SOP for Antimicrobial Effectiveness Test: EP/USP
Learn how to write a SOP for Antimicrobial Effectiveness Test (AET) on pharmaceutical products to ensure the efficacy of antimicrobial preservatives as per Pharmacopeia guidelines.
1.0 PURPOSE
To describes a standardized procedure for performing Antimicrobial Effectiveness Test (AET) on pharmaceutical products in order to evaluate the efficacy of antimicrobial preservatives used in formulations.
2.0 SCOPE
This SOP is applicable to all sterile and non-sterile pharmaceutical products manufactured and/or tested in the Quality Control Laboratory that require antimicrobial preservation.
3.0 RESPONSIBILITY
3.1 Microbiologist/QC Analyst is responsible to perform the test as per approved procedures.
3.2 QA Officer is responsible to verify compliance with the SOP and regulatory guidelines.
3.3 Department Head is responsible to ensure training and implementation of the SOP.
4.0 ACCOUNTABILITY
Head of Quality Assurance is accountable for ensuring the integrity, accuracy, and compliance of antimicrobial effectiveness testing data with applicable regulatory standards.
5.0 ASSOCIATED DOCUMENTS
5.1 USP <51>: Antimicrobial Effectiveness Testing
5.3 EP 5.1.3 – Test for Efficacy of Antimicrobial Preservation
5.4 SOP for Microbiological Media Preparation
6.0 PRECAUTIONS
6.1 Perform testing in aseptic conditions using a biosafety cabinet.
6.2 Use only validated microbial cultures for the test.
6.3 Ensure media sterility and proper environmental conditions.
6.4 Dispose of biohazardous waste as per SOP on Waste Disposal.
Related: SOP for Settle Plates of Environmental Monitoring
SOP for Maintenance of Stock Cultures in Microbiology
7.0 PROCEDURE
7.1 Test Organisms and Culture Media Used
7.1.1 Use the following test organisms ATCC reference strains as specified in the test:
–Escherichia coli ATCC 8739
–Pseudomonas aeruginosa ATCC 9027
–Staphylococcus aureus ATCC 6538
–Candida albicans ATCC 10231
–Aspergillus niger ATCC 16404
7.1.2 Use the following media as specified in the test:
–Soybean Casein Digest Agar (SCDA) – for bacterial growth and maintenance.
–Sabouraud Dextrose Agar (SDA) – for fungal growth and maintenance.
–Soybean Casein Digest Broth (SCDB) or Fluid Soybean Casein Digest Medium (TSB) – for preparation of inoculum suspensions.
7.3.1 Use the following Materials and Equipment as specified in the test:
–Sterile Saline / Phosphate Buffer – for dilution of cultures
–Polysorbate 80 (for fungal suspensions)
–Neutralizing agents (validated)
–Sterile pipettes, syringes, dilution tubes
–Sterile containers for test samples
–Incubators (22.5 ± 2.5 °C and 32.5 ± 2.5 °C)
–Colony counter and vortex mixer
–AET Data Recording Sheet
7.2 Media Preparation Procedure
7.2.1 Weigh the required quantity of dehydrated media as per manufacturer’s instructions.
7.2.2 Dissolve in the recommended volume of purified/distilled water with gentle heating and stirring.
7.2.3 Adjust pH if necessary (pH 7.2 ± 0.2 for SCDA, pH 5.6 ± 0.2 for SDA) before sterilization.
7.2.4 Dispense into suitable containers (e.g., flasks, bottles, or tubes).
7.2.5 Sterilize by autoclaving at 121°C ± 2°C for 15–20 minutes.
7.2.6 Cool the medium to 45–50°C and pour into sterile Petri plates in aseptic conditions (for solid media).
7.2.7 Label each lot with:
–Name of media
–Preparation date
–Batch number
–Expiry date (as per validated shelf life)
7.2.8 Inspect the solidified media for any contamination, cracks, or dehydration before use.
7.2.9 Store the prepared media at 2–8°C in a refrigerator, protected from light and drying.
7.3 Revival of Stock Cultures
7.3.1 Retrieve lyophilized or preserved culture from storage (–20°C or as recommended).
7.3.2 Revive by streaking onto respective agar medium (SCDA for bacteria, SDA for fungi).
7.3.3 Incubate at: 30–35°C for bacteria (24–48 hours) and 20–25°C for fungi (3–5 days)
7.3.4 Select typical colonies and transfer to fresh agar slants to obtain working cultures.
7.3.5 Verify purity and typical colony morphology before use in the Antimicrobial Effectiveness Test.
7.4 Preparation of Inoculums
7.4.1 Prepare microbial inoculums following the respective SOP for the preparation of culture suspensions using sterile normal saline or phosphate buffer solution (pH 7.2).
