Institution
Laboratory name
Location
Head/Responsible person
Standard Operating Procedure (SOP)
Preparation of PBS
Code:
Version: no.
Date: of release
Page: 1 of 4
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.
PBS
NaOH
SOP
MGIT
NALC
Techs
Not applicable
Phosphate buffered saline
Sodium Hydroxide
Standard Operating Procedure
Mycobacteria Growth Indicator Tube
N-acetyl-L-Cysteine
Technicians/Technologists
4. Tasks, responsibilities and accountabilities
Task
Preparation of PBS
Quality control of PBS
Responsible
Techs in Reagent
Preparation section
Techs in Reagent
Preparation section
Accountable
Lab Manager
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)
Auto clave
Autoclave tape
Balance sensitive to 0.1g
Flat bottomed conical flask
Refrigerator
Kraft paper/foil
Non absorbent cotton wool
Source: GLI Stepwise Process towards TB Laboratory Accreditation
Institution
Laboratory name
Location
Head/Responsible person
•
•
•
•
•
Standard Operating Procedure (SOP)
Preparation of PBS
Code:
Version: no.
Date: of release
Page: 2 of 4
pH paper/pH meter
Potassium phosphate monobasic anhydrous KH2PO4
Sodium phosphate dibasic hepathydrate Na2HPO4.7H2O
Spatula
Weighing paper
6.2 Stepwise 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.
6.3 Storage
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
Source: GLI Stepwise Process towards TB Laboratory Accreditation
Institution
Laboratory name
Location
Head/Responsible person
Standard Operating Procedure (SOP)
Preparation of PBS
Code:
Version: no.
Date: of release
Page: 3 of 4
6.5 Reference Tables
Na2HPO4
KH2PO4
pH
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
Final volume
pH 6.8 PBS
in mL
1
200
1000
2000
4000
4500
5000
M/15
Na2HPO4.7
H2O
in mL
0,495
99
495
990
1980
2227,5
2475
M/15
KH2PO4
Na2HPO4.7
H2O
KH2PO4
in mL
0,505
101
505
1010
2020
2272,5
2525
in g
0,0088
1,77
8,84
17,69
35,38
39,80
44,22
in g
0,0046
0,92
4,58
9,16
18,31
20,60
22,89
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:____________
2. Bacterial contamination Check: incubate the aliquots at 37 oC 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:____________
2. Bacterial contamination Check: incubate the aliquots at 37 oC 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