Receiving, Issuance and Verification of Biological Indicators-SOP

Biological indicators SOP | Biological Indicator Testing Procedure |

• Biological indicators SOP covers below points :
  • Identification of Biological indicator used for steam sterilization (Strips and Ampoule)
  • Identification of Biological indicator used for sterilization
  • Total Viable Spore Count in Biological Indicator
  • Biological Indicator spore population determination format

1.0 OBJECTIVE :

• To lay down the procedure for receiving, issuance and determination of spore population of biological indicator which are commercially available and are used in validation process of steam sterilizer.

2.0 SCOPE :

• The scope of this SOP is applicable for receive, issue and verify spore population of biological indicator used in validation process of steam sterilizer.

3.0 RESPONSIBILITY :

• Microbiologist

4.0 ACCOUNTABILITY:

• Head of Department

5.0 PROCEDURE :

5.3 Identification of Biological indicator used for sterilization:

• Take one strip of biological indicator Baciilus atrophaeus ATCC 9372
• Aseptically remove the glass line paper from the biological indicator and inoculate strip of the biological indicator into 10 ml of Soyabean casein Digest Medium (SCDM) under LAF.
• Incubate the tube containing the biological indicator at 30ºC to 35ºC for 18 to 24 hours.
• After incubation pipette 1 ml from the biological indicator suspension into each of the four sterile Petri plate.
• Pour approximately 15 ml to 20 ml of autoclaved media Soyabean casein Digest agar Media (SCDA), previously cooled to 40ºC to 45ºC.
• Swirl to mix the media in the Petri plate.
• Allow the media to solidify and incubate two media Petri plate at 55ºC to 60ºC for 24 to 48 hours and other two media Petri plate at 30ºC to 35ºC for 24 hours.
• The media Petri plate incubated at 55ºC to 60ºC shall show no evidence of growth, media Petri plate incubated at 30ºC to 35ºC shall show evidence of growth.
• Prepare the slide of the organism and observe microscopically.
• The microscopic examination of the organism shall show Gram-positive rods with central oval endospores.
• Record the result in the Record for Biological Indicator spore population determination.

5.4 Total Viable Spore Count in Biological Indicator:

• Randomly select approximately four spore strips of Biological Indicator from its original individual container.
• Place each spore strip in a sterile, tube with three  approximately 6 mm glass beads, and 5.0 ml of sterile purified water.
• Vortex approximately 4 to 7 minutes until the paper carrier is macerated to pulp.
• Add 5.0 ml sterile purified water. Vortex again.
• Transfer 1 ml of the suspension to a test tube containing 9 ml of sterile purified water & vortex.
• Place the dilution tube in a preheated bath at 95ºC – 100ºC for 15±1 minutes for Geobacillus stearothermophilus and  for Bacillus atrophaeus  80ºC – 85ºC for 10 minutes.
• Remove tubes and cool rapidly in an ice bath (0ºC – 4ºC).
• Vortex each heat-shocked tube for at least ten seconds.
• Now serially dilute these tubes to yield 30 to 300 colonies. Vortex each dilution tube for at least ten seconds.
• Prepare a separate series of plates for each aliquot.
• Place 1.0 ml of each selected dilution in each of two Petri dishes.
• Pour approximately 20 ml of melted Soybean Casein Digest Agar cooled to 45º to 50ºC into the petri plates. Swirl to assure adequate mixing and  allow the agar to solidify.
• Invert and incubate plates at 55º – 60ºC for Geobacillus stearothermophilus & for Bacillus atrophaeus 30ºC – 35ºC for 48 to 72 hours.
• Units incubated at 55ºC to 60ºC may be placed inside a plastic bag to prevent the plates from excessive drying.
• After incubation count CFU.
• Average counts and then multiply by the dilution factor to calculate population per original unit.
• Record the result in  “Record for Biological Indicator spore population” determination.
• For Biological indicators, sealed ampoule, take approximately 4 ampoules in a sterile 250 ml conical flask and break the ampoules by shaking against the sides of the flask.
• Add sufficient sterile purified water to make 100 ml & vortex vigorously to achieve a homogeneous suspension.
• Pipette out 10 ml of the aliquot from the conical flask into a sterile test tube.
• Place the dilution tube in a preheated bath at 95ºC – 100ºC for 15±1 minutes for Geobacillus stearothermophilus and  for Bacillus atrophaeus  80ºC – 85ºC for 10 minutes.
• Remove tubes and cool rapidly in an ice bath (0ºC – 4ºC).
• Vortex each heat-shocked tube for at least ten seconds.
• Now serially dilute these tubes to yield 30 to 300 colonies. Vortex each dilution tube for at least ten seconds.
• Prepare a separate series of plates for each aliquot.
• Place 1.0 ml of each selected dilution in each of two Petri dishes.
• Pour approximately 20 ml of melted Soybean Casein Digest Agar cooled to 45º to 50ºC into the petri plates. Swirl to assure adequate mixing and  allow the agar to solidify.
• Invert and incubate plates at 55º – 60ºC for Geobacillus stearothermophilus & for Bacillus atrophaeus 30ºC – 35ºC for 48 to 72 hours.
• Units incubated at 55ºC to 60ºC may be placed inside a plastic bag to prevent the plates from excessive drying.
• After incubation count CFU.
• Average counts and then multiply by the dilution factor to calculate population per original unit.
• Record the result in  “Record for Biological Indicator spore population” determination.

5.5 The frequency of the testing shall be as follow:

• The analysis for each lot of newly received spore strips / ampoule shall be Carried out.
• If the newly received lot is not used within six months, the analysis shall be carried out before usage.

5.6 The requirements for biological spore count test are met if the log average number of viable spores per carrier is not less than 0.3 log of the labeled spore count per carrier and does not exceed the log labeled spore count per carrier by 0.48.

5.7 After completion of Identification and Verification Biological Indicator can be issued when required for validation activities. Record the details of the issuance “Record for Receiving and Issuance of Biological Indicator”.

6.0 ABBREVIATIONS:

Abbreviation	Expanded form
SCDM	Soyabean Casein Digest Medium
SCDA	Soyabean Casein Digest Agar
SCA	Sabouraud Chloramphenicol Agar
SOP	Standard Operating Procedure
°C	Degree Celsius
ml	Milliliter
cfu	Colony Forming Unit
LAF	Laminar Air Flow
BI	Biological Indicator
ATCC	American Type Culture Collection

7.0 ANNEXURES:

Annex. No.	Title
01	Record for Biological Indicator spore population determination
02	Record for Receiving and Issuance of Biological Indicator

8.0 SOP REFERENCES:

• Indian Pharmacopeia

END OF THE SOP

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ANNEXURES :

Annex. No. 01 Record for Biological Indicator spore population determination

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Annex. No. 02 Record for Receiving and Issuance of Biological Indicator

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