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Total Viable Spore Count Test for Biological Indicators

Total Viable Spore Count Test for Biological IndicatorsObjective

To lay down the procedure for Total Viable Spore Count and Resistance Performance test for Biological Indicators

 Scope

This SOP is applicable for Total Viable Spore Count and Resistance Performance test for Biological Indicators of (Pharmaceutical Company Name).

  • Responsibility
    • Chemist or above of QC laboratory.
    • Head – Microbiology Section.
  • Accountability
    • Head – Quality Control.
  • Abbreviations and Definitions

SOP                             : Standard operating procedure

No.                              : Number

QC                              : Quality Control

QA                              : Quality Assurance

BI                                : Biological Indicator

  • Procedure
    • Biological Indicator organisms: Geobacillus stearothermophillus, ATCC 7953 for steam sterilization validation, Bacillus subtilis, ATCC 9372 for dry heat sterilization validation.
    • On receipt of Biological indicators, check for the certificate of analysis, manufacturing date, expiry date, labeled spore count and ‘D’ value. Store as per the manufacturer’s instructions.
    • Total viable spore count:
      • Strips:
        • Aseptically remove three specimens of the relevant biological indicator strips from their original individual containers using a sterile forceps.
  • Disperse the strips into component fibers by placing the test specimens in a sterile 250 ml screw capped bottle containing 100 ml of chilled, sterile purified water and mixing.
  • Ampoules:
    • Take three ampoules of the relevant biological indicators.
    • Open and add the liquid containing BI spores in a sterile 250 ml screw capped bottle containing 100 ml of chilled, sterile purified water.
  • Stir it on vortex mixer for 15 minutes to achieve a homogenous suspension.
  • Transfer a 10 ml aliquot of the suspension to a sterile, screw-capped test tube.
  • For Geobacillus stearothermophillus, heat the tube containing the suspension in the water bath at 95oC to 100oC for 15 minutes (heat shock), starting the timing when the temperature reaches 950 C.
  • For Bacillus subtilis, heat tube containing the suspension in the water bath at 80oC to 85oC for 10 minutes (heat shock), starting the timing when the temperature reaches 800 C.
  • Cool rapidly in ice water.
  • Transfer 1 ml aliquots in duplicate to tubes containing 9 ml of sterile purified water.
  • Make appropriate 10 fold serial dilutions in sterile purified water.
  • The dilutions being selected as calculated to yield preferably 30-300 colonies, but not less than 6, on each of a pair of agar plates.
  • Prepare a separate series of plates for each aliquot.
  • Place 1 ml of each selected dilution in each of two sterile petri dishes.
  • Within 20 minutes, add to each plate 20ml of Soybean-Casein Digest Agar Medium, which has been melted and cooled to 40 oC – 45oC. Swirl to attain homogenous suspension, and allow to solidify.
  • Incubate the plates in an inverted position at 55oC-60oC for Geobacillus stearothermophillus and 30 oC – 35oC for Bacillus subtilis.
  • Examine the plates after 24 and 48 hrs. in inverted position.
  • Record the number of colonies for each plate and use the dilution of plate, which shows 30 – 300 colonies for calculating the results.
  • Calculate the average No. of spores per carrier from the results.
  • The requirements of the tests are met if the average number of viable spores per carrier is not differ by a factor greater than 2 from the labeled spore count per carrier
  • The test is valid if the log number of spores per Carrier at 48 hours is equal to or greater than the log number after 24 hours in each case.
  • Record all the test results in the format given as Annexure No.
  • Decontaminate and dispose the used plates by following SOP No.
  • Forms and Records (Annexures)
    • Not applicable
  • Distribution
    • Master copy       –       Quality Assurance
    • Controlled copies  –       Quality Assurance, Production, Quality Control, Stores, Engineering and Human Resource
  • History:
Date Revision Number
Reason for Revision
00   NEW SOP

 

 

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About Pharmaceutical Guidanace

Mr. Shiv Kumar is the Author and founder of pharmaceutical guidance, he is a pharmaceutical Professional from India having more than 14 years of rich experience in pharmaceutical field. During his career, he work in quality assurance department with multinational company’s i.e Zydus Cadila Ltd, Unichem Laboratories Ltd, Indoco remedies Ltd, Panacea Biotec Ltd, Nectar life Science Ltd. During his experience, he face may regulatory Audit i.e. USFDA, MHRA, ANVISA, MCC, TGA, EU –GMP, WHO –Geneva, ISO 9001-2008 and many ROW Regularities Audit i.e.Uganda,Kenya, Tanzania, Zimbabwe. He is currently leading a regulatory pharmaceutical company as a head Quality. You can join him by Email, Facebook, Google+, Twitter and YouTube

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