Media and Reagent Preparation for Polio virus detection-Part-2

     I.          Hepes Buffer

·       Materials required: Hepes powder, Flask, Sterile 0.22µ membrane filter with receptor, Weighing machine, Bottles, Peptone broth, Thioglycollate broth & Sabraud agar slant, D.D.W, pipette.

·       Procedure:

o   Take 150ml D.D.W in one sterile Flask.

o   Weight 59.4gm of Hepes and dissolve it and make total volume 250ml with DDW.

o   Filter by sterile Millipore Membrane filter of porosity 0.22µ.

o   Put the sterility in peptone broth, Thioglycollate broth and Sabouraud agar slant. 

o   Distribute 20ml media in sterile bottles. Keep it at 2°C to 8°C.

·                 Documentation: Following information is noted in PR-32

• Manufacturer, Catalogue No. and  Expiry of Media
• Date used    

                                     

  II.          7.5% NaHCO3    

·       Materials required: NaHCO3 powder, 500 ml Flask, Sterile 0.22µ membrane filter with receptor, Weighing machine, Bottles, pipette, Peptone broth, Thioglycollate broth, sabraud agar slant & D.D.W.

·       Procedure:

o   Take 500ml D.D.W in one flask, weigh 37.5gm powder of NaHCO3.

o   Filter by sterile Millipore Membrane filter of porosity 0.22 µ and put the sterility in peptone broth, Thioglycollate broth and sabouraud agar slant.

o   Distribute 25ml and 15ml in bottles. Keep it at 2⁰C to 8⁰C.

·       Documentation: Following information is noted in PR-32

• Manufacturer, Catalogue No. and  Expiry of Media
• Date used                                          

     I.          Phenol Red (0.4%)

·       Materials required: Phenol red powder, Glass Bottles, Pipette, Weighing machine and Autoclave.

·       Procedure:

o   Take 100ml D.D.W in sterile bottle. Weigh 400mg powder and Dissolve.

o   Autoclave it at 121°C for 30min.

o   After autoclaving, cool it and keep it at 2°C to 8°C.

·       Documentation: Following information is noted in PR-32

• Manufacturer, Catalogue No. and  Expiry of Media
• Date used  

                                       

  II.          FBS -Ready to use

·       Materials required:   FBS, Pipette, Peptone broth, Thioglycolate broth, and Sabouraud agar slant.

·       Procedure: Check the sterility of each bottle before using it. Put the sterility in peptone broth, Thioglycolate broth, Sabouraud agar slant and plain tube.

·       Documentation: Following information is noted in PR-32

• Manufacturer, Catalogue No. and  Expiry of Media
• Date used 

III.          P+S (Penicillin + Streptomycine): Ready to use

·       Documentation: Following information is noted in PR-32

• Manufacturer, Catalogue No. and  Expiry of Media
• Date used  

IV.          PBS without Ca⁺⁺ and Mg⁺⁺ for cell-line:

·       Materials required: 1 litre Glass Bottle, Pipette, D.D.W., Autoclave, PBS 10x without Ca & Mg, P+S Antibiotics, Spirit swab, measuring cylinder, Peptone broth, Thioglycolate broth, and Sabouraud agar slant.

·       Procedure:

o   Take sterile 1 litre bottle and add 900ml D.D.W.

o   Autoclave it at 121⁰C for 30min.

o   After cooling, add 100ml PBS (10× without Ca & Mg).

o   Add 20ml Penicillin+ Streptomycin & put sterility of media in peptone broth, Thioglycollate broth and sabouraud agar. Keep it at 2 to 8⁰C in refrigerator.

·       Documentation: Following information is noted in PR-32

• Manufacturer, Catalogue No. and  Expiry of Media
• Date used  

     I.          PBS with Ca⁺⁺ and Mg⁺⁺ for sample process (Ready to use) 100ml

·       Materials required: Pipette, Spirit swab, PBS with Ca⁺⁺ and Mg⁺⁺ and  Penicillin + Streptomycin

·       Procedure: Add 2 ml penicillin ₊ Streptomycin in each 100ml PBS Bottle. Keep it at 2-8°C_.

·       Documentation: Following information is noted in PR-32

• Manufacturer, Catalogue No. and  Expiry of Media
• Date used  

  II.          Trypsin-EDTA (Ready to use )

·       Material: Trypsin-EDTA (SIGMA), Pipette, Peptone broth, Thioglycolate broth, Sabraud agar slant.

·       Sterility testing Procedure:

o   Put the sterility in peptone, Thioglycolate broth & Sabraud agar slant before use.

o   Store it at -20⁰C in Deep freeze no.DF-8.

·       Documentation: Following information is noted in PR-32

• Manufacturer, Catalogue No. and  Expiry of Media
• Date used  

2.    Safety conditions

·       To avoid contamination, every time wash hands properly before and after preparing media.

·       Check glassware for proper washing and autoclaving to reduce chances of toxicity or contamination.

·       Pour ingredients carefully, mix properly and close lid tightly and wrap Para film.

·       Store media at proper temperature.

3.    Documentation

·       Media Preparation register PR-32.

4.    References used to draw SOP

·       WHO Polio Laboratory Manual.

