SOP for Bio-Burden Test

PURPOSE

To check the Bio-burden level on the non-sterile product.

SCOPE

This SOP is applicable for testing of non-sterile components and non-sterile primary packaged/finished product.

RESPONSIBILITY 

QC Microbiologist - To carry out bio-burden in the microbiology laboratory.

ACCOUNTABILITY 

Department Head & QA Head shall be accountable for the implementation of this SOP.

PROCEDURE 

Note: Use membrane filtration method if estimated CFU is low and pour plate if estimated CFU is high.

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Membrane Filtration Method 

• Prepare and sterilize the media as per respective SOP.
• Collect the samples for microbiological examination in pre-sterilized glass bottles.
• Testing is to be performed in the biosafety cabinet.

• Sterilize the required articles and keep them in the dedicated area for analysis.
• Arrange all the required materials in the bio-safety cabinet before starting the test.
• Place the sterile 47mm, 0.45-u membrane filters in the filtration cups.
• For solid products dissolve approximately lg of the sample in 100ml of suitable sterile diluents(Buffered Peptone Water) and filter through a sterile 47mm, 0.45-u membrane filter, by applying vacuum.
• For the samples having anti-microbial activity rinse the membrane with 3 x 100ml of sterile 0.1% peptone water.
• Aseptically place the filtered membrane on the surface of Nutrient Agar / or Soyabean Casein Digest Agar plates.
• Prepare the - ve control late by filtering not less than 100ml sterile water through the membrane and put the membrane on the surface of Nutrient Agar / or Soyabean Casein Digest Agar plates and incubate along with the test samples.
• Incubate the plates at 30℃ -35 ℃ for 48 hours followed by 20℃ -25 ℃ for 72 hours.
• Observe the plates for CFU counts after the incubation period and interpret the result as the number of CFU per 100ml/ gram as applicable.

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Pour Plate Method

• Prepare and sterilize the media as per respective SOP.
• Collect the samples for microbiological examination in pre-sterilized glass bottles.
• Testing is to be performed in a bio-safety cabinet.
• Sterilize the required articles and keep them in the dedicated area for analysis.

• Prepare single plates for each sample to be tested.
• For the solid products dissolved or suspend approximately 1 0g of the sample in 100ml of suitable sterile diluents mark the dilution as 1: 10.
• For the samples estimated to have more microbial load (more than 300 CFU/ml), do the required dilution.
• Pipette out 1.0ml of sample into a sterilized petri plate before adding molten medium.
• Maintain molten medium at about 55℃ and cool to about 40℃ to 45℃ just before use.
• Pour approximately 20ml sterilized molten Nutrient Agar / or Soyabean Casein Digest Agar medium into each plate.
• Smoothly rotate the petri dish clockwise and anti-clockwise wise or do the spread plate method using an L-spreader to mix the sample evenly with the molten agar. Allow the medium to solidify.
• Count all the colonies on the plates after the incubation period with the help of a colony counter and record the counts as several CFU/ml or gm for each sample.
• Record the results.
• Calculation: Count per gm/ml= Dilution Factor x Number of CFU

Safety and Precaution 

• Follow the entry, exist procedure of respective areas for sampling.
• Wear sterile gloves, cap, and facemask while collecting the samples.

Test Limit for Bio-Burden

Bacterial Count: NMT 20 CFU/30 c㎡

Fungal Count: NMT 2 CFU/30 c㎡

ABBREVIATIONS

CFU-Colony Forming unit

SOP-Standard Operating Procedure

NMT-National Microbiological Test

G-Grams

ML-Milliliter

MM- Millimeter

QC-Quality Control

QA-Quality Assurance

ANNEXURE

Nil

REVISION HISTORY

Nil

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