SOP for PROCEDURE FOR MICROBIAL LIMIT TEST

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SOP for PROCEDURE FOR MICROBIAL LIMIT TEST

1.0 OBJECTIVE

To establish a standardized procedure for determining microbial load, including specified pathogenic microorganisms, in raw materials and finished products using the Microbial Limit Test (MLT).

2.0 SCOPE

This procedure applies to the analysis of microbial contamination and specified pathogens in raw materials and finished pharmaceutical products tested in the Microbiology Laboratory.

3.0 RESPONSIBILITY

3.1 Trainee Officer / Officer – Microbiology

  • Perform microbial limit testing as per this SOP.

  • Record observations and sign all relevant documents.

3.2 Senior Officer / Executive – Microbiology

  • Provide training to microbiology personnel.

  • Review related documents for accuracy and completeness.

  • Ensure procedures are correctly performed.

3.3 QC Head / Above Executive

  • Review overall activities and documentation.

  • Verify technical correctness and approve records.

  • Ensure adequacy and compliance with this SOP.

3.4 QA Head

  • Approve the SOP.

4.0 ACCOUNTABILITY

The Head of the Department shall be accountable for effective implementation of this SOP.

5.0 DEFINITIONS & ABBREVIATIONS

5.1 Definition

Microbial Limit Test (MLT): A test performed to determine whether a product complies with established microbiological quality specifications.

5.2 Abbreviations

SOP – Standard Operating Procedure
QC – Quality Control
QA – Quality Assurance
OOS – Out of Specification
MLT – Microbial Limit Test
TAMC – Total Aerobic Microbial Count
TYMC – Total Yeast and Mold Count
CFU – Colony Forming Units

6.0 PRECAUTIONS, SAFETY & EHS

  • Perform all tests under aseptic conditions to avoid contamination.

  • Ensure precautions taken do not inhibit the growth of microorganisms to be detected.

  • Follow laboratory safety procedures and wear appropriate PPE.

7.0 MATERIALS AND EQUIPMENT

7.1 Equipment

  • Laminar Air Flow (LAF)

  • Incubators (20–25°C, 30–35°C, 42–44°C)

  • Vortex mixer

  • Colony counter

  • Water bath

  • Anaerobic jar (for Clostridia testing)

7.2 Culture Media (As Applicable)

  • Soybean Casein Digest Agar (SCDA)

  • Sabouraud Dextrose Agar (SDA)

  • Sabouraud Dextrose Broth

  • Fluid Soybean Casein Digest Medium

  • MacConkey Agar & Broth

  • Mannitol Salt Agar

  • Cetrimide Agar

  • Xylose Lysine Deoxycholate (XLD) Agar

  • Rappaport Vassiliadis Salmonella Enrichment Broth

  • Violet Red Bile Glucose Agar

  • Enterobacteria Enrichment Broth (Mossel)

  • GN Broth

  • Reinforced Medium for Clostridia

  • Columbia Agar

  • Levine Eosin Methylene Blue (EMB) Agar

  • Peptone Water

8.0 PROCEDURE

8.1 Sample Preparation

Use 10 g or 10 ml of the product unless otherwise specified.

8.1.1 Water-Soluble Products

Dissolve 10 g/ml in sterile Soybean Casein Digest Medium and make up to 100 ml.
If antimicrobial activity is suspected, add Polysorbate 80 as neutralizer.
Prepare serial tenfold dilutions as required.

8.1.2 Water-Insoluble / Non-Fatty Products

Prepare as above. Add 1 ml Polysorbate 80 per 100 ml if required.

8.1.3 Fatty Products

Homogenize with sterile Polysorbate 80 (not more than half the product weight) at ≤40°C.
Add Soybean Casein Digest Medium to obtain 1:10 dilution.
Maintain temperature for the shortest possible time (not more than 30 minutes).
Adjust pH to 6.0–8.0 if necessary.

8.2 Neutralization of Antimicrobial Activity

Neutralizers may be added to diluent or medium before sterilization.

