Sterilizer Microbial Challenge Test

NegotiableUpdate on 01/04

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Overview

Microbial Challenge Test in Sterilizer Equipment Validation is one of the key experiments to verify the effectiveness of the sterilization process. It is mainly used to confirm that the sterilization method used (such as wet heat sterilization, dry heat sterilization, ethylene oxide sterilization, irradiation sterilization, or steam sterilization, etc.) can effectively kill microorganisms under certain conditions, thereby achieving the predetermined level of sterility assurance (usually requiring SAL ≤ 10 ⁻⁶, that is, the probability of surviving microorganisms after sterilization of each item does not exceed one in a million)

Product Details

Sterilizer Microbial Challenge TestCore Content Overview Table
classification	specific project	detailed description
Core purpose of the experiment	1. Verify the effectiveness of sterilization process	Under the confirmed set parameters, even if the microorganisms are in“The most difficult sterilization location”(such as the coldest point and areas where gases are difficult to penetrate) can also be killed.
	2. Confirm the adequacy of the conditions	By using highly resistant biological indicators（BI）Simulation“worst-case”Prove that sterilization is effective for all products/All loads are reliable.
	3. Support verification and revalidation	It's a sterilizerIQ/OQ/PQThe core components of verification and periodic revalidation.
Core tools:BI	1. definition	Carriers containing known quantities and specific resistant microbial spores (spore strips, ampoules, filter papers, etc.), representing“The most difficult to kill microorganisms”.
Core tools:BI	2. Selection of bacterial strains (by sterilization method)	-Wet heat sterilization: thermophilic Bacillus subtilis -Dry heat sterilization: Bacillus subtilis var. niger -Ethylene oxide sterilization: Bacillus subtilis var. niger/Thermophilic fat loving Bacillus subtilis -Radiation sterilization: Thermophilic Bacillus subtilis/Bacillus subtilis
	3. key parameters	Spore quantity: usually 10⁵~10⁶CFU /Carrier; Each batch needs to be providedBIof“Live bacteria count report”Ensure accurate initial bacterial count.
BIPlacement strategy	1. Placement principle	simulation“condition”Prioritize coverage of sterilizers/Under load“The most difficult sterilization location”.
BIPlacement strategy	2. Typical placement point	-Sterilizer chamber: cold spots (such as near exhaust ports, corners of chambers), dead corners of airflow -Load inside: packaging center, highest density area, stacking bottom layer
	3. quantity requirement	Multiple tests need to be placed in a single experimentBI(Usually)≥3One), covering different key positions to avoid“Single point misjudgment”.
Experimental practical operation process	1. Test preparation	confirmBIActivity, sterilizer parameter calibration, load simulation (consistent with actual production).
	2. Perform sterilization	According to the set parameters (temperature)/pressure/time/ EORun the sterilization program with concentration, etc., and record key parameters (such as chamber temperature, pressure curve) during the process.
	3. Cultivation and control settings	-Experimental group: Remove sterilized samplesBICultivate according to the instructions（24~48Hour, depending on the strain) -Positive control: unsterilizedBISynchronous cultivation (requires good growth, proofBIEffective on its own) -Negative control: synchronous cultivation in sterile medium (sterile, excluding contamination interference)
	4. Result determination	experimental groupBIAll negative (sterile growth), positive control positive, negative control negative→Test qualified; Otherwise, it is necessary to investigate the sterilization parametersBIPlace the issue and try again.
Test frequency requirements	1. Verification（PQStage)	Must be executed as a sterilization process“release”The core basis.
	2. Periodic revalidation	Usually every year 1 Secondly; If the sterilizer undergoes major maintenance (such as replacing heating tubes and seals), it needs to be re validated in advance.
	3. Verification after Change	Sterilization parameter adjustment, load type/Change in loading methodBIAfter batch replacement, the test needs to be re executed.
Core regulations and standards	International Standard	- ISO 17665(Wet heat sterilization) - ISO 20857(Dry heat sterilization) - ISO 11135(Ethylene oxide sterilization) - ISO 11137(Radiation sterilization)
	Domestic regulations	-Chinese Pharmacopoeia (Guidelines for Validation of Aseptic Inspection and Sterilization Methods) - NMPAGuidelines for Sterilization Validation of Medical Devices - GMP(Drug)/Specification for Quality Management of Medical Device Production
	International Regulatory Guidelines	FDAGuidelines for Sterilization Process ValidationEU GMPGuidelines for the Production of Aseptic Drugs
Core objective:SAL	Sterility assurance level requirements	Must achieveSAL ≤ 10⁻⁶The probability of surviving microorganisms after sterilization of each item does not exceed one in a million, which is the core indicator for passing the test.

Sterilizer Microbial Challenge Test Comparison Table of Differences in Microbial Challenge Tests with Different Sterilization Methods

Sterilization method	commonly usedBIstrain	Monitoring of key experimental parameters	Characteristics of cultivation conditions	Core precautions
Wet heat sterilization (steam)	Thermophilic fat loving Bacillus subtilis	Temperature（121℃/132℃）Pressure, sterilization time	37~40℃aerobic cultivation	Need to ensure steam saturation（≥95%）To avoid“Dry saturated steam”Resulting in sterilization failure.
dry heat sterilization	Bacillus subtilis var. niger var. niger	Temperature（160~180℃）Constant temperature time	30~35℃aerobic cultivation	The load needs to be evenly distributed to avoid stacking too densely, which may cause problems“Insufficient local temperature”.
Ethylene oxide sterilization	Bacillus subtilis var. niger var. niger	EOConcentration and temperature（37~55℃）Humidity and exposure time	30~35℃aerobic cultivation	Need to control chamber humidity（40%~80%）Insufficient humidity will decreaseEOSterilization effect; After sterilization, residual substances need to be fully analyzedEO.
Irradiation sterilization	Bacillus subtilis	Radiation dose (usually25~50 kGy）	30~35℃aerobic cultivation	BINeed to closely adhere to the surface of the load/Internally, ensure that the product receives the same dose of radiation.