Institution
Laboratory name
Location
Head/Responsible person / Standard Operating Procedure (SOP)
Preparation of PBS / Code:
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1. Preparation of Phosphate Buffered Saline (PBS) pH 6.8
2. Objectives and scope

During the digestion/decontamination process of sputum samples for culture on Löwenstein Jensen media or MGIT, NACL-NaOH solution is added to digest and loosen the sputum as well kill other bacteria except Mycobacterium. After optimal incubation, the reaction is stopped and neutralized by addition of Phosphate Buffered Saline (PBS) to create the slightly acidic environment optimal for Mycobacterium growth. This SOP describes the method for laboratory preparation of PBS of pH 6.8.

This SOP applies to all laboratory personnel involved in the preparation of reagents.

3. Abbreviations, definitions and terms

•N.A.Not applicable

•PBSPhosphate buffered saline

•NaOHSodium Hydroxide

•SOP Standard Operating Procedure

•MGITMycobacteria Growth Indicator Tube

•NALCN-acetyl-L-Cysteine

•TechsTechnicians/Technologists

4. Tasks, responsibilities and accountabilities

Task / Responsible / Accountable
Preparation of PBS / Techs in Reagent Preparation section / Lab Manager
Quality control of PBS / Techs in Reagent Preparation section / Lab Manager

5. Safety and environment

Wear laboratory coat and gloves when performing this procedure.

6. Procedure

6.1 Reagents and Materials

•De-ionized/Distilled water

•500 mL thick glass bottles ( Schott® type)

•Autoclave

•Autoclave tape

•Balance sensitive to 0.1g

•Flat bottomed conical flask

•Refrigerator

•Kraft paper/foil

•Non absorbent cotton wool

•pH paper/pH meter

•Potassium phosphate monobasic anhydrous KH2PO4

•Sodium phosphate dibasic hepathydrate Na2HPO4.7H2O

•Spatula

•Weighing paper

6.2Stepwise Procedure (1000 ml)

  1. Measure 4500 mL (4.5 liters) of distilled water into a sterile, dry 5 Liter flat bottomed flask.
  2. Weigh 39.8 g of Na2HPO4.7H2O.
  3. Weigh 20.6 g of KH2PO4.
  4. Dissolve the two salts in the 5 L flat bottomed conical flask.
  5. Mix till complete dissolution of the salts occurs.
  6. Aliquot the solution into 500 mL in the 500 mL thick glass bottles, and check the pH,using a pH metre or paper.
  7. Autoclave at 1210C for 30minutes.
  8. Label appropriately including the name of solution, date prepared, expiration date and initials of person who prepared.

6.3Storage

Store properly labeled PBS bottles at 4ºC up to 3 months.

6.4 Quality Control

6.4.1 pH Check

Check the pH of the new PBS stock by using pH paper or pH meter. The pH should be in between an acceptable range of 6.5 to 7.2. Record on Appendix 1.

6.4.2 Bacterial contaminants check

Before the first day of the use of new PBS stock, incubate a small aliquot overnight at 37ºC. Discard the whole solution if it becomes cloudy.Record on Appendix 1

6.5 Reference Tables

pH / Na2HPO4 / KH2PO4
at M/15 / at M/15
5,6 / 10,0 ml / 190,0 ml
5,8 / 16,5 ml / 183,5 ml
6 / 25,0 ml / 175,0 ml
6,2 / 36,0 ml / 164,0 ml
6,4 / 53,5 ml / 146,5 ml
6,6 / 74,5 ml / 125,5 ml
6,8 / 99,0 ml / 101,0 ml
7 / 122,0 ml / 78,0 ml
7,2 / 143,0 ml / 57,0 ml
7,4 / 161,0 ml / 39,0 ml
7,6 / 172,5 ml / 27,5 ml
7,8 / 182,5 ml / 17,5 ml
8 / 189,0 ml / 11,0 ml

7. Related documents

N.A.

8. Related forms

N.A.

9. References

N.A.

10. Attachments/Annexes

Appendix 1, Preparation of phosphate buffered saline (PBS) pH 6,8

Source: GLI Stepwise Process towards TB Laboratory Accreditation

Preparation of Phosphate Buffered Saline (PBS) pH 6.8

Date Prepared: ______Prepared by: ______

Ingredients / Lot: Number / Expiration date / Amount Used
Na2HPO4.7H2O
KH2PO4
Distilled Water

Quality Control Check:

  1. pH Check (6.5 to 7.2)

pH:______Pass/Fail:______

  1. Bacterial contamination Check: incubate the aliquots at 37oC overnight should not be cloudy.

Cloudy/Not Cloudy: ______

PBS BATCH NO:______Expiration Date:______

NB: Storage at 4oC for 3 months.

Date Prepared:______Prepared by:______

Ingredients / Lot: Number / Expiration date / Amount Used
Na2HPO4.7H2O
KH2PO4
Distilled Water

Quality Control Check:

  1. pH Check (6.5 to 7.2)

pH:______Pass/Fail:______

  1. Bacterial contamination Check: incubate the aliquots at 37oC overnight should not be cloudy.

Cloudy/Not Cloudy:______

PBS BATCH NO:______Expiration Date:______

NB: Storage at 4oC for 3 month Page No: ______

Source: GLI Stepwise Process towards TB Laboratory Accreditation