7.4.2 For harvesting bacterial cultures and Candida albicans, use sterile peptone saline to wash the surface growth.
7.4.3 Collect the suspension in appropriate sterile glassware, adjusting the final volume with sterile peptone saline to achieve a microbial load of approximately 1 × 10⁸ CFU/mL. For Aspergillus niger, harvest the spores using sterile peptone saline containing 0.05% polysorbate 80. Adjust the volume with sterile peptone saline to achieve a spore count of approximately 1 × 10⁸ CFU/mL.
7.4.4 Determine the colony-forming units (CFU) per milliliter in each microbial suspension using the media and incubation conditions listed in the table below. This value will be used to calibrate the inoculum size.
–Bacterial and yeast suspensions must be used within 24 hours of preparation.
–Fungal suspensions (e.g., A. niger) can be stored at 2–8°C for up to 7 days.
7.4.5 Table of Culture Conditions for Inoculum Preparation as per USP ˂51˃
| Organism | Suitable Medium | Incubation Temperature | Inoculum Incubation Time | Microbial Recovery Incubation Time |
| Escherichia coli ATCC No. 8739; NCIMB 8545; CIP 53.126 | Soybean–Casein Digest Broth; Soybean–Casein Digest Agar | 32.5 ± 2.5°C | 18 to 24 hours | 3 to 5 days |
| Pseudomonas aeruginosa ATCC No. 9027; NCIMB 8626; CIP 82.118. | Soybean–Casein Digest Broth; Soybean–Casein Digest Agar | 32.5 ± 2.5°C | 18 to 24 hours | 3 to 5 days |
| Staphylococcus aureus ATCC No. 6538; NCTC 10788; NCIMB 9518; CIP 4.83. | Soybean–Casein Digest Broth; Soybean–Casein Digest Agar | 32.5 ± 2.5°C | 18 to 24 hours | 3 to 5 days |
| Candida albicans ATCC No. 10231 NCPF 3179; IP 48.72. | Sabouraud Dextrose Agar; Sabouraud Dextrose Broth | 22.5 ± 2.5°C | 44 to 52 hours | 3 to 5 days |
| Aspergillus niger ATCC No. 16404; IMI 149007; IP 1431.83 | Sabouraud Dextrose Agar; Sabouraud Dextrose Broth | 22.5 ± 2.5°C | 6 to 10 days | 3 to 7 days |
7.4.6 Table Test Microorganisms and Culture Conditions as per (EP 5.1.3)
| Test Organism | Reference Culture No. | Suitable Culture Medium | Incubation Temperature | Inoculum Incubation Time | Microbial Recovery Incubation Time |
| Pseudomonas aeruginosa | ATCC 9027; NCIMB 8626; CIP 82.118 | Soybean–Casein Digest Agar (Medium B); Soybean–Casein Digest Broth | 30–35 °C | 18–24 hours | 3–5 days |
| Staphylococcus aureus | ATCC 6538; NCTC 10788; NCIMB 9518; CIP 4.83 | Soybean–Casein Digest Agar (Medium B); Soybean–Casein Digest Broth | 30–35 °C | 18–24 hours | 3–5 days |
| Escherichia coli (recommended for oral preparations) | ATCC 8739; NCIMB 8545; CIP 53.126 | Soybean–Casein Digest Agar (Medium B); Soybean–Casein Digest Broth | 30–35 °C | 18–24 hours | 3–5 days |
| Candida albicans | ATCC 10231; NCPF 3179; IP 48.72 | Sabouraud Dextrose Agar (Medium C); Sabouraud Dextrose Broth | 20–25 °C | 44–52 hours (≈48 h) | 3–5 days |
| Aspergillus niger | ATCC 16404; IMI 149007; IP 1431.83 | Sabouraud Dextrose Agar (Medium C); Sabouraud Dextrose Broth | 20–25 °C | 6–10 days (or until good sporulation) | 3–7 days |
| Zygosaccharomyces rouxii (for sugar-based oral prep, if applicable) | NCYC 381; IP 2021.92 | Sabouraud Dextrose Agar (Medium C); Sabouraud Dextrose Broth | 20–25 °C | 2–3 days | 3–5 days |
7.5 Test Procedure
7.5.1 Select five (5) test units for each product to be tested:
–Either five original unopened containers that can be aseptically entered, or
–Five sterile bacteriological containers into which the product has been aseptically transferred.
