Sterility – Stasis Test: Purpose, Procedure, and Interpretation as per GMP Guidelines

Sterility testing is one of the most critical microbiological tests in the pharmaceutical industry, designed to confirm that sterile drug products are free from viable microorganisms. To ensure reliability of the sterility test results, a Stasis Test (also known as a Hold Time or Incubation Hold Test) is performed as part of method validation. The Stasis Test confirms that the sterility testing system, media, and environment remain sterile during the pre-incubation or hold period before the actual test begins.

What Is a Sterility Stasis Test?

The Stasis Test is a microbiological control step conducted to verify that the test system remains sterile during the interval between the completion of test setup and the beginning of the incubation period. It is essentially a pre-incubation holding validation carried out to demonstrate that the sterility test environment, transfer process, and media are not compromised by any contamination.

Purpose of the Stasis Test

The main objective of the Stasis Test is to ensure that no microbial contamination occurs during the period between:

• Preparation of the sterility test setup, and
• Transfer of inoculated media to the incubator for incubation.

It validates the aseptic integrity of the testing environment and process by confirming that any delay in incubation (commonly referred to as "stasis") does not result in contamination.

Regulatory Reference

• USP <71> – Sterility Tests
• Ph. Eur. 2.6.1 – Sterility
• EU GMP Annex 1 (2022 Update) – Guidance on sterility test and incubation conditions
• WHO TRS 1025 – Sterility testing of pharmaceutical preparations

Importance of the Stasis Test in GMP Environment

In a Good Manufacturing Practice (GMP) compliant facility, sterility testing is conducted under Grade A conditions (usually in a Laminar Air Flow or Isolator). Any unvalidated delay between filtration or sample transfer and incubation could allow contamination. Hence, the stasis test provides assurance that aseptic practices, personnel techniques, and environmental controls are effective even during the pre-incubation hold period.

Typical Scenarios Where Stasis Test Is Required

• When performing initial validation of a sterility test isolator or LAF setup
• During qualification of new media or new batch of sterility testing units
• During revalidation or periodic sterility testing method verification
• After major maintenance or modifications in the sterility test area
• During audit observations to demonstrate aseptic assurance

Procedure for Performing Stasis Test

The stasis test procedure may vary slightly depending on the test setup (Isolator or LAF), but the general GMP-compliant method is as follows:

1. Preparation of Media

Prepare sterile media such as Fluid Thioglycollate Medium (FTM) and Soybean Casein Digest Medium (SCDM) according to pharmacopeial specifications. Verify sterility of the media before use.

2. Setup of Test Units

Transfer a sufficient number of sterile media units (FTM and SCDM) into the sterility test area under normal aseptic working conditions. The number of units should be statistically representative—usually a minimum of 10 units for each medium type.

3. Simulate Routine Sterility Test Conditions

Perform all operations such as container handling, filtration setup, transfer, and sealing—just as in the actual sterility test—but without introducing any product sample or microorganism.

4. Hold Period (Stasis Period)

Keep the test setup (unincubated media units) under normal working conditions or within the isolator for a predefined duration, usually:

• Minimum: 4 to 6 hours
• Maximum: 24 hours (depending on laboratory procedure)

During this time, no incubation is performed. The purpose is to simulate potential delays before incubation in real test conditions.

5. Incubation

After completion of the hold period, transfer the media units to the incubators:

• FTM: 30–35°C for 14 days
• SCDM: 20–25°C for 14 days

6. Observation

Examine all test units daily for any visible sign of microbial growth (turbidity, sediment, color change, or pellicle formation). If all media remain clear after 14 days, the stasis test is considered passed.

Acceptance Criteria

• All FTM and SCDM media units shall remain clear and free from any turbidity or microbial growth after 14 days of incubation.
• Environmental monitoring data during the stasis period shall remain within alert and action limits.
• No procedural deviations or contamination incidents shall occur during the test.

Interpretation of Results

A clear medium after incubation indicates that:

• The aseptic environment and procedures are validated and reliable.
• Personnel technique during sample setup does not introduce contamination.
• The media and equipment are sterile and properly handled.

If any turbidity or microbial growth appears, it indicates a potential aseptic breach or procedural lapse, and an investigation must be initiated.

Investigation Steps for Stasis Test Failure

• Review environmental monitoring data during the stasis period.
• Check personnel aseptic technique records and training history.
• Review media preparation and sterilization records.
• Examine equipment or isolator integrity (HEPA filter leak, glove tear, etc.).
• Perform Gram staining or subculture of turbid media to identify contaminant type.

Corrective and Preventive Actions (CAPA)

• Retrain personnel in aseptic technique if operator error is suspected.
• Review gowning procedures and environmental controls.
• Perform isolator or LAF integrity verification (smoke study, pressure decay test).
• Replace contaminated media and repeat the stasis test after corrective action.
• Document all findings and justifications in the sterility validation report.

Documentation Requirements

A detailed report must be prepared containing:

• Purpose and scope of the stasis test
• Test setup description (media type, number of units, environmental data)
• Hold duration and conditions
• Incubation results with daily observations
• Signatures of microbiologist and QA reviewer
• Conclusion and CAPA (if applicable)

Common Audit Observations Related to Stasis Test

• Stasis test not performed during sterility validation
• Hold duration not justified or documented
• Results not linked with environmental monitoring data
• Absence of clear acceptance criteria in SOP
• Failure investigation not adequately documented

Best Practices to Ensure Compliance

• Include stasis test as a part of every sterility method validation protocol.
• Define maximum hold time in SOP and validation protocol.
• Perform stasis test whenever major environmental or process changes occur.
• Maintain traceable batch records for each test setup.
• Review all data by qualified QA personnel before release.

Conclusion

The Sterility – Stasis Test plays a crucial role in ensuring the integrity and reliability of sterility testing results. It confirms that the testing environment, media, and aseptic procedures can maintain sterility even during hold periods. By performing stasis testing as per USP, Ph. Eur., and GMP guidelines, pharmaceutical laboratories can ensure accurate sterility testing, minimize false positives, and demonstrate compliance during regulatory inspections.

💬 About the Author

Siva Sankar is a Pharmaceutical Microbiology Consultant and Auditor with extensive experience in sterility testing, validation, and GMP compliance. He provides consultancy, training, and documentation services for pharmaceutical microbiology and cleanroom practices.

📧 Contact: siva17092@gmail.com
📱 Mobile: 09505626106

Disclaimer: This article is for educational purposes and does not replace your laboratory’s SOPs or regulatory guidance. Always follow validated methods and manufacturer instructions.