This is a ready-to-use protocol to establish method suitability (bacteriostasis and fungistasis) for a product sterility test under USP <71>, so a later “no growth” result can be read as “no contamination” rather than “growth was inhibited.” Replace every <<FILL: ...>> placeholder, set your document numbers and dates, and route it through approval before execution. A worked filled specimen follows. The compendial chapter is copyrighted; this protocol describes the method in original wording and cites the chapter by number, and you must read the current chapter before use. This is general guidance to adapt and verify, not legal or regulatory advice.
Approval page
| Role | Name | Signature | Date |
|---|---|---|---|
| Author (QC Micro SME) | <<FILL>> | ||
| Reviewer (Validation) | <<FILL>> | ||
| Approver (QA) | <<FILL>> |
| Field | Entry |
|---|---|
| Protocol number | <<FILL: e.g. VAL-MS-071-001>> |
| Version | <<FILL>> |
| Product | <<FILL: product, strength, container>> |
| Effective / execution date | <<FILL>> |
1. Objective
Demonstrate that the product and the full sterility test setup (membrane filtration and rinse, or direct inoculation, plus any neutralizer) do not inhibit the growth of a low inoculum of the compendial challenge organisms, so the sterility test method is suitable for this product.
2. Scope
This protocol covers method suitability for <<FILL: product / strength / container>> by <<FILL: membrane filtration or direct inoculation>>. It is repeated on any change to the product, formulation, container, filter type, rinse fluid, neutralizer, or media supplier that could affect inhibition, and periodically per <<FILL: SOP-ID>>.
3. System / method description
Describe the sterility test method being qualified: the filtration and rinse scheme or the direct-inoculation ratio, the media (FTM and SCDM), the rinsing fluids, and any neutralizer or inactivating enzyme. <<FILL: describe the specific method under test>>.
4. Prerequisites
- Challenge organism suspensions prepared and standardized to not more than 100 CFU per inoculum portion, confirmed by parallel plate count.
- Media lots within their growth promotion validity, with negative controls available.
- Analysts qualified for aseptic technique.
- Instruments (incubators, canister system) qualified and within calibration.
5. Roles
| Role | Responsibility |
|---|---|
| QC Micro analyst | Executes the arms, records reads. |
| QC Micro SME | Interprets results, decides corrective actions on inhibition. |
| Validation | Manages protocol execution and deviations. |
| QA | Approves the protocol and the conclusion. |
6. Challenge organisms
Use the compendial panel spanning aerobes, an anaerobe, a yeast, and a mold. The strains commonly used (confirm assignments against the current chapter):
| Organism | Type | ATCC | Assigned medium |
|---|---|---|---|
| Staphylococcus aureus | Gram-positive aerobe | 6538 | SCDM |
| Bacillus subtilis | Aerobe, spore-former | 6633 | SCDM |
| Pseudomonas aeruginosa | Gram-negative aerobe | 9027 | SCDM |
| Clostridium sporogenes | Anaerobe | 19404 (or 11437) | FTM |
| Candida albicans | Yeast | 10231 | SCDM |
| Aspergillus brasiliensis | Mold | 16404 | SCDM |
The inoculum is not more than 100 CFU per vessel; a low challenge is the point, because the method must catch low-level contamination.
7. Acceptance criteria
- For each organism, growth in the product arm is visually comparable to growth in the positive-control arm within the read window (not more than 3 days for bacteria, not more than 5 days for fungi).
- Every positive-control arm shows growth (if a control fails, the run is invalid and is repeated).
- If any product arm shows absent or clearly poorer growth, the product is inhibitory; the method is modified and the affected organism (at minimum) is repeated until growth is comparable.
