DEFINITION:
Biological Indicator: A biological indicator is defined as a characterized preparation of specific microorganism that provides a defined and stable resistance to a specific sterilization process..
Biological Indicator for steam sterilization: Biological indicator for steam sterilization is a defined preparation of viable spores made from a culture derived from a specified strain of Geobacillus stearothermophilus ATCC 7953(earlier known as Bacillus stearothermophilus) in the form of paper carrier, self-contained liquid spore suspensions (ampoule) or self-contained vials.
Biological Indicator for dry heat sterilization: Biological indicator for dry heat sterilization is a defined preparation of viable spores made from a culture derived from a specified strain of Bacillus atrophaeus ATCC 9372 (Earlier known as Bacillus subtilis var. niger) in the form of paper carrier.
Biological Indicator for vapour-phase hydrogen peroxide (VPHP) decontamination: Biological indicator for VPHP decontamination is a defined preparation of viable spores made from a culture derived from a specified strain of Geobacillus stearothermophilus ATCC 7953 (earlier known as Bacillus stearothermophilus) in the form of glass/ metal carriers (gas impervious carrier systems).
PROCEDURE:
Procedure for Receipt and Storage of Biological Indicators :
Procure the biological indicators, to the extent possible, based on the regular consumption in such a way that the indicators shall not last for more than one year.
On receipt of biological indicators, ensure that the Certificate of analysis (COA) is received along with each lot of the biological indicators.
Verify the details given in the COA with that of the label information on the biological indicators. Also verify the indicators physically for the packing condition of primary overwrap and for integrity of the container especially in case of self-contained ampoules.
Review the COA for the correctness and completeness of information like Manufacturer’s name, name of the organism, ATCC strain number from which the spores are derived, Lot number / batch number, total viable spore count or mean population per indicator, D-value and the method used to determine D-value, Z value, expiry date, storage conditions, recovery media, survival time/ kill time data and the disposal procedure.
Verify the information like the spore counts, D-value, survival times/ kill times given in the COA against the data given in Table-I.
If the information given on the COA and label information of the indicators complies with the data given in Table-I, accept the lot of biological indicators. After verification of COA stamp “Verified by with sign and date”
Count the number of Biological Indicators (BI) and enter the details of receipt in the ‘Biological Indicator Stock Card’. File the COA along with the ‘Biological Indicator Stock Card’.
Note: After receipt of biological indicator count the number of biological indicators from all the packs and enter the total quantity onto the stock card.
In case of any discrepancy in the information given on the COA and on the label, or any packing integrity issues, inform the manufacturer/ supplier and reject the consignment.
Based on the manufacturer/supplier’s comments and written justification, and based on the risk assessment, the consignment of indicators may be accepted. Documents containing the written justification from the manufacturer/ supplier and the risk assessment shall be filed along with the stock card of indicators.
Paste a ‘BI Receipt and Enumeration details’ label as below given label on the outer cover of the indicators. Update the applicable details like ‘Lot number of BI’, ‘Name of the microorganism/ ATCC strain number’, ‘Expiry date’ and ‘Date of receipt and signature of the analyst’.
After completion of enumeration, update the date of enumeration and sign on the same.
Store all the biological indicators as per the manufacturer/ supplier’s instructions
Table-1: Typical Characteristics of Commercially Supplied Biological Indicators
Sterilization Mode
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Range of Spore Count per Indicator
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Range of
D-values (minutes)
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Limits for a Suitable Resistance
(depending on the particular D-value) (minutes)
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Survival Time
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Kill Time
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Moist Heat at 121°C
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1.0 × 105
to 5.0 × 106
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Min. 1.5
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Min. 4.5
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13.5
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Max. 3.0
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Max. 14.0
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32.0
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Dry heat at
160 ° C
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1.0 × 106
to 5.0 × 106
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Min. 1.0
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Min. 4.0
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10.0
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Max. 3.0
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Max. 14.0
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32.0
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VPHP
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1.0 × 105
to 5.0 × 106
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Min. 1.0
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Min. 4.0
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10.0
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Max. 3.0
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Max. 14.0
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32.0
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Note: Low population and D value level biological indicators also can be used manufacturing process / validation and based upon the product nature. However the receipt, enumeration, identification and usage shall be followed as same as other biological indicators.
