Amphotericin B)

(250 g/ml) Gibco 15290-026

Penicillin-Streptomycin 10,000 U/ml Penicillin G sodium

10,000 g/ml Streptomycin sulfate

Gibco 15140-122

Gentamicin Reagent Solution 50 mg/ml gentamicin sulfate in distilled water

Gibco 15750-060

Tube, Quality Control

1. Visual Inspection

Media should be slightly turbid and yellow in color.

Pass Fail By: Date:

Notes:

Disposition

Accepted – this lot has been accepted and can be released to customers / clients.

Rejected – this lot has failed quality control standards and cannot be released. Document final disposition.

Accepted with addendum – this lot has been accepted despite some issues with production or quality control.

Attach supporting documentation or annotate on this sheet.

BSS Supervisor Signature Date

APPENDIX B

Phosphate Buffered Saline (PBS) Cell Washing Solution

Product No.: P503 Page: 1 of 7 Effective Date: 18 Oct 2012 Revision: 2

C:\Users\apjcchandler\Desktop\SPV bait titration\Appendix B.docx

Biological Support Services Section Media Lab Protocol

1.0 Purpose, Scope and Background

1.1 This protocol covers the production of Phosphate Buffered Saline (PBS) Cell Washing Solution (CWS) by the Biological Services Section at the National Wildlife Health Center. This solution is used during cell culture operations to wash cells to remove media, debris, cell waste and dead cells. The PBS-CWS is prepared without calcium (Ca++) or magnesium (Mg++) to prevent possible inactivation of trypsin in cell dissolution.

2.0 Safety

2.1 Wear gloves, eye protection and laboratory smock.

3.0 Materials and Recipe

Component Vendor Product Number For 1 L** For 5 L For 8 L For 10 L

Sodium chloride, NaCl Any various 8.00 g 40.00 g 64.00 g 80.00 g

Potassium chloride, KCl Any various 0.20 g 1.00 g 1.60 g 2.00 g Sodium phosphate, dibasic, Na2HPO4 anhydrous*

Any various 1.146 g 5.73 g 9.168 g 11.46 g

Potassium phosphate, monobasic KH2PO4

Any various 0.20 g 1.00 g 1.60 g 2.00 g

RODI water N/A N/A QS to 1L QS to 5L QS to 8L QS to 10L

Sodium phosphate, dibasic, Na2HPO4 - 7 hydrate *

Any various 2.13 g 10.65 g 17.04 g 21.30 g

Sodium phosphate, dibasic, Na2HPO4 12 hydrate * Any various 2.89 g 14.45 g 23.15 g 28.90 g

* Anhydrous or 7 hydrate Sodium Phosphate can be substituted for the 12 hydrate in the listed quantities.

reagent name

Formula weight BSS recipe final concentration/L g/mol grams/liter millimolar

Potassium phosphate monobasic

KH2PO4 136.09 0.2 1.5mM

Sodium chloride, NaCl 58.44 8.0 140mM

Sodium phosphate, dibasic, Na2HPO4 anhydrous 141.6 1.146 8.1mM

APPENDIX B

Phosphate Buffered Saline (PBS) Cell Washing Solution

Product No.: P503 Page: 2 of 7 Effective Date: 18 Oct 2012 Revision: 2

C:\Users\apjcchandler\Desktop\SPV bait titration\Appendix B.docx

Biological Support Services Section Media Lab Protocol reagent name

Formula weight BSS recipe final concentration/L

Potassium chloride, KCl 74.5513 0.2 3mM

Sodium phosphate, dibasic, 12 waters 358.14 2.89 8.1mM

Sodium phosphate, dibasic, 7 waters 268.07 2.13 8.0mM

4.0 Procedure

4.1 Prepare Phosphate Buffered Saline - CWS

4.1.1 Prepare solution in approximately 80% of final volume.

4.1.2 Add components in order. Ensure each chemical completely dissolves before adding the next chemical.

4.1.3 pH to 7.3.

4.1.4 QS to the appropriate volume with RODI water

4.2 Bottling

4.2.1 Add 500mL of PBS-CWS to each 500mL bottle

4.2.2 Loosely cap bottle. Do not tighten cap.

4.2.3 Apply a small piece of autoclave tape on each cap

4.3 Sterilize

4.3.1 Autoclave at 121°C, 15psi for 20 minutes under the liquid (slow exhaust) cycle.

4.3.2 Allow PBS-CWS to cool until the bottles can be easily handled.

4.3.3 Tighten caps

4.4 Labeling

4.4.1 Label bottles and seal with a strip of parafilm.

5.0 Quality Control

5.1 Visual Inspection

5.1.1 Pass / Fail criteria: solution should be clear and colorless.

5.2 Test pH

5.2.1 pH should be between 7.2 and 7.4.

Phosphate Buffered Saline (PBS) Cell Washing Solution

Product No.: P503 Page: 3 of 7 Effective Date: 18 Oct 2012 Revision: 2

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Biological Support Services Section Media Lab Protocol

5.3 Sterility Testing

5.3.1 Randomly select an appropriate number of bottles for QC testing at a rate of x = √(n-1). See table for selection quantities.

5.3.2 From each bottle to be tested, remove 1ml of media and transfer to a sterile tube containing 5mL of BHI medium (BSS # B605).