Interfering Substance Neutralizing Agent
Glutaraldehyde, Mercurials Sodium Sulphite
Phenolics, Alcohol, Sorbates Dilution
Aldehydes Glycine
Quaternary Ammonium Compounds Lecithin
Iodine, Parabens Polysorbate
Mercurials Thioglycollate
Halogens, Aldehydes Thiosulphate
EDTA Magnesium or Calcium ions

8.3 Total Aerobic Microbial Count & Total Yeast and Mold Count

8.3.1 Pour Plate Method

  1. Aseptically transfer 1 ml of prepared sample into sterile Petri plates (duplicate).

  2. Add 15–20 ml SCDA (40–45°C) for TAMC.

  3. Add 15–20 ml SDA (40–45°C) for TYMC.

  4. Allow to solidify and incubate:

    • SCDA: 30–35°C for 3–5 days

    • SDA: 20–25°C for 5–7 days

  5. Count colonies and calculate CFU/g or CFU/ml.

  6. Report:

    • SCDA → TAMC

    • SDA → TYMC

8.3.2 Membrane Filtration Method

  1. Use membrane filter (≤0.45 µm pore size).

  2. Filter 10 ml sample through membrane.

  3. Wash with sterile peptone solution (3 × 100 ml).

  4. Transfer membranes to SCDA and SDA plates.

  5. Incubate as above and calculate CFU.

8.4 Test for Specified Pathogens

8.4.1 Bile-Tolerant Gram-Negative Bacteria

  • Enrich sample in Enterobacteria Enrichment Broth.

  • Subculture on Violet Red Bile Glucose Agar.

  • Incubate 30–35°C for 18–24 hrs.

  • Absence of growth indicates compliance.

8.4.2 Escherichia coli

  • Enrich in MacConkey Broth (42–44°C).

  • Subculture on MacConkey Agar.

  • Brick red colonies indicate possible presence.

  • Confirm using EMB Agar and Indole test.

8.4.3 Staphylococcus aureus

  • Streak on Mannitol Salt Agar.

  • Yellow colonies with yellow zones indicate presence.

8.4.4 Pseudomonas aeruginosa

  • Streak on Cetrimide Agar.

  • Greenish colonies with fluorescence indicate presence.

  • Confirm by pigment production and oxidase test.

8.4.5 Salmonella spp.

  • Enrich in Rappaport Vassiliadis Broth.

  • Subculture on XLD Agar.

  • Red colonies with/without black centers indicate presence.

8.4.6 Clostridia

  • Heat treatment at 80°C for 10 minutes (one portion).

  • Inoculate in Reinforced Medium.

  • Incubate anaerobically.

  • Subculture on Columbia Agar.

  • Gram-positive bacilli with negative catalase confirm presence.

8.4.7 Candida albicans

  • Enrich in Sabouraud Dextrose Broth.

  • Subculture on SDA.

  • Cream-colored colonies indicate presence.

8.4.8 Shigella

  • Enrich in GN Broth.

  • Subculture on XLD Agar.

  • Red translucent colonies with black center indicate presence.

8.5 OOS (Out of Specification) Investigation

8.5.1 Laboratory Investigation

  • Review analyst training.

  • Check media, glassware, environment, equipment calibration.

  • Verify positive and negative controls.

  • Store OOS plates at 2–8°C until disposition.

8.5.2 Repeat Testing

  • Repeat test once using resample.

  • If assignable cause identified → invalidate original OOS.

  • Document corrective and preventive actions.

8.6 Frequency

Perform for each lot of incoming active raw material and as per product specification.

8.7 Acceptance Criteria

If not specified in monograph:

1. Non-Sterile Substances

Parameter Limit
TAMC ≤ 10³ CFU/g or ml
TYMC ≤ 10² CFU/g or ml

2. Non-Sterile Dosage Forms

Must comply with specified TAMC, TYMC limits and absence of pathogens such as:

  • E. coli

  • Salmonella spp.

  • Shigella spp.

  • Staphylococcus aureus

  • Pseudomonas aeruginosa

  • Clostridia

Negative controls must show no growth.

