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1. PT .....
VALIDATION PROTOCOL
Protocol No.
Replaced
Prepared
Date
ANALYTICAL METHOD VALIDATION
MICROBIAL CONTAMINATION TEST
(FILTRATION AND POUR PLATE)

2. PT .....
VALIDATION PROTOCOL
Protocol No.
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APPROVAL SHEET
Analytical Method Validation Protocol for microbial contamination test has
been reviewed and approved by:
QUALITY CONTROL MANAGER DATE
QUALITY ASSURANCE MANAGER DATE

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I. LIST OF CONTENT
Topic Chapter Page
Title N/A N/A
Approval Sheet N/A N/A
List of Content I 1
Revision History II 2
Background III 2
Purpose IV 2
References V 2
Scope VI 3
Responsibility VII 3
Procedure:
- Equipment and material
- Preparing inoculums
- Inspection LAF (Laminar Air Flow)
- Fertilities Test
- Effectiveness Test
- Recovery Test
- Limit
VIII 4-6
II. REVISION HISTORY
No. Revision No. Revision History
1. Original Version

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III. BACKGROUND
Pharmaceutical of raw material, finished product and packaging material
needs to be checked microbiological contamination test. These tests
have to be performed aseptically under laminar air flow (LAF class 100)
and consist of inspection of aerobic microbial contamination and
Identification test (ID Test). The aerobic microbial contamination
performed with filtration and pour plate method.
IV. PURPOSE
To prove and ensure that analytical method has been validated
conform to specification.
V. REFERENCES
Microbiological examination of non sterile products (Total viable
aerobic count) (2.6.1.2) and Microbiological examination of non-
sterile products (Test for specified micro-organisms) (2.6.1.3)
European Pharmacopoeia 5th edition 2005 ( Lab Kimia)
SOP Cara Mengoperasikan Inkubator (Microbiology Laboratory).
Validation of Aseptic Pharmaceutical Processes, Frederick J. Carleton
& James P. Agalloco (Microbiology Laboratory).
Aseptic Pharmaceutical Manufacturing Technology for the 1990s
edited by Wayne P Olson, page 315 – 354 (Microbiology Laboratory).
VI. SCOPE
Analytical Method Validation of microbial contamination test was
performed on existing analytical method that has been used for period
time in microbiology laboratory PT Wira.
Analytical method validation consists of following parameter:
Fertility test of testing media (Growth Promotion Test)
Effectiveness of testing media (to be used) and ID test of the testing
bacteria.
VII. RESPONSIBILITY
7.1. Analyst/assigned operator
- Responsible in preparing media and equipment/tools and also
conduct the analysis.

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7.2. Microbiology Lab. Supervisor (Microbiologist)
- Responsible to prepare validation protocol.
- To train the analyst regarding method of validation.
- Reviewing to the validation report
7.3. QC Manager and QA Manager
- Responsible in evaluating, reviewing and approving of the
validation protocol and validation report.
- To ensure all activities of validation have been conducted
precisely and correctly according to validation protocol.
VIII. PROCEDURE
8.1. Equipment and Material
8.1.1. Equipments:
- A set of filtration holder
- 2 litres erlenmeyer
- pincers
- sterile membrane filter 0,45 μm
- pipette
8.1.2. Materials:
- Tryptone Soy Agar (TSA) media
- Tryptone Soy Agar (TSA) media with penase
- Saboroud Dextrose Agar (SDA) media
- Saboroud Dextrose Agar (SDA) media with penase
- Mac-Conkey Broth
- Mac-Conkey Agar
- Tetrathionate Broth (TB)
- Massal Broth (MB)
- Tryptone Soy Broth (TSB)
- Xylose Lysine Deoxycholate (XLD) agar
- Brilliant Green Phenol Red Lactose (BPLS) agar
- Baird Parker Agar (BPA)
- Vogel Johnson Agar (VJA)
8.2. Inoculum Preparation

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8.2.1. Bacteria and fungi are used as follows:
- Staphylococcus aureus ATCC ...
- Bacillus subtilis ATCC ...
- Pseudomonas aeroginosa ATCC ...
- Candida albican ATCC ...
- Aspergillus niger ATCC ...
- Escherichia coli ATCC ...
- Salmonella abony ATCC ...
8.2.2. Making series of inoculums dilution as according to SOP
“Pembuatan dan Pemantauan Seri Pengenceran Inokulum“
(SOP Making and Monitoring of Inoculums Dilution) from
each bacteria and fungi as mentioned above with contain
not more from 100 cfu/ml.
8.2.3. Concentration from final dilution that used for testing
should be noted at validation worksheet.
8.3. Environmental Monitoring of LAF
8.3.1. Conduct the environmental monitoring during validation of
microbial contamination test in LAF ...(class 100) by using
settle plate.
8.3.2. Incubate of settle plate and rodac plate at temperature 30-
35o
C for 5 days.
8.3.3. Read out the microorganism growth and noted at validation
worksheet.
8.4 Fertility Test of Media (Growth Promotion Test)
8.4.1. Prepare each 4 settle plate of TSA media, add 0,1 ml
suspension of subculture of Staphylococcus aureus ATCC
6538, Bacillus subtilis ATCC 6633, Pseudomonas aeroginosa
ATCC 9027, Candida Albican ATCC 10231, Aspergilus niger
ATCC 16404 (each contain ±100 cfu microorganism) to
indvidual of settle plate.
8.4.2. To streak the microorganism to the certain media smoothly.
8.4.3. Incubate of TSA Media at temperature 30-35o
C for 5 day and
read out the microorganism growth of bacteria and fungi.
8.4.4. Read out and noted the grow colony of bacteria at validation
worksheet.
8.4.5. Prepare the 4 settle plate of SDA media, add 0,1 ml (contain
±100 cfu microorganism) suspension of subculture of Candida

