Standard Operating Procedure for Testing of population count of Biological Indicator
1.0 Purpose:
To lay down the standard operating procedure for testing of Biological indicator and used for sterilization validation.
To lay down the standard operating procedure for testing of Biological indicator and used for sterilization validation.
2.0 Scope:
This procedure is applicable for testing of Biological indicator (Spore Population Verification) and testing of Biological indicator exposed to sterilization validation in Microbiology Laboratory.
3.0 Responsibility:
Officer/ Designee Microbiology:
•To responsible for verification of spore population on receipt as per procedure.
•To responsible for use of Biological indicator for sterilization validation as per procedure.
•To maintain format as per procedure.
Executive/ Designee Microbiology:
•To check the format as per procedure.
•To check the format as per procedure.
•To review, implement and compliance of SOP.
Head/ Designee QA:
•To approve, implement and compliance of SOP.
•To approve, implement and compliance of SOP.
4.0 Procedure:
4.1 Receipt:
4.1.1 On receipt of the biological indicator, check the Lot no. and Exp. date on container and record the details in format.
4.1.2 After receipt, record all the details on label as per format.
4.1.3 Ensure that the Certificate of analysis is received along with the lot of biological indicators and check the certificate for the following information:
4.1.3.1 The name of the organism along with the ATCC number from which the spores are derived.
4.1.3.2 Batch number/Lot number
4.1.3.3 The total viable spore count or mean population per unit of biological indicator.
4.1.3.4 ‘D’ value and the method used to determine ‘D’ value (Decimal Reduction Value)
4.1.3.5 ‘Z’ value.
4.1.3.6 Manufacturing date
4.1.3.7 Expiry date
4.1.3.8 Storage specifications
4.1.3.9 Disposal procedure
4.1.3.10 Bacteriological medium which will meet the requirement for growth promoting ability.
4.1.3.11 Conditions under which reproducible resistance characteristics are obtained.
4.1.3.12 Survival time and kill time under specified conditions.
4.1.4 Store the biological indicators between 15 to 25°C
4.2 Performance Check:
4.2.1 Randomly pick 3 ampoules from each Batch received. Under aseptic conditions, cut open these ampoules with ampoule cutter. Pool the contents in a sterile 250 ml conical flask. Add chilled sterilized purified water to make 100 ml and mix for 3 to 5 minutes to achieve a homogeneous suspension.
OR
Randomly pick 3 strips from each Batch received. Under aseptic conditions, remove the strips from the individual envelope. Place these strips in 250 ml sterile conical flask containing 100 ml of chilled sterilized purified water and sterile glass beads. Sonicate until the paper carrier is macerated to achieve a homogeneous suspension
4.2.2 Transfer a 10 ml aliquot to a sterile tube and place the tube in a water bath at 95°C to 100°C for 15 minutes (Heat Shock), starting the timing when the temperature reached 90°C. Cool rapidly in an ice water bath (0°C to 4°C).
4.2.3 Transfer to 1 ml aliquots to suitable tubes, and make appropriate serial dilution in sterilized purified water to yield 30 to 300 colonies when plated. The serial dilutions are prepared as per the label claim.
4.2.4 Place 1 ml of each selected dilution into 2 sterile petriplates. Within 20 minutes, add to each plate 15-20 ml of Soyabean Casein Digest Agar Medium which has been melted and cooled to 45°C to 50°C. Mix well and allow to solidify.
4.2.5 Incubate the plates in an inverted position at 55°C to 60°C for 24 to 48 hours. Examine the plates for evidence of contamination with other microorganisms.
4.2.6 Count the number of colonies.
4.2.7 Calculate the average of the number of viable spores per container by multiplying by dilution factor to the total colonies.
4.2.8 Calculate the average of the number of viable spores per ml by this formula.
AV x DF = CFU /ml.
Where,
AV = average no. of spore & DF = dilution factor.
AV = average no. of spore & DF = dilution factor.
AV X DF
Therefore , CFU /ml = ---------------------------------------------
Total quantity in the container.
Therefore , CFU /ml = ---------------------------------------------
Total quantity in the container.
4.2.9 Frequency:
4.2.9.1 Check the performance of each fresh lot of received biological indicators.
4.2.9.2 Retest for total viable count per ampoule should be carried out after one year ± 30 days.
4.3 Use of Biological Indicator for Sterilization Validation:
4.3.1 Place three biological indicator strip /ampoules at different positions within the autoclave chamber and carry out the sterilization cycle.
4.3.2 On completion of sterilization, remove the biological indicator strips aseptically take out the strip, transfer into sterile 100 ml Soyabean Casein Digest medium and incubate at 55-60°C for 7 days along with an unexposed strip as positive control OR ampoules direct incubate at 55-60°C for 7 days along with an unexposed ampoule as positive control.
4.3.3 After completion of incubation period should not show turbidity in SCDM medium and positive control shows turbidity.
4.3.4 After completion of incubation period ampoule should not show a change in colour from purple to yellow and positive control change in colour purple to yellow.
4.3.3 Record the observations in the format.
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