Docstoc

Analysis SOP (DOC)

Document Sample
Analysis SOP (DOC) Powered By Docstoc
					Institution                                                                    Code:
Laboratory name             Standard Operating Procedure (SOP)                 Version: no.
Location                                                                       Date: of release
Head/Responsible person               Preparation of PBS                       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.     Not applicable
      •    PBS      Phosphate buffered saline
      •    NaOH     Sodium Hydroxide
      •    SOP      Standard Operating Procedure
      •    MGIT     Mycobacteria Growth Indicator Tube
      •    NALC     N-acetyl-L-Cysteine
      •    Techs    Technicians/Technologists


  4. Tasks, responsibilities and accountabilities

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


  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                                                                     Code:
Laboratory name           Standard Operating Procedure (SOP)                    Version: no.
Location                                                                        Date: of release
Head/Responsible person               Preparation of PBS                        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                                                                   Code:
Laboratory name                 Standard Operating Procedure (SOP)            Version: no.
Location                                                                      Date: of release
Head/Responsible person                       Preparation of PBS              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              M/15          M/15              Na2HPO4.7   KH2PO4
   pH 6.8 PBS             Na2HPO4.7       KH2PO4                H2O
                              H2O
       in mL                 in mL         in mL                 in g      in g
          1                  0,495         0,505               0,0088    0,0046
        200                    99           101                  1,77      0,92
       1000                   495           505                  8,84      4,58
       2000                   990          1010                 17,69      9,16
       4000                  1980          2020                 35,38     18,31
       4500                 2227,5        2272,5                39,80     20,60
       5000                  2475          2525                 44,22     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

				
DOCUMENT INFO
Shared By:
Categories:
Tags:
Stats:
views:9
posted:12/10/2011
language:English
pages:4