REVITAL HEALTHCARE (EPZ) LTD.

T I T L E :SOP FOR BIO-BURDEN TEST

PROCEDURE NO.: ISSUE DATE: EFFECTIVE DATE: NEXT REVIEW DATE:

RHEL/QA/SOP/13 04/02/2022 07/02/2022 07/02/2024

REVISION NO.:03

BIO-BURDEN TEST PROCEDURE.

APPROVED BY: SIGN & DATE

Mariannah Kimatu
(Quality control-Microbiologist)
(Prepared By)

Kevin Mochama
(Quality Control Manager)
(Reviewed By

Nancy Mwangi
(Ass. Quality Assurance Manager)
(Reviewed By)

LucyCaster Muoti
(Quality Assurance Manager)
( Approved By )

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 REVITAL HEALTHCARE (EPZ) LTD.
T I T L E :SOP FOR BIO-BURDEN TEST

PROCEDURE NO.: ISSUE DATE: EFFECTIVE DATE: NEXT REVIEW DATE:

RHEL/QA/SOP/13 04/02/2022 07/02/2022 07/02/2024

REVISION NO.:03

REVISION HISTORY
Revision No. Revised Page(s) Description Date
00 Original issue 01.01.2017
01 05 Test limits for bio-burden 01.09.2019

02 All Footer, Procedure, 01/02/2021

Header, Responsibility

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 REVITAL HEALTHCARE (EPZ) LTD.
T I T L E :SOP FOR BIO-BURDEN TEST

PROCEDURE NO.: ISSUE DATE: EFFECTIVE DATE: NEXT REVIEW DATE:

RHEL/QA/SOP/13 04/02/2022 07/02/2022 07/02/2024

REVISION NO.:03

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.

RESPONSIBILITIES:
QC Microbiologist – To carry out bio burden in microbiology laboratory.

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

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

MEMBRANE FILTRATION METHOD

 Prepare and sterilize the media as per respective SOP.

 Collect the samples for microbiological examination in pre sterilized glass bottles.
 Testing to be performed in bio safety cabinet.
 Sterilize the required articles and keep it in the dedicated area for analysis.
 Arrange all the required materials in 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 1g of the sample in 100ml of suitable sterile
diluents(Buffered Peptone Water) and filter through a sterile 47mm, 0.45-u membrane filters, by
applying vaccum.
 For the samples having anti microbial activity rinse the membrane with 3×100ml of sterile 0.1%
peptone water.
 Asseptically place the filtered membrane on the surface of Nutrient Agar / or Soyabean Casein
Digest Agar plates.
 Prepare the –ve control plate by filtering not less than 100ml sterile water through the membrane

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 REVITAL HEALTHCARE (EPZ) LTD.
T I T L E :SOP FOR BIO-BURDEN TEST

PROCEDURE NO.: ISSUE DATE: EFFECTIVE DATE: NEXT REVIEW DATE:

RHEL/QA/SOP/13 04/02/2022 07/02/2022 07/02/2024

REVISION NO.:03

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˚C-35˚C for 48 hours followed by 20˚C -25˚C for 72 hours.
 Observe the plates for CFU counts after incubation period and interpret the result as number of
CFU per 100ml/ gram as applicable.

POUR PLATE METHOD

 Prepare and sterilize the media as per respective SOP.
 Collect the samples for microbiological examination in pre sterilized glass bottles.
 Testing to be performed in bio safety cabinet.
 Sterilize the required articles and keep it in the dedicated area for analysis.
 Prepare single plates for each sample to be tested.
 For the solid products dissolved or suspend approximately 10g of the sapmle in 100ml of
suitable sterile diluents mark the dilution as 1:10.
 For the samples estimated to be having more microbial load(more than 300CFU/ml), do the
required dilution.
 Pipette out 1.0ml of sample into sterilized petri plate before adding molten medium.
 Maintain molten medium at about 55˚C and cool to about 40˚C to 45˚C just before use.
 Pour approximately 20ml sterilized molten Nutrient Agar / or Soyabean Casein Digest Agar
medium into each plate.
 Smoothly rotate the petri dish clock wise and anti clock wise or do spread plate method using L-
spreader to mix the sample evenly with the molten agar.Allow the medium to solidify.
 Count all the colonies on the plates after incubation period with the help of a colony counter and
record the counts as number of CFU/ml or gm for each sample.
 Record the results.

Calculation: Count per gm/ml= Dilution Factor×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.

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 REVITAL HEALTHCARE (EPZ) LTD.
T I T L E :SOP FOR BIO-BURDEN TEST

PROCEDURE NO.: ISSUE DATE: EFFECTIVE DATE: NEXT REVIEW DATE:

RHEL/QA/SOP/13 04/02/2022 07/02/2022 07/02/2024

REVISION NO.:03

TEST LIMITS FOR BIO BURDEN

Bacterial Count Fungal Count

NMT 20 CFU/30CM² NMT 2 CFU/30CM²

REFERENCE:
QA/FM-016
Reference standard used- ENISO 11737 Part 1 and part 2 -2006

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

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