SOP for Qualification of Biological Indicators
This Document describes (Standard Operating procedure) SOP for Qualification of Biological Indicators
SOP for Qualification of Biological Indicators
I. Purpose & Scope:
- The purpose of this SOP is to lay down the procedure for Qualification of Biological Indicators
- This Standard operating procedure shall be applicable for Qualification of Biological Indicators
II. Responsibilities:
- All Quality Control and Quality Assurance personnel’s shall be responsible to follow and implement this SOP.
III : Introduction and Procedural Part :
Introduction :
- Steam heat sterilization depends upon the exposure of microorganisms to the saturated steam at temperatures sufficient to destroy it.
- The Biological indicators are used for qualification of Steam Heat Sterilizer. To evaluate its performance, for providing greater than 6 Log reduction in challenged microbial population of resistant strain, to achieve Sterility Assurance Level (SAL) of 10-6.
- Achieving greater than 6 Log reduction in biological population of resistant strain thereby resulting in sterile load.
- The spore population determination of the Biological Indicator (BI) strips (Geobacillus stearothermophilus ATCC 7953) shall be performed prior to the bacterial challenge activities.
- Confirm the D value of biological indicators in certificate of analysis (COA) of supplier. Proceed only if D value of biological indicator is Not more than 2.5 minutes. The BI strips shall be tested for spore population determination to ensure that each BI is having a minimum spore population of 1×106
- The Biological Indicators to be challenged must have minimum 1.0 x 106 spores/unit population of viable spores of Geobacillus stearothermophilus ATCC 7953.
- Biological Indicators must meet the In-house standards, so determine the Inhouse spore population of Biological indicator strips/ampoules.
Procurement :
- Procure the required biological indicator strips/ampoules of Geobacillus stearothermophillus (ATCC 7953) from any of the sources.
- Procured biological indicator strips/ampoules should be checked for its Spore population details, Date of manufacturing, Date of Expiry, D-Value, Storage condition.
- After Procurement, perform the Qualification of biological indicator strips/ampoules. At first perform Growth promotion characteristics and identification of strain and then determine the spore population of given biological indicator strips.
Requirement :
- Sterile Soybean casein digest medium (SCDM).
- Sterile Soybean casein digest Agar (SCDA).
- Sterile Purified water/sterile saline
- Vortex mixer
- Heating block
- Sterile pipette tips and sterile test tubes.
Growth promotion characteristics and Identification:
- Perform the Growth promotion test of biological indicator strips in Preincubated Soybean casein digest medium.
- Clean the LAF work station with 70% IPA.
- Take the one strip and inoculate in Preincubated Soybean caseine digest medium(10ml or 100ml) aseptically.
- Incubate this inoculated medium at 55-60°C for minimum 24-48 hours. Keep negative control of that medium along with test.
- After incubation, observe the medium for any growth in the form of turbidity.
- For identification purpose, streak from inoculated medium on the preincubated Soybean casein digest agar plate. Incubate the SCDA plate at 55-60°C for minimum 48 hours.
- After observing the growth characteristics perform the Gram staining of the observed colonies.
- Record the observation and results in Annexure-I.
- Acceptance criteria: Luxuriant growth within 48 hours.
Procedure for determination of spore population :
- After Growth promotion characteristics and identification of strain, perform the spore population determination.
- Take 03 number of biological indicator strips/ampoules and inoculated into 100ml of sterilized purified water/saline.
- Vortex whole contents on vortex mixer for 15-20minutes for dislodge and disperse spores.
- After mixing, take 10ml stock solution in sterile test tube aseptically.
- Give the heat shock to this stock solution at 95-100°C in heating block/water bath for minimum 15 minutes.
- Perform the serial dilution of this stock solution for enumeration of spore population.
- Aseptically pipette out 1ml of stock solution and inoculate in 9ml sterile saline in test tube, vortex the tube to get homogenized suspension. This shall be treated as 10-1
- Serially dilute the microbial suspension by taking 1ml into another tube containing 9ml of sterile saline solution .
- Continue the dilution process by following the below mentioned steps up to 10-6 Vortex each dilution for approximately 30 seconds.
1 ml stock solution + 9 ml saline → 10-1
1 ml 10-1 + 9 ml saline → 10-2
1 ml 10-2 + 9 ml saline → 10-3
1 ml 10-3 + 9 ml saline → 10-4
1 ml 10-4 + 9 ml saline → 10-5
1 ml 10-5 + 9 ml saline → 10-6
- Pour 1ml each in two sterile petri plates from 10-2 to 10-5
- Add 20-25 ml sterile soybean casein digest agar medium in each plate (at temperature NMT 45°C) and swirled for uniform mixing. Allow the media to solidify. Keep the negative control plate of SCDA media.
- After solidification, Incubate the plates at 55° – 60°C for 24-48 hrs.
- After incubation period count the number of colonies on each dilution. Record the mean results of two plates for each dilution in Annexure-I.
- Obtain the spore population of stock solution per 10ml, per 100ml and finally per strip or per ampoule.
- Acceptance criteria: Spore population should be ±1 log of the label claim.
Precautions :
- Maintain the aseptic condition through the process to avoid contamination.
- After completion of qualification, destruct the plates and suspensions in media destruction autoclave.
Annexures :
Annexure-I : Report for Qualification of Biological Indicator
Annexure I
Report for Qualification of biological indicator
Page No.: 1 of 2
- I) Details of Biological Indicator:
| Date of Receipt | Name of Biological Indicator/Strain No. | ||
| Date of Mfg. | Type of Biological Indicator | ||
| Date of Expiry | Lot No/D Value | ||
| Manufacturer/
Storage condition |
Spore Population
(As per COA) |
- II) Growth Promotion Characteristics and Identification:
(Acceptance Criteria : Luxuriant growth within 48 hours)
| Purpose and Date
of Inoculation |
Inhouse Media
Lot No. |
Date of Observation after incubation at
(55-60°C) |
Observation |
| Growth Promotion: | |||
| Identification: |
III) Spore Population Determination :
| Date of Testing : | Done By : |
| No. of indicators/ampoules used : | Volume of Sterile Purified water/saline used and sterilization Inhouse lot No.: |
| Blending Time : | Volume of Stock Solution : |
| Heat Shock Temperature : | Heat Shock Time : |
| Inhouse lot No. of used Media : | Incubation Condition : |
| Incubator I.D : | Observed On/By : |
Annexure I
Report for Qualification of Biological Indicator
Page No.: 2 of 2
Observation:
| Dilution Used | 10-2 | 10-3 | 10-4 | 10-5 | ||||
| Observation after | 24 hrs | 48 hrs | 24 hrs | 48 hrs | 24 hrs | 48 hrs | 24 hrs | 48 hrs |
| Plate-1 (cfu/ml) | ||||||||
| Plate-1(cfu/ml) | ||||||||
| Average | ||||||||
Average of Obtained Population of Stock suspension:————————–
= ——————— (X)
Obtained Spore Population in Stock solution (10ml) = ———————-
= ———————-
= ——————– (Y)
Obtained Spore Population in Stock solution (100ml) = ———————
= ——————— (Z)
Obtained Spore Population/Strip = ————————
= ————————
| Acceptance Criteria for spore Population Determination: |
| Spore Suspension : ±1 log value of the label claim. |
| Obtained Spore Population : Meeting the Acceptance Criteria/Not Meeting the Acceptance Criteria. |
Remark: The Biological Indicator lot No.——————— to be used for Steam sterilization Qualification stands qualified/disqualified for its intended use.
Analysed By: Checked By:
Date : Date :