7.5.2 Record the container identification numbers in the test worksheet.
7.5.3 For each of the five containers, prepare a separate test sample to be inoculated with a single microorganism (one organism per container).
7.5.4 Label each container clearly with the organism name and the sampling schedule.
7.5.5 Add an inoculum volume equal to 0.5%–1.0% (v/v) of the product volume.
7.5.6 The chosen volume must yield a final microbial concentration after inoculation as follows:
–Product Categories 1, 2, and 3: 1 × 10⁵ to 1 × 10⁶ CFU/mL
–Product Category 4 (Antacids): 1 × 10³ to 1 × 10⁴ CFU/mL
7.5.7 Calculate the exact inoculum volume based on the verified inoculum concentration to achieve the required range.
7.5.8 Gently but thoroughly mix the inoculated product by slow inversion or brief vortexing to ensure uniform distribution of microorganisms. Avoid foaming or excessive aeration.
7.5.9 Record physical observations such as appearance, color, odor, and any phase separation immediately after inoculation.
7.6 Incubation Conditions
7.6.1 Incubate all inoculated containers at 22.5 ± 2.5 °C for the full duration of the test unless otherwise justified by product characteristics.
7.6.2 Protect samples from direct light and excessive agitation during incubation.
Record environmental conditions and any unusual changes in the product during the test period.
7.7 Sampling Intervals and Sampling Schedule:
7.7.1 Collect samples from each inoculated container at the intervals specified for its product category (refer to Section 7.11).
7.7.2 Typical sampling points include Day 7, 14, and 28, depending on the category.
7.7.3 At each sampling interval, aseptically withdraw a representative aliquot from each test container using sterile instruments.
7.7.4 Observe and record any visual or physical changes in the product prior to sampling.
7.8 Neutralization and Dilution:
7.8.1 Perform appropriate serial dilutions in a validated neutralizing diluent that effectively inactivates the preservative system without affecting microbial viability.
7.8.2 Use the neutralizer validated previously for this product.
7.9 Plating and Incubation:
7.9.1 Plate suitable dilutions using either the spread plate or pour plate method according to the related SOP.
7.9.2 Incubate plates using the same recovery media, temperature, and time defined for each microorganism in Section 7.4 (Preparation of Inoculums).
7.10 Plate Count, Colony Enumeration, and Calculations:
7.10.1 After incubation, select plates containing 30–300 CFU for accurate enumeration.
7.10.2 Count the colonies and calculate CFU/mL in the test sample, taking into account the dilution factor and sample volume.
7.10.3 Determine the log₁₀ concentration of viable microorganisms at each sampling point.
7.10.4 Calculate the log₁₀ reduction relative to the initial concentration immediately after inoculation using the following formula:
Log10 Reduction=log10(Initial CFU/mL)−log10(CFU/mL at Time point)
7.10.5 If the microbial count is below the detection limit, record as “< LOD” and calculate the minimum possible log reduction based on that limit.
7.10.6 Where recovery tests show incomplete neutralization or neutralizer toxicity, the results are invalid and the test must be repeated after correction.