Determination of Total Viable Spore Count of Biological Indicators:
Biological Indicator for Steam Sterilization (Self-Contained-Liquid Spore Suspensions -Ampoules):
Randomly pick 4 ampoules from each lot of the indicators received. Place in 250 mL conical flask. Aseptically break the ampoules with sterile spatula under laminar air flow.
Add chilled sterilized purified water to make 100 mL and mix for about 15 minutes to achieve a homogeneous suspension.
(1)Transfer a 10 mL aliquot to a sterile screw cap 16 × 125 mm tube and place the tube in a water bath at 95°C to 100°C for 15 minutes for (heat shock),starting the time when the temperature reaches 95°C and 10 minutes at 80 to 85 ºC for Bacillus subtilis (heat shock) starting the time when the temperature reaches 80°C .
Cool rapidly in an ice water bath (0°C to 4°C).Transfer 1 mL aliquot to suitable tube in duplicate and prepare appropriate serial dilutions in sterilized purified water to yield 30 to 300 colonies(but not less than 6,) when plated. Prepare a separate series of plates for each aliquot. Prepare the serial dilutions as per the label claim.Transfer 1 mL each from the selected dilution into each of the two 15 × 100 mm sterile Petri dishes. Within 20 minutes, add about 20 mL of Soyabean Casein Digest Agar (pre-sterilized and cooled to about 45°C to 50°C) to each of the plate. Swirl to attain homogeneous suspension and allow it to solidify.
Incubate the plates in an inverted position at 55 to 60°C for Geobacillus stearothermophilus and 48 º to 52º C for Bacillus subtilis for 48 hours or at the optimal recovery conditions as specified by the Manufacturer. Examine the plates for evidence of any contamination with other microorganisms.Count the number of colonies after 48 hours, Calculate the average of the number of viable spores per container using an appropriate dilution factor. The requirements of the tests are met if the average number of viable spores per carrier should be not less than 50% of the labeled spore count per carrier and does not exceed the labeled spore count per carrier by 300%.
After completion of observation Calculate the Result as per below:
(Aliquot 1) Count of plate 1 + plate 2 + (Aliquot 2) Count of plate 1 + plate 2
1) Average CFU = ---------------------------------------------------------------------------------------------
4
2) CFU per 0.1 mL spore suspension (For Self-Contained-Liquid Spore Suspensions: Ampoules) = Average CFU X Dilution factor
3) CFU per unit = Average CFU X Dilution factor
---------------------------------------
Number of BIs tested
Calculated Average Population of Sample
4) Percent recovery = -------------------------------------------------- ×100
Labeled population on certificate
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Biological Indicator for Steam Sterilization (Paper Carrier):
Randomly pick 3 strips from each lot of the indicators received. Remove the strips from the individual overwraps aseptically and place them in a 250 mL sterile conical flask containing 100 mL of chilled sterilized purified water and sterile glass beads.
Vortex until the paper carrier is macerated to achieve a homogeneous suspension.
Proceed further as per the (1)
Biological Indicator for Steam Sterilization (Self-Contained Paper Carrier):
Randomly pick 4 containers from each lot of the indicators received. Remove the spore strip from the containers aseptically and place them in a 250 mL sterile conical flask containing 100 mL of chilled sterilized purified water and sterile glass beads.
Vortex until the paper carrier is macerated to achieve a homogeneous suspension.
Proceed further as per (1)
Biological Indicator for vapor-phase hydrogen peroxide (VPHP) decontamination (Stainless Steel Carrier)
Randomly pick 4 units from each lot of the indicators received. Remove the strips from the individual overwraps aseptically and place them in a 250 mL sterile conical flask containing 100 mL of chilled sterilized purified water and sterile glass beads.
Sonicate or Vortex about 15 minutes to achieve a homogeneous suspension.
Proceed further as per the (1)
Biological Indicator for Dry Heat Sterilization
Randomly pick 4 ampoules from each lot of the indicators received. Place in 250 mL conical flask. Aseptically break the ampoules with sterile spatula under laminar air flow.
Add chilled sterilized purified water to make 100 mL and mix for about 15 minutes to achieve a homogeneous suspension.
Transfer a 10 mL aliquot to a sterile screw cap tube and place the tube in a water bath at 80°C to 85°C for 10 minutes. Cool rapidly in an ice water bath (0°C to 4°C).