5.3.3 Incubate at 37°C for 3 to 5 days. Media should remain clear and free of microbiological growth.

5.3.4 If any tested samples demonstrate growth, discard the entire lot.

5.3.5 Include a negative control consisting of a 5mL tube of BHI Medium

6.0 Labeling

6.1 Use Avery 5160 labels.

7.0 Storage Conditions

7.1 Store at 4°C.

8.0 Shelf Life

8.1 12 months. NOTE: Estimate. The PBS is likely stable at 4°C for several years as long as no evaporation occurs to change salt concentrations. The 12 mo shelf life should ensure product remains sterile.

9.0 References

9.1 R. Ian Freshney. Culture of Animal Cells – A manual of basic technique. 4th Ed. 2000. Wiley-Liss

9.2 Use of Muscovy Duck Embryo Fibroblasts for the Isolation of Viruses from Wild Birds, Docherty, D.E.

and P.G. Slota Journal of Tissue Culture Methods 11(3): 165-170

10.0 Revision History

# Bottles Samples 1 - 3 1 4 - 8 2

9 - 13 3 14 - 21 4 22 - 31 5 32 - 43 6

P503 Phosphate Buffered Saline

Cell Washing Solution Lot:

Store at 4°C Exp:

Phosphate Buffered Saline (PBS) Cell Washing Solution

Product No.: P503 Page: 4 of 7 Effective Date: 18 Oct 2012 Revision: 2

C:\Users\apjcchandler\Desktop\SPV bait titration\Appendix B.docx

Biological Support Services Section Media Lab Protocol

11.0 Approval

DOCUMENT REVISION HISTORY

Edition

No. Brief Description of Changes Effective Date of Document

0 Document Initiation 12 Apr 2000

1 Update 13 Nov 2006

2 Update to current format. Change sterilization from filtration to autoclave. Add COGS 18 Oct 2012

APPROVAL BLOCK

POSITION SIGNATURE DATE

BSS Group Leader

Phosphate Buffered Saline (PBS) Cell Washing Solution

Product No.: P503 Page: 5 of 7 Effective Date: 18 Oct 2012 Revision: 2

C:\Users\apjcchandler\Desktop\SPV bait titration\Appendix B.docx

Biological Support Services Section Media Lab Protocol

Costs of Goods Sold (COGS)

Component Vendor Product Number

Purchase Size Cost

S/H Cost

Total Cost Cost / Unit

Sodium chloride, NaCl Any* - 500 g $24.67 N/A $24.67 $0.04934 / g

Potassium chloride, KCl Any* - 500 g $23.51 N/A $23.51 $0.04702 / g

Sodium phosphate, dibasic, Na2HPO4 12 hydrate * Any* - 500 g $34.26 N/A $34.26 $0.06852 / g

Potassium phosphate, KH2PO4

Any* - 500 g $37.38 N/A $37.38 $0.07476 / g

Label, main Avery 5160 3000/pk $27.00 Unk $27.00 $0.009 ea

* Biological grade. product costs from Fisher Scientific

Cost for one unit 500ml Bottle of media with labels

Component Cost / unit Amount Cost Sodium chloride, NaCl $0.04934 / g 0.5 L $0.1974

Potassium chloride, KCl $0.04702 / g 5.00 mL $0.0047 Sodium phosphate, dibasic, Na2HPO4 12 hydrate * $0.06852 / g 5.00 mL $0.0991

Potassium phosphate, KH2PO4 $0.07476 / g 5.00 mL $0.0075

Label, main $0.009 ea 10.00 mL $0.00900

Total Cost Per Unit: $0.3177

Price to Charge Per Unit: $0.33

Prices Updated: 18 October 2012 by MDB

Phosphate Buffered Saline (PBS) Cell Washing Solution

Product No.: P503 Page: 6 of 7 Effective Date: 18 Oct 2012 Revision: 2

C:\Users\apjcchandler\Desktop\SPV bait titration\Appendix B.docx

Biological Support Services Section Media Lab Protocol

Batch Production Log Production Lot Number Amount Produced Produced By Date Produced

Phosphate Buffered Saline (PBS) Cell Washing Solution

Product No.: P503 Page: 7 of 7 Effective Date: 18 Oct 2012 Revision: 2

C:\Users\apjcchandler\Desktop\SPV bait titration\Appendix B.docx

Biological Support Services Section Media Lab Protocol

Batch Production Record Lot Number: Quantity: Produced By:

Production Date: Expiration Date:

Materials Vendor Product # Lot Number Exp. Date Quantity

Sodium chloride, NaCl Fisher

Potassium chloride, KCl Sigma

Sodium phosphate, dibasic, Na2HPO4 anhydrous Fisher

Potassium phosphate, KH2PO4 , monobasic Mallinckrodt

BHI Broth BSS

Quality Control

1. Visual Inspection

Solution should be clear and colorless.

Pass Fail By: Date:

2. pH Testing Test the pH of the final solution. It should read between 7.2 and 7.4.

Record pH:

Pass Fail By: Date:

3. Sterility Testing

Start Date: End Date:

# Days Incubated: Read By:

Notes:

Disposition Accepted – this lot has been accepted and can be released to customers / clients.

Rejected – this lot has failed quality control standards and cannot be released. Document final disposition.

Accepted with addendum – this lot has been accepted despite some issues with production or quality control.

Attach supporting documentation or annotate on this sheet.

BSS Supervisor Signature Date

Sample ID Result

Pass / Fail control

APPENDIX C

APPENDIX C