7.11 Acceptance Criteria as per guidelines:
7.11.1 Apply the following acceptance criteria depending on the product category.
7.11.2 Each category represents a specific product type as defined in USP <51>.
7.11.3 Follow USP and EP acceptance limits for log reduction or absence of microorganisms.
Table 1: Acceptance Criteria of Antimicrobial Effectiveness Test as per USP <51>
| Categories | Acceptance Criteria (Bacterial Log Reduction) | |||
| Initial Count | 7th Days | 14th Days | 28th Days | |
| Category-1: Products for Topical Use, Oral Use (other than antacids), Nasal Use, and Products for Mucous Membranes | Baseline | Not less than 2.0 log₁₀ reduction from the initial count at 14 days, and no increase at 28 days | ≥ 2.0 log₁₀ reduction | No increase |
| Category-2: Antacids and Products for Topical Use Made with Aqueous Bases | Baseline | No increase from initial count at 14 days | No increase | No increase |
| Category-3: Oral Products Containing Aqueous Bases (Other than Antacids) | Baseline | ≥ 1.0 log₁₀ reduction from initial count at 14 days | ≥ 3.0 log₁₀ reduction from initial count at 28 days | ≥ 3.0 log₁₀ reduction |
| Category-4: Antimicrobial-Containing Products for Oral Use (Antibiotics, etc.) | Baseline | No increase from initial count at 14 days | No increase | No increase |
| Acceptance Criteria (Fungal Log Reduction) | ||||
| Categories | Initial Count | 7th Days | 14th Days | 28th Days |
| Category-1: Products for Topical Use, Oral Use (other than antacids), Nasal Use, and Products for Mucous Membranes | Baseline | No increase from initial count at 14 days | No increase | No increase |
| Category-2: Antacids and Products for Topical Use Made with Aqueous Bases | Baseline | No increase from initial count at 14 days | No increase | No increase |
| Category-3: Oral Products Containing Aqueous Bases (Other than Antacids) | Baseline | No increase from initial count at 14 days | No increase | No increase |
| Category-4: Antimicrobial-Containing Products for Oral Use (Antibiotics, etc.) | Baseline | No increase from initial count at 14 days | No increase | No increase |
Notes (USP <51> Reference Summary):
–Initial count: Determined immediately after inoculation.
–“No increase” means ≤ 0.5 log₁₀ increase from the previous value.
–Log₁₀ reduction:
Log Reduction= log 10 (Initial Count) – log 10 (Surviving Count)
Table 2: Acceptance Criteria of Antimicrobial Effectiveness Test as per EP 5.1.3
| Parenteral and Ophthalmic Preparations | Acceptance Criteria (Log Reduction) | ||||||||
| Organisms | Criteria | 2 Hours | 24 Hours | 7th Days | 14th Days | 28th Days | |||
| Bacteria | A | 2 | 3 | — | — | NR | |||
| B | — | 1 | 3 | — | NI | ||||
| Fungi | A | — | — | 2 | 1 | NI | |||
| B | — | — | — | — | NI | ||||
| Topical Preparations | Acceptance Criteria ( Log Reduction) | ||||||||
| Categories | Criteria | 2nd Days | 7th Days | 14th Days | 28th Days | ||||
| Bacteria | A | 2 | 3 | — | NI | ||||
| B | — | — | 3 | NI | |||||
| Fungi | A | — | — | 2 | NI | ||||
| B | — | — | 1 | NI | |||||
| Oral Preparations | Acceptance Criteria ( Log Reduction) | ||||||||
| Categories | 2nd Days | 7th Days | 14th Days | 28th Days | |||||
| Bacteria | — | — | 3 | NI | |||||
| Fungi | — | — | 1 | NI | |||||
Note: **NR: No Recover
**NI: No Increase
Clarification of Acceptance Criteria (A and B)
The A criteria express the recommended efficacy to be achieved for antimicrobial preservation.
In justified cases where the A criteria cannot be attained — for example, due to the increased risk of adverse reactions arising from higher concentrations of preservatives — the B criteria may be applied as acceptable alternative limits, provided that the product demonstrates satisfactory antimicrobial protection under the conditions of use.
A criteria = Recommended (standard) preservative efficacy
B criteria = Acceptable only if A cannot be met for valid safety reasons (e.g., irritation, toxicity) be satisfied.
8.0 ABBREVIATIONS/DEFINITIONS
8.1 SOP – Standard Operating Procedure
8.2 AET – Antimicrobial Effectiveness Testing
8.3 ATCC – American Type Culture Collection
8.4 NCTC – National Collection of Type Cultures
8.5 NCIMB – National Collection of Industrial, Food and Marine Bacteria
8.6 CIP – Collection de l’Institut Pasteur
8.7 CFU – Colony Forming Unit
8.8 USP – United States Pharmacopeia
8.9 EP – European Pharmacopeia
8.10 QC – Quality Control
8.11 QA – Quality Assurance
8.12 SCDB – Soybean Casein Digest Broth
8.13 SCDA – Soybean Casein Digest Agar
8.14 SDA – Sabouraud Dextrose Agar
9.0 ANNEXURES
9.1 Annexure-1: Format for Antimicrobial Effectiveness Test Report
9.2 Annexure-2: Media and Strain Preparation Log
10. REVISION HISTORY
| Version | Date | Description of Change | Reviewed By | Approved By |
| 1.0 |