Microbial Limit Test
Purpose
- The procedure applies to testing the microbiological quality of raw materials and batches of non-sterile pharmaceutical products such as tablets, capsules, ointments, and semi-solid dosage forms.
Materials and Equipment
- Personal protective equipment: Head cap, full-sleeve apron, slippers, disposable sterilized gloves, safety goggles, liquid hand sanitizer (70% IPA).
- Media and solutions for microbial testing include:
- Soyabean Casein Digest Agar, Sabouraud Dextrose Agar, Mac Conkey Broth/Agar, XLD Agar, Rappaport Vassiliadis Salmonella Enrichment Broth, Cetrimide Agar, Mannitol Salt Agar, GN Broth.
- Instruments: Incubator, autoclave, colony counter, microscope.
- Testing environment: Laminar Air Flow or Biosafety Cabinet.
Procedure Overview
Sample Preparation
- Water soluble products: Dissolve 1 g or dilute 1 ml in Soyabean Casein Digest Medium (SCDM), volume adjusted to 10 ml (Solution A).
- Water insoluble products: Disperse 1 g or dilute 1 ml in SCDM with 1 g/L Polysorbate 80, volume adjusted to 10 ml (Solution A).
- Fatty products: Homogenize 10 g or 10 ml sample with 5 g sterilized Polysorbate 80, heat if necessary (≤40°C), add buffered sodium chloride peptone solution to total 100 ml (Solution A).
- Alternative diluents: Phosphate buffer pH 7.2 or buffered sodium chloride peptone solution pH 7.0 may be used for all product types.
Inoculation and Controls
- Add prepared sample to test media.
- Prepare positive controls with ≤100 cfu of inoculum.
- Prepare negative controls without sample or inoculum.
Inactivation of Antimicrobials
- If antimicrobial substances are present, inactivate using Polysorbate 80 (30 g/l), Lecithin (3 g/l), Sodium Lauryl Sulphate (4 g/l), or dilution techniques.
Microbiological Tests
1. Microbial Enumeration Tests
Total Aerobic Microbial Count (TAMC):
- Sample aliquots plated in Soyabean Casein Digest Agar, incubated at 30-35°C for 3-5 days.
- Controls: Bacillus subtilis ATCC 6633 (positive), no inoculum (negative).
- Colony forming units (cfu) counted and calculated per g or ml of product.
Total Yeast and Mould Count (TYMC):
- Sample aliquots plated in Sabouraud Dextrose Agar, incubated at 20-25°C for 5-7 days.
- Controls: Candida albicans ATCC 10231 (positive), no inoculum (negative).
- Colony counts are reported similarly to TAMC.
Acceptance Criteria for Enumeration Tests ( as per IP 2018):
| Product Type | TAMC (cfu/g or ml) | TYMC (cfu/g or ml) |
|---|---|---|
| Non-aqueous oral preparations | ≤ 10³ | ≤ 10² |
| Aqueous oral preparations | ≤ 10² | ≤ 10¹ |
| Raw materials & products | As per individual specifications |
2. Tests for Specified Organisms
For each specified microorganism, the procedure involves sample enrichment, selective subculture, incubation, and colony identification based on morphology and color. Positive and negative controls are run simultaneously.
| Organism | Sample Prep & Enrichment | Selective Medium & Incubation | Colony Characteristics | Compliance Criteria |
|---|---|---|---|---|
| Escherichia coli | 1 g/1 ml in SCDM, incubate 18-24h at 30-35°C | Mac Conkey broth (42-44°C, 24-48h), then Mac Conkey agar (30-35°C, 18-72h) | Pink, non-mucoid colonies | Absent per g or ml (or as per specification) |
| Salmonella spp. | 10 g/10 ml in SCDM, incubate 18-24h at 30-35°C | Rappaport Vassiliadis Salmonella Enrichment Broth (30-35°C, 18-24h), XLD agar (30-35°C, 18-48h) | Red colonies with/without black center | Absent per 10 g or 10 ml (or as per specification) |
| Pseudomonas aeruginosa | Same as E. coli enrichment | Cetrimide agar (30-35°C, 18-72h) | Greenish colonies | Absent per g or ml (or as per specification) |
| Staphylococcus aureus | Same as E. coli enrichment | Mannitol Salt Agar (30-35°C, 18-72h) | Yellow/white colonies with yellow zone | Absent per g or ml (or as per specification) |
| Shigella spp. | Same as Salmonella, then 1 ml to GN Broth (30-35°C, 24-48h) | XLD Agar (30-35°C, 24-48h) | Red translucent colonies without black center | Absent per 10 g or 10 ml (or as per specification) |
Annexure - Media Details (per sample)
| Day | Media | Volume Prepared | Purpose |
|---|---|---|---|
| Day I | Soyabean Casein Digest Agar (SCD Agar) | 100 ml | TAMC |
| Sabouraud Dextrose Agar | 100 ml | TYMC | |
| Soyabean Casein Digest Medium / Buffered Peptone Water pH 7.0 | 10 ml | Diluent for TAMC and TYMC | |
| Soyabean Casein Digest Medium | 10 ml | Sample prep and pre-incubation for E. coli, S. aureus, P. aeruginosa | |
| Soyabean Casein Digest Medium | 200 ml | Sample prep and pre-incubation for Salmonella and Shigella | |
| Day II | Mac Conkey Broth | 300 ml | Enrichment of E. coli |
| Rappaport Vassiliadis Salmonella Enrichment Broth | 30 ml | Enrichment of Salmonella | |
| Gram Negative Broth | 300 ml | Enrichment of Shigella | |
| Cetrimide Agar | 75 ml | Enumeration of P. aeruginosa | |
| Mannitol Salt Agar | 75 ml | Enumeration of S. aureus | |
| Day III | Mac Conkey Agar | 75 ml | Enumeration of E. coli |
| XLD Agar | 75 ml | Enumeration of Salmonella | |
| XLD Agar | 75 ml | Enumeration of Shigella |
Key Insights
- The Microbial Limit Test (MLT) ensures safety and quality of non-sterile pharmaceutical products through quantitative and qualitative microbial assessments.
- It integrates aseptic techniques, sample-specific preparation methods, and well-defined acceptance criteria aligned with global pharmacopeial standards.
- The procedure includes controls and inactivation methods to manage potential antimicrobial interference in samples.
- Rigorous testing for specified pathogens (E. coli, Salmonella, P. aeruginosa, S. aureus, Shigella) ensures compliance with safety standards.
- Documentation and traceability are emphasized via specific forms and revisions, supporting quality assurance and regulatory compliance.
This summary encapsulates the full scope of the microbial limit test procedure as detailed in the source document without any external assumptions.
References
- USP 41
- Indian Pharmacopoeia (IP) 2018
- European Pharmacopeia 9.0
- British Pharmacopoeia (BP) 2020
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