8. Test cases
Run, for each organism, a product arm (full method plus not more than 100 CFU) and a positive-control arm (media plus the same inoculum, no product, or membrane and rinse without product).
| TC ID | Organism | Arm | Inoculum (CFU) | Expected | Actual | Day of growth | Pass/Fail | Tester / date |
|---|---|---|---|---|---|---|---|---|
| MS-01 | S. aureus | Product | <<FILL: <=100>> | Growth comparable to control | <<FILL>> | <<FILL>> | <<FILL>> | <<FILL>> |
| MS-02 | S. aureus | Control | <<FILL>> | Growth | <<FILL>> | <<FILL>> | <<FILL>> | <<FILL>> |
| MS-03 | P. aeruginosa | Product | <<FILL>> | Growth comparable | <<FILL>> | <<FILL>> | <<FILL>> | <<FILL>> |
| MS-04 | P. aeruginosa | Control | <<FILL>> | Growth | <<FILL>> | <<FILL>> | <<FILL>> | <<FILL>> |
| MS-05 | B. subtilis | Product | <<FILL>> | Growth comparable | <<FILL>> | <<FILL>> | <<FILL>> | <<FILL>> |
| MS-06 | C. sporogenes (FTM) | Product | <<FILL>> | Growth comparable | <<FILL>> | <<FILL>> | <<FILL>> | <<FILL>> |
| MS-07 | C. albicans | Product | <<FILL>> | Growth comparable | <<FILL>> | <<FILL>> | <<FILL>> | <<FILL>> |
| MS-08 | A. brasiliensis | Product | <<FILL>> | Growth comparable | <<FILL>> | <<FILL>> | <<FILL>> | <<FILL>> |
(Add the matching control arms for each organism.)
9. Deviation handling
Record any deviation (a control that failed to grow, an inhibited product arm, an inoculum out of range) with its investigation and impact on the conclusion. An inhibited product arm is not a protocol failure to hide; it is the expected trigger to modify the method (more rinse volume, a different rinsing fluid, a neutralizer or inactivating enzyme, a larger media volume) and repeat.
10. Summary and conclusion
State the method conditions found suitable (rinse count, rinsing fluid, neutralizer, media volumes) and declare the method suitable for the product, or list the corrective actions and the re-executed result that achieved suitability. <<FILL: conclusion>>.
11. Attachments
- Plate-count records establishing the inoculum.
- Raw read records for each arm.
- Any deviation records and re-execution data.
Filled specimen
An antibiotic injectable that first failed and then passed after method modification.
| TC ID | Organism | Arm | Inoculum | Actual | Day | Pass/Fail |
|---|---|---|---|---|---|---|
| MS-01 (attempt 1) | S. aureus | Product (3 x 100 mL Fluid A) | 62 CFU | No growth at day 3 | none | Fail |
| MS-02 (attempt 1) | S. aureus | Control | 62 CFU | Growth | day 2 | Pass |
| MS-01 (attempt 2) | S. aureus | Product (5 x 100 mL Fluid A + beta-lactamase) | 58 CFU | Growth | day 2 | Pass |
| MS-06 (attempt 2) | C. sporogenes (FTM) | Product | 44 CFU | Growth | day 3 | Pass |
| MS-08 (attempt 2) | A. brasiliensis | Product | 71 CFU | Growth | day 4 | Pass |
Conclusion (specimen): the residual antibiotic on the membrane inhibited growth with three rinses; with five 100 mL Fluid A rinses plus beta-lactamase added to the media, all challenge organisms grew comparably to their controls. The method is suitable for this product with five rinses plus beta-lactamase, and those conditions become the validated product sterility method. The failed first attempt is documented, not discarded.
Common inspection findings this protocol prevents
- A product sterility test run with no demonstrated suitability, so a no-growth result cannot be trusted.
- Suitability not repeated after a formulation, filter, rinse fluid, or neutralizer change.
- An inhibited arm quietly ignored rather than driving a method modification and re-test.
- Inoculum not confirmed at not more than 100 CFU, so the sensitivity of the challenge is unknown.
How to adapt this protocol
- Fill the product identity, method, and the challenge assignments to your media.
- Expand the test-case table to include every control arm and any replicate scheme you use.
- Reference your organism source, passage control, and suspension standardization method.
- Tie repeat triggers to your change control SOP.
- Confirm the organism list and read windows against the current USP <71> before execution.