Preparation of Sterility testing Media

1.     Scope

·       This sop explains preparation of sterility testing media.

2.     Definition

·       D.D.W             =          double distilled water

·       ml                    =          milliliter

·       gm                   =          gram

3.    Material

·       Peptone powder,

·       Thioglycollate powder,

·       Sabouraud’s agar powder,

·       Pipette (10ml)

·       Double distilled water,

·       whatman filter paper,

·       test tubes with cap,

·       weighingMachine,

·       Autoclave,

·       Test tube rack & flask.

4.     Procedure

A.    Peptone Broth:

·       Composition:

o   Peptone           -           7.5gm

o   D.D.W             -           500ml

o   Filter paper

·       Preparation

o   Take 500ml D.W. in a sterile glass flask.

o   Weight 7.5gm of peptone powder.

o   Dissolve peptone powder into D.D.W

o   Filter it with whatman filter paper.

o   Distribute 5 ml media with pipette into screw cap test tubes.

o   Keep the caps slightly loose.

• Autoclave it at 121°Ć and 15 1bs pressure for 30min.
• After  autoclaving tighten the screw cap, cool it & keep in refrigerator (4Ċ to 8Ċ)
• Keep one tube of Peptone broth at room temperature and one at 36Ċ for sterility test.
•  Observe if for seven days for appearance for turbidity.
•  If there is no turbidity after seven days the batch is ready for sterility testing of media.
• If turbidity appears discard the whole batch by autoclaving and prepare fresh media.

A.    Thioglycollate Broth:

·       Composition:

o   D.D.W                                     500ml

o   Thioglycollate Powder           14.9gm

·       Preparation

• Take 500ml D.D.W in a sterile glass flask.
• Weigh 14.9 gm of Thioglycollate powder, add to DDw and mix well.
• Heat on a burner till the color changes from blue to orange yellow.
• Cool it and distribute 5ml with pipette in screw cap test tubes.
• Keep the cap slightly loose.
• Autoclave at 121°Ċ and 15 lbs pressure for 30 min.
• Keep one tube of thioglycollate broth at room temperature and one at 36°Ċ for sterility test.
• Observe it for seven days for appearance of turbidity.
• If no turbidity appears after 7 days the batch is ready for use.
• If turbidity appears discard the whole batch by autoclaving and prepare fresh media.

B.    Sabouraud’s Agar:

·                 Composition:

o   D.D.W             -           500ml

o   Saboraud powder -      32.5gm

·       Preparation

• Take 500ml D.D.W in a flask.
• Weigh the Saboraud Agar powder.
• Dissolve it in flask containing D.D.W.
• Heat on a burner till the Agar is dissolved.
• Distribute 4ml with pipette in screw cap test tubes.
• Keep the cap slightly loose.
• Autoclave at 121 C for 30 min.
• After autoclaving tighten the screw cap and keep the tubes in a slating position till the medium solidifies.
• Keep the medium in refrigerator. (4⁰C to 8⁰C)
• Keep one tube of sabouaraud agar at room temperature and one at 36 C
• For seven days.
• Observe daily for appearance of fungus.
• If there is no growth of fungus the batch is ready for use.
• If fungus grows discard the whole batch and prepare fresh medium.

1.    Safety conditions

·        Add required amount of media in each tube.

·        Check the autoclave cycle is proper.

2.    Documentation

·        Media preparation register PR-32

3.    References used to draw SOP

·       WHO Polio Laboratory Manual.