SOP Title: Receiving, Storage and Qualification of Biological Indicator
- Objective:
To lay down the procedure for Receiving, storage and qualification of biological indicator.
- Scope
This procedure is applicable for procedure for receiving, storage and qualification of biological indicator in the Microbiology Section.
- Responsibility:
3.1 Quality Control: To prepare and review the SOP. To follow the procedures for Receiving, storage and qualification of biological Indicators as per this SOP.
- Quality Assurance Department: To review and approve the SOP and Annexure.
- Accountability:
Head Quality Control Department, Head Quality Assurance Department
- Procedure:
| 5.1 | Safety Precautions: | |
| 5.1.1 | Wear personal protective equipments such as protective hand gloves, nose masks and over gown during operation. | |
| 5.1.2 | Microbiologist should disinfect the hand gloves with 70% v/v IPA before carrying out operation. | |
| 5.2 | Procedure for Receiving the Biological Indicators (BI) | |
| 5.2.1 | Check the physical condition of biological indicator strip/ampoules at the time of receiving. | |
| 5.2.2 | Record shall be maintained as per Annexure. | |
| 5.3 | Storage :
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| 5.3.1 | Biological indicators shall be stored in refrigerator at 2-8 0C after receiving the BI or as per suggested by the Supplier. | |
| 5.3.2 | Opened biological indicators shall be stored in refrigerator wrapped with parafilm/Aluminium foil. | |
| 5.3.3 | Record shall be maintained as per Annexure. | |
| 5.4 | Spore count (Label Claim) determination of Biological indicator: | |
| 5.4.1 | Take randomly three paper strips or ampoules of Biological Indicator
Geobacillus stearothermophilus ATCC 7953 (BI) which is used to confirm the efficiency of Steam Sterilization at 121 0C. |
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| 5.4.2 | Tear off the outer covering of BI if it is a paper carrier and transfer it into a single glass tube/bottle containing 100 ml sterile purified water with help of a sterile forceps. In case of Glass ampoules, break the neck under LAF using a SS rod/glass rod which is sterile and transfer the contents of ampoules into a single glass tube/bottle containing 100 ml sterile purified water. | |
| 5.4.3 | Gently vortex the tube about 15 minutes so as to achieve a homogenous suspension with 100 ml sterile purified water. | |
| 5.4.4 | Heat the tube/bottle containing the suspension with the help of water bath at 95–1000 C for about 15 minutes. This shall be considered as Stock suspension. | |
| 5.4.5 | Prepare 0.9% sterile saline solution and dispense in 10 tubes with 9 ml in each tube, Refer SOP No. 012 for reagents preparation. | |
| 5.4.6 | Take 1 ml from stock suspension tube and inoculate into the tube containing 9 ml sterile 0.9% sterile normal saline solution under LAF. This constitute dilution is considered as 10 -1. Shake the tube well. | |
| 5.4.7 | From tube 10 –1, transfer 1 ml into tube 10-2 using a pipette and shake well. Serially transfer 1 ml from tube 10-2 to tube marked as 10-3 and prepare further serial dilutions as 10-3 to 10-4, 10-4 to 10-5 upto 10-7. Record in Annexure No. 01. Refer Annexure No. 03 for dilutions. | |
| 5.4.8 | Prepare Soyabean casein digest agar as per SOP No. 034 | |
| 5.4.9 | Inoculate 1.0 ml suspension from each dilution into plates in duplicate by pour plate method under LAF. | |
| 5.4.10 | After inoculation of 1 ml of suspension into plates, pour 15-20 ml soyabean casein digest agar-medium cooled to 45-50°C in each petriplate. | |
| 5.4.11 | Each Plate has to be marked with dilution number, SCDA Media lot number, Date and Sign. | |
| 5.4.12 | Swirl gently to mix and allow it to solidify. | |
| 5.4.13 | Incubate the plates at 55-60 ºC for 24-48 hrs. Check count at 24 hours and final count after 48 hours. | |
| 5.4.14 | Note: Adjust the Incubator set at 20-25ºC to 55-60ºC for the above incubation as it is validated for 55-60ºC. Monitor the temperature of incubator twice in a day. | |
| 5.4.15 | Examine the plates after 24 hours and 48 hours and record the number of CFU in each plate in Annexure. Take observations of 48 hours for final calculation. | |
| 5.4.16 | Select the dilution for calculation which gives 10-100 CFU count per ml on petriplates. | |
| 5.4.17 | Calculate average number of spores per specimen by dividing the CFU with dilution factor.
Example.: Calculation for BI spore count:- Results of 3 strips of BI = Average spore count Dilution Factor Suppose, Average spore count = 67 CFU for serial dilution 10-3 for 3 strips of BI
Then, Results of spore count for = 67 = 67 x 103 3 strips of BI 10-3
Therefore, Results per BI = 67 x 103 3 = 22.33 x 103
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| 5.4.18 | Divide the results obtained by 3 to get the spore count (Population) for single strip as the analysis is performed using three strips. | |
| 5.4.19 | After completion of analysis and result recording, all the petriplates and glasswares used for the test to be Decontaminated as per procedure given in SOP for Decontamination of culture media and microbial waste. | |
| 5.4.20 | Acceptance Criteria: The log of average spore count obtained should not be less than the theoretically calculated value by substracting 0.3 from the log of suppliers label claim value and should not be more than theoretically calculated value by adding 0.48 to the log of suppliers label claim value.
For e.g:- 1.Suppliers label claim spore count = 3.8 x 104 2.Log of supplier label claim spore count = log (3.8x 104) = 4.5798 3. Lower limit of label claim = 4.5798 – 0.3 = 4.2798 3. Higher limit of label claim = 4.5798 + 0.48 = 5.0598 4. Acceptance criteria = 4.2798 to 5.0598 |
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| 5.4.21 | If the Biological Indicators does not show proper count within the labelled claim, then inform the Department head and discard the Biological Indicators and procure a new lot. | |
- Definitions / Abbreviations:
- Definitions:
- Quality Control Associate: Executive or Officer Quality Control Department or any personnel trained to perform duties as per this SOP.
- Quality Assurance Associate: Executive of Quality Assurance Department or any personnel trained to perform duties as per this SOP.
- Abbreviations:
| Abbreviation | Expansion |
| LAF | Laminar Air Flow |
| °C | Degree centigrade |
| ml | mililiter |
| CFU | Colony Forming Unit |
| % | Percentage
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| BI | Biological Indicator |
| IPA | Isopropyl Alcohol |
| ATCC | American Type Culture Collection. |
*Note – Ready to use SOP available in “DOWNLOAD” Section.