Proceed further as per(1)
Determination of Cultural Characteristics and Identification:
Observe the colonies on the plates for homogeneity of the culture and for any signs of contamination.
Identification shall be performed as by MICROBIAL IDENTIFICATION SYSTEM..
In case of any discrepancy observed during identification of microorganism, Investigation shall be carried out..
Handling of Biological Indicators during Validation Studies:
Use the biological Indicators for the validation purposes from the lots for which the spore counts (enumeration) are verified.
Note: The spore counts of biological indicator to be done simultaneously if any validation is plan before releasing of spore count results. The validation results are valid after completion of spore counts results only.
Follow the First in First out procedure (FIFO) during the issuance as far as possible.
Issue the required number of BIs against the requirement from the User department. Verify whether all the relevant details like the type of biological indicators, the quantity required and name of equipment to be qualified are entered on the requisition.
Enter the issuance details like lot number, quantity issued, and expiry date of the lot on the requisition and also enter the issuance details immediately on the relevant columns of the “Biological Indicator Stock Record”.
Issue the indicators only if the Lot has adequate number of indicators sufficient for the one validation run plus at least one for positive control. If a lot has sufficient indicators only for a validation run and no stock for positive control, do not issue these indicators for the validation purpose.
After completion of sterilizing procedure, ensure the exposed indicators are tested (inoculated in medium and incubated) within 4 hours.
During recovery testing, use one unexposed biological indicator from the same lot as a positive control. Enter the issuance of positive control indicator separately in the issuance columns of the ‘Biological Indicator Stock Record’.
Recovery of Exposed Biological Indicator
Receive the exposed indicators in the microbiology lab as quickly as possible.
Verify the location numbers marked on the biological indicators and tally the number of indicators issued versus the number of indicators received after exposure.
Carry out the recovery test in aseptic condition under Biosafety cabinet.
In case paper carrier Mark the sterile Soybean casein digest medium tubes with the location numbers as marked on the exposed biological indicators. Mark one tube as positive control and one tube as negative control. In case of paper carriers, aseptically remove the outer wrap from each of the indicators, transfer the paper carrier in to the tube marked with the corresponding number. Ensure that the paper carriers are immersed in the media completely. Transfer one unexposed indicator in to the tube marked as positive control. Keep the un-inoculated media tube as negative control.
In case of self-contained liquid spore suspensions (ampoules), verify the location numbers on the indicators and directly transfer to the incubator/oven along with an unexposed indicator as positive control. Incubate one ampoule supplied with the lot for the purpose of negative control.
In case of self-contained (paper carrier) indicators, verify the location numbers on the indicators, squeeze the containers gently so as to allow the contact of the exposed indicator with the recovery medium. Squeeze one unexposed container and incubate as a positive control.
Incubation Condition:
Incubate the tubes containing paper carriers/ S.S.carrier of Geobacillus stearothermophilus at 55°C to 60ºC for a period of 7 days or at optimum recovery conditions as specified by the Manufacturer..
In case of Self-contained liquid spore suspensions of Geobacillus stearothermophilus, incubate at 55°C to 60ºC for a period of 48 hours or at optimum recovery conditions as specified by the Manufacturer.
In case of Self-contained paper carriers of Geobacillus stearothermophilus, incubate at 55°C to 60ºC for a period of 24 hours or at optimum recovery conditions as specified by the Manufacturer.
In case of paper carriers of Bacillus atrophaeus, incubate at 30°C to 35ºC for a period of 7 days or at optimum recovery conditions as specified by the Manufacturer.
Interpretation of the results:
Observe the tubes/ containers daily during incubation for evidence of change in colour or turbidity which is indicative of growth.
If growth is observed at any particular observation time, further incubation of the particular specimen is not required.
Note the number of specimens showing no evidence of growth at any observation.
The negative controls shall not show any growth.
Positive controls shall show the evidence of growth.
Read the tubes for evidence of growth/ no evidence of growth in terms of colour change or turbidity or as per the instructions of the Manufacturer.
Handling of abnormal results:
In any of the below cases, raise an incident(Or Proper QMS action) for investigation and disposal.
If growth is observed in the negative control.
If growth is not observed in the positive control.
If the exposed indicators show evidence of growth.
Disposal of Used and/or Expired Biological Indicators
Decontaminate the used and/or expired biological indicators and dispose as per the Manufacturer’s instructions.