9.0 REFERENCES

  • Indian Pharmacopoeia (IP)

  • British Pharmacopoeia (BP)

  • United States Pharmacopeia (USP)

  • European Pharmacopoeia (EP)

10.0 ENCLOSURES

Annexure I – Microbial Limit Test Record for Pathogens

ANNEXURE – I

MICROBIAL LIMIT TEST REPORT – PATHOGENS

1. Product Details

Product Name : ________________________________________________

Batch No. : ___________________  Batch Size : ___________________

Manufacturing Date : ___________________  Expiry Date : ___________________

Sample Quantity : ___________________  Date of Sampling : ___________________

Date of Testing : ___________________  Date of Release : ___________________

Sampled By : ___________________  Report No. : ___________________

(A) Sample Preparation:

_____ g / _____ ml of sample was dissolved / suspended in _____ ml of Soybean Casein Digest Medium (Solution A).

1.0 Test for Total Aerobic Microbial Count (TAMC) & Total Yeast and Mold Count (TYMC)

_____ ml of Solution A was aseptically transferred in quadruplicate Petri plates.

  • 15–20 ml Soybean Casein Digest Agar (SCDA) was poured into two plates for TAMC.

  • 15–20 ml Sabouraud Dextrose Agar (SDA) was poured into two plates for TYMC.

Observation Table:

Parameter Media Lot No. Incubation Conditions Date of Observation Observation I Observation II Average × Dilution Factor Result
TAMC SCDA: ______ 30–35°C / 3–5 days ______ ______ ______ ______ ______
TYMC SDA: ______ 20–25°C / 5–7 days ______ ______ ______ ______ ______

2.0 Test for Specified Pathogenic Microorganisms

(a) Test for Bile-Tolerant Gram-Negative Bacteria

The previously enriched Solution A was mixed and incubated at 20–25°C for 2–5 hours for resuscitation.

Sr. No. Test Description Incubation (°C) Duration (hrs) Specification Observation Result
A _____ ml of Solution A added to 100 ml Enterobacteria Enrichment Broth (Mossel) 30–35 24–48 Growth observed Yes / No ______
B Streak on Violet Red Bile Glucose Agar 30–35 18–24 Red / Reddish colonies Yes / No ______

(b) Test for Escherichia coli

Sr. No. Test Description Incubation (°C) Duration (hrs) Specification Observation Result
A _____ ml of Solution A added to 100 ml MacConkey Broth 42–44 24–48 Acid & gas formation Yes / No ______
B Streak on MacConkey Agar 30–35 18–72 Brick red colonies Yes / No ______

(c) Test for Pseudomonas aeruginosa

Sr. No. Test Description Incubation (°C) Duration (hrs) Specification Observation Result
A Streak enriched culture on Cetrimide Agar 30–35 18–72 Greenish colonies with fluorescence Yes / No ______
B Oxidase Test — — Purple color on oxidase disc Yes / No ______

(d) Test for Staphylococcus aureus

Sr. No. Test Description Incubation (°C) Duration (hrs) Specification Observation Result
A Streak enriched culture on Mannitol Salt Agar 30–35 18–72 Yellow/white colonies with yellow zone Yes / No ______
B Coagulase Test — — Plasma coagulation observed Yes / No ______

(e) Test for Salmonella spp.

Sr. No. Test Description Incubation (°C) Duration (hrs) Specification Observation Result
A _____ ml Solution A added to Rappaport Vassiliadis Broth 30–35 24–48 Growth observed Yes / No ______
B Subculture on XLD Agar 30–35 24–48 Red colonies ± black centers Yes / No ______

(f) Test for Shigella

Sr. No. Test Description Incubation (°C) Duration (hrs) Specification Observation Result
A _____ ml Solution A added to 100 ml GN Broth 30–35 24–48 Growth observed Yes / No ______
B Subculture on XLD Agar 30–35 24–48 Red translucent colonies without black center Yes / No ______

(g) Test for Candida albicans

Sr. No. Test Description Incubation (°C) Duration Specification Observation Result
A _____ ml Solution A added to 100 ml Sabouraud Dextrose Broth 20–25 3–5 days Growth observed Yes / No ______
B Subculture on Sabouraud Dextrose Agar 20–25 3–5 days Creamy colonies Yes / No ______

(h) Test for Clostridia

Sr. No. Test Description Incubation (°C) Duration (hrs) Specification Observation Result
A _____ ml Solution A added to Reinforced Medium for Clostridia 30–35 48 Growth observed Yes / No ______
B Subculture on Columbia Agar 30–35 48 Slightly yellowish colonies Yes / No ______

Final Remark

The sample Complies / Does Not Comply with the harmonized microbiological specification.

Done By Checked By
Name: ___________________ Name: ___________________
Date: ___________________ Date: ___________________
Signature: _____________ Signature: _____________
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