·       Documentation: Following information is noted in PR-32

• Manufacturer, Catalogue No. and  Expiry of Media
• Date used  

     I.          PBS with Ca⁺⁺ and Mg⁺⁺ for sample process (Ready to use) 100ml

·       Materials required: Pipette, Spirit swab, PBS with Ca⁺⁺ and Mg⁺⁺ and  Penicillin + Streptomycin

·       Procedure: Add 2 ml penicillin ₊ Streptomycin in each 100ml PBS Bottle. Keep it at 2-8°C_.

·       Documentation: Following information is noted in PR-32

• Manufacturer, Catalogue No. and  Expiry of Media
• Date used  

  II.          Trypsin-EDTA (Ready to use )

·       Material: Trypsin-EDTA (SIGMA), Pipette, Peptone broth, Thioglycolate broth, Sabraud agar slant.

·       Sterility testing Procedure:

o   Put the sterility in peptone, Thioglycolate broth & Sabraud agar slant before use.

o   Store it at -20⁰C in Deep freeze no.DF-8.

·       Documentation: Following information is noted in PR-32

• Manufacturer, Catalogue No. and  Expiry of Media
• Date used  

2.    Safety conditions

·       To avoid contamination, every time wash hands properly before and after preparing media.

·       Check glassware for proper washing and autoclaving to reduce chances of toxicity or contamination.

·       Pour ingredients carefully, mix properly and close lid tightly and wrap Para film.

·       Store media at proper temperature.

3.    Documentation

·       Media Preparation register PR-32.

4.    References used to draw SOP

·       WHO Polio Laboratory Manual.

·       Documentation: Following information is noted in PR-32

• Manufacturer, Catalogue No. and  Expiry of Media
• Date used  

     I.          PBS with Ca⁺⁺ and Mg⁺⁺ for sample process (Ready to use) 100ml

·       Materials required: Pipette, Spirit swab, PBS with Ca⁺⁺ and Mg⁺⁺ and  Penicillin + Streptomycin

·       Procedure: Add 2 ml penicillin ₊ Streptomycin in each 100ml PBS Bottle. Keep it at 2-8°C_.

·       Documentation: Following information is noted in PR-32

• Manufacturer, Catalogue No. and  Expiry of Media
• Date used  

  II.          Trypsin-EDTA (Ready to use )

·       Material: Trypsin-EDTA (SIGMA), Pipette, Peptone broth, Thioglycolate broth, Sabraud agar slant.

·       Sterility testing Procedure:

o   Put the sterility in peptone, Thioglycolate broth & Sabraud agar slant before use.

o   Store it at -20⁰C in Deep freeze no.DF-8.

·       Documentation: Following information is noted in PR-32

• Manufacturer, Catalogue No. and  Expiry of Media
• Date used  

2.    Safety conditions

·       To avoid contamination, every time wash hands properly before and after preparing media.

·       Check glassware for proper washing and autoclaving to reduce chances of toxicity or contamination.

·       Pour ingredients carefully, mix properly and close lid tightly and wrap Para film.

·       Store media at proper temperature.

3.    Documentation

·       Media Preparation register PR-32.

4.    References used to draw SOP

·       WHO Polio Laboratory Manual.

·       Documentation: Following information is noted in PR-32

• Manufacturer, Catalogue No. and  Expiry of Media
• Date used  

     I.          PBS with Ca⁺⁺ and Mg⁺⁺ for sample process (Ready to use) 100ml

·       Materials required: Pipette, Spirit swab, PBS with Ca⁺⁺ and Mg⁺⁺ and  Penicillin + Streptomycin

·       Procedure: Add 2 ml penicillin ₊ Streptomycin in each 100ml PBS Bottle. Keep it at 2-8°C_.

·       Documentation: Following information is noted in PR-32

• Manufacturer, Catalogue No. and  Expiry of Media
• Date used  

  II.          Trypsin-EDTA (Ready to use )

·       Material: Trypsin-EDTA (SIGMA), Pipette, Peptone broth, Thioglycolate broth, Sabraud agar slant.

·       Sterility testing Procedure:

o   Put the sterility in peptone, Thioglycolate broth & Sabraud agar slant before use.

o   Store it at -20⁰C in Deep freeze no.DF-8.

·       Documentation: Following information is noted in PR-32

• Manufacturer, Catalogue No. and  Expiry of Media
• Date used  

2.    Safety conditions

·       To avoid contamination, every time wash hands properly before and after preparing media.

·       Check glassware for proper washing and autoclaving to reduce chances of toxicity or contamination.

·       Pour ingredients carefully, mix properly and close lid tightly and wrap Para film.

·       Store media at proper temperature.

3.    Documentation

·       Media Preparation register PR-32.

4.    References used to draw SOP

·       WHO Polio Laboratory Manual.

·       Name of the reference material:

o   NIBSC – Poliovirus reference sabin type 1 (01/528).

o   NIBSC – Poliovirus reference sabin type 2 (01/530).

o   NIBSC – Poliovirus reference sabin type 3 (01/532).

·       Characterization: - Standard sabin virus P1, P2, & P3.

·       Handling: - Handled by Laboratory technicians

·       Receiving: - Date of receiving entered in stock register.(30/01/2003)

·       Storage : - 20⁰C

·       Use:- These are used in preparation of laboratory quality control standards mentioned in

SOP No.5.2, which is further used for evaluating cell culture sensitivity by titration of        these standard prepared.