Environment, Health and Safety
In case of spillage of the cultures, follow the procedure detailed in the relevant SOP for handling of the spillage. Decontaminate all the used culture suspensions by autoclave cycle before disposal. Wear goggles, hand gloves, during analysis.
ABBREVIATIONS:
Sr. No.
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Abbreviation Used
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Full Form of Abbreviation
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1.
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ATCC
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American Type Culture Collection
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2.
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BI
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Biological Indicators
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3.
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cfu
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Colony Forming Unit
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4.
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°C
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Degree Centigrade
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5.
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COA
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Certificate Of Analysis
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6.
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ID
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Identification
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7.
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IOCP
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Instrument operation & calibration procedure
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8.
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mL
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Milliliter
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9.
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NA
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Not Applicable
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10.
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NMT
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Not More Than
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11.
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Min
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Minimum
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12.
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Max
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Maximum
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13.
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No.
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Number
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14.
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QA
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Quality Assurance
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15.
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QC
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Quality Control
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16.
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SOP
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Standard Operating Procedure
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17.
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Sr.
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Serial
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18.
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TAMC
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Total Aerobic Microbial Count
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19.
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USP
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United State Pharmacopoeia
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20.
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SCDM
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Soyabean casein digest medium
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SPECIMENN LOG
Stock Record
Date of Receipt:
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Lot No./ Batch No.:
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Expiry Date:
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Type of the BI:
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Manufacturer’s Name:
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Storage Temperature:
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Received quantity
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Labelled Spore Count:
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Labelled D-Value:
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Received By:
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Enumeration done on:
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Observed Spore Count:
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Retest Date:
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Reference document No.:
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Recorded By:
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Date
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Quantity Issued
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Issued for the purpose
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Balance Quantity
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Issued By
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Checked By
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Remarks
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Spore Count Report
1. Biological Indicator Details:
Type :
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Manufacturer’s Name:
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Name of the organism:
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ATCC No:
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Lot No / Batch No:
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Expiry Date:
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Mean population labeled
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Date of Testing:
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2. Enumeration details:
Name of the media used:
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Media Lot No.:
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Use Before Date :
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Used Sterile Purified water:
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Sterilization lot No.:
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Number of Units Taken:
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Water bath ID:
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Heat Shock Details:
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Cold shock details:
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Thermometer ID:
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Dilutions Performed Up To :
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3. Media and Incubation Details:
Name of the media used:
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Media Lot No.:
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Use Before Date of Media
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Incubation Temp. (ºC):
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Equipment Id.
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Incubation Start Date/
Time:
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Incubation End Date /
Time:
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Incubation Initiated By: Sign/date
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4. Observations:After 48 Hours
Dilution factor
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Aliquot 1
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Aliquot 2
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Observed By
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Plate 1
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Plate 2
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Plate 1
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Plate 2
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Verified By Sign/Date:
5. Calculations:
Result:
CFU per unit:
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CFU per 0.1 mL spore suspension:
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Percent recovery:
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Remark (If Any):
Conclusion: As per above result Biological indicator comply /does not comply for routine use.
Done By (Sign& Date)
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Checked By (Sign& Date)
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Lable on BI
1)
BB.I. RECEIPT & ENUMERATION DETAILSI
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Lot No. of B.I. :
Name of the Organism/ ATCC No. :
Expiry Date :
Date of Receipt :
S ig n .
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2)
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Recovery of BI.
Date :
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Tested by:
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Name of the Equipment:
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Equipment Id:
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Run No.:
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Type of Load:
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Type of Biological Indicator:
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Lot No. / B.No.:
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Labelled Spore Count:
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Expiry Date:
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Name of the Media Used:
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Media Lot No./Batch No.:
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Incubation Temp. (ºC):
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Incubator Id.:
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Incubation Start Date/Time:
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Incubation End Date /Time
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Incubated By:
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Sr.
No.
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Location of Biological Indicators
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Observation (Day Wise)
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1
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2
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3
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4
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5
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6
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7
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1.
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2.
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NC
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PC
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Observations Taken By:
Sign & Date
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Note: (-Ve) indicates no change in colour (remains purple)/ no turbidity (No growth observed)
(+Ve) indicates change in colour from purple to yellow / turbidity (Growth observed)
(H) Indicates holiday/ weekly off
NC: Negative Control PC: Positive Control
Result: Complies / does not comply.
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