Media and Reagent Preparation for Polio virus detection-Part-1

1.     Scope

·       This SOP describes the preparation of Media from their contents and reagents used in the lab.

Media and reagents used in polio laboratory are,

o   Minimum Essential Media – Growth medium (10%) and Maintenance medium (2%)

o   Phosphate buffered saline without Ca++ and Mg++ for cell line

o   Phosphate buffered saline with Ca++ and Mg++ for sample processing

o   L-glutamine

o   HEPES Buffer (N-2 hydroxyethyl piperazine N-2 etnanesulphonic acid)

o   7.5% NaHCO₃

o   Phenol Red

o   MEM 10% is used as a growth medium in both cell lines and MEM 2% is used as maintenance medium in both cell lines.

o   PBS without CaCl₂ and MgCl₂ is basic salt solution and is used to remove PBS from cells prior to cell detachment. It makes cell detachment easier.

o   PBS with CaCl₂ and MgCl₂ is used in sample processing to stabilize virus.

o   L-glutamine is amino acid and is used in MEM for growth of cells in tissue culture flask, tubes and plate.

o   HEPES buffer is used in MEM growth medium and maintenance medium. It maintains the pH of  the medium ( pH range- 7.2 to 7.4 )

o   7.5% NaHCO3 together with gaseous CO₂ provides buffering substance for many cell culture media. It’s also an essential metabolite.

o   Phenol Red is used as pH indicator in cell culture media. 

2.     Definition

·       MEM              -           Minimum Essential Medium

·       PBS                 -           Phosphate buffer saline

·       HEPES            -           N-2 hydroxyethyl piperazine N-2 ethenesulphonic acid

·       NaHCO₃          -           Sodium bicarbonate

·       Ml                   -           Milliliter

·       DDW               -           Double Distilled Water

·       ⁰C                    -           Degree centigrade

·       Min.                -           Minutes

·       P+S                 -           Penicillin + streptomycin 

·       FBS                 -           Fetal bovine serum

1.    Procedure

     I.          MEM(Minimum essential medium Eagle-with Earle’s salts) 

·       Materials: Media bottle, Pipettes, Distilled water, Ingredients, Biological safety cabinet, Spirit swab, measuring cylinder, Peptone broth, Thioglycolate Broth, Sabouraud agar slant.

1.     10% growth media:-

a.     Take one-liter Media bottle and add 855ml-distilled water. (18.2 M cm )

b.     Weigh 9.39 gms MEM powder and dissolve contents in 855ml double distilled water and autoclave at 121⁰C for 30 min.

c.      Allow it to cool, add ingredients as below by pipette. Use different pipette for each ingredient.

o                Penicillin + Streptomycin                                         10ml

o                L – Glutamine (3%)                                                  10ml 

o                Hepes Buffer                                                             10ml

o                7.5% NaHCo3                                                           15ml

o                Phenol red                                                                  02ml

o                FBS                                                                           100ml

d.     After adding these ingredients put the sterility test of the prepared media in Peptone broth, Thioglycolate broth, Saboraud‘s agar slant & plain tube as in sop No.4.

e.     Check the sterility of media till 7 days at 37° C and Room temp.  After 7 days, if sterility is O.K, then medium is ready for use.

f.      Documentation – Following information is noted for MEM:

·            Manufacturer, Catalogue No. and  Expiry of Media             

·            Date used                                  

                  

2.     2% growth media:-

a.     Take one liter Media bottle and add 925ml distilled water ( 18.2 M Ωcm )

b.     Weigh 9.39 gm powder and dissolve contents in 925ml double distilled water and autoclave at 121°C for 30 min.

c.      After cooling, add ingredients as below by pipette. Use different pipette for each ingredient.

o   Penicillin + Streptomycin      10ml            

o   L- Glutamine (3%)                 10ml

o   Hepes Buffer                          10ml

o   7.5% NaHCO₃                        25ml

o   Phenol red                               02ml

o   FBS                                         20ml

b.     After adding these ingredients, put the sterility test of the prepared media in Peptone, broth, Thioglycolate broth, Saboraud’s agar slant & plain tube as in sop No. 6.3

c.      Check the sterility of media till 7 days at 37°C and room temperature. After 7 days, if sterility is O.K, then medium is ready for use.

d.     Documentation – Following information is noted in PR-32

·       Manufacturer, Catalogue No. and  Expiry of Media             

·       Date used                                  

     I.          L-Glutamine (3%)

·       Materials: L – Glutamine powder, Weighing machine, Brown bottles,sterile cap, Oven, Sterile 0.22µ membrane filter with receptor, Peptone broth, Thioglycolate Broth, Sabouraud agar slant, Flask & D.D.W.

·       Procedure:

o   Take 100ml D.D.W in sterile flask.

o   Weigh 3gm powder of L-Glutamine and dissolve it.

o   After dissolving, filter using sterile Millipore Membrane filter of porosity 0.22 µ.

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

o   Distribute 10ml media in sterile brown bottle, cap it and cover with foil.

o   Freeze it at -20° temp.

·       Documentation: Following information is noted in PR-32

• Manufacturer, Catalogue No. and  Expiry of Media             

Date used