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albican ATCC 10231, Aspergilus niger ATCC 16404 to
individual of settle plate.
8.4.6. To streak the microorganism to the certain media smoothly.
8.4.7. Incubate of TSA SDA Media at temperature 20-25o
C for 5 day
and read out the microorganism growth of bacteria and
fungi.
8.4.8. Read out and noted the grow colony of bacteria at validation
worksheet.
8.5. Effectiveness of Testing Media
8.5.1. Each a number of 3 batches of sample that used in the form
of finished product, material and both of penicillin and non
penicillin raw material.
8.5.2. Conduct the test of sample microbial contamination as
according to SOP “Uji Kontaminasi Mikroba” (SOP Test of
Microbial Contamination).
8.5.3. Add 0.1 ml following microorganism:
- Staphilococcus aureus,
- Pseudomonas aeroginosa,
- Salmonella abony,
- Bacillus subtilis,
- Ccandida albican,
- Escherichia coli dan
- Aspergillus niger.
to 100 ml diluted sample.
8.5.4. Pipette 1ml sample solution which has been added with
microorganism, and then pour with 20-25 ml TSA or 25-30 ml
SDA for the non penicillin sample and pour with 20-25 ml TSA
penase or 25-30 ml SDA penase for the penicillin sample into
4 Petri dish.
8.5.5. For the primary packaging sample, filled and shake with 100
ml Saline and filtered by using sterile membrane filter 0,45
μm.
8.5.6. Incubate of TSA media/TSA media with penase at
temperature 30-35o
C during 5 day.
8.5.7. Incubate of TSA media/TSA media with penase at
temperature 20-25o
C during 5 day.
8.5.8. Read out and noted of microorganism observation at
validation worksheet.

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8.6. ID Test
8.6.1. Identify the microorganism of that sample which have been
added with certain of microorganism as according to SOP The
Microbial Contamination ...
8.6.2. Read out and noted the result at validation worksheet.
8.7. Limit
8.7.1. Environmental Monitoring by Settle Plate
8.7.2. Acceptance Criteria:
Fertility Test of media meets test if:
- Negative control media test shown negative result (No
growth microorganism).
- Positive control media test shown positive result
(growth microorganism).
Effectiveness test of testing media meets test if:
- Positive control test media shown positive result
(growth microorganism).
- Positive control media of product test shown positive
result (growth microorganism) and the recovery test
not less than 50%.
Ruangan Class
Limit (cfu/plate)
Alert Action
Bakteri Jamur Bakteri Jamur
LAF 06.116 100 <1 <1 1 1

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(Worksheet)
A. Reagent end Equipment
1. Dry Heat Sterilizer Heraeus
Calibration Date :
Recalibration :
2. Autoclave ...
Calibration Date :
Recalibration :
3. Inkubator ...
4. Calibration Date :
Recalibration :
5. Inkubator ...
Calibration Date :
Recalibration :
6. Balance ...
Calibration Date :
Recalibration :
7. Inkubator ...
Calibration Date :
Recalibration :
8. Inkubator ...
Calibration Date :
Recalibration :

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9. Tryptone Soya Agar
Batch No. :
Expired Date :
Merck :
10.Saboraoud Dextrose Agar
Batch No. :
Expired Date :
Merck :
11.Mac-conkey Agar
Batch No. :
Expired Date :
Merck :
12.Mac-conkey Broth
Batch No. :
Expired Date :
Merck :
13.Tryptone Soya Broth
Batch No. :
Expired Date :
Merck :
14.XLD Agar
Batch No. :
Expired Date :
Merck :

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15.BPLS Agar
Batch No. :
Expired Date :
Merck :
16.Cetrimide Agar
Batch No. :
Expired Date :
Merck
17.Staphylococus aureus ATCC ...
Batch No. :
Expired Date :
Merck :
18. Bacillus subtillis ATCC ...
Batch No. :
Expired Date :
Merck :
19. Pseudomonas aeroginosa ATCC ...
Batch No. :
Expired Date :
Merck :
20. Escherichia coli ATCC ...
Batch No. :
Expired Date :

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Merck :
21. Salmonella abony ATCC ...
Batch No. :
Expired Date :
Merck :
22. Candida albican ATCC ...
Batch No. :
Expired Date :
Merck :
23. Aspergillus niger ATCC ...
Batch No. :
Expired Date :
Merck :
B. Microbial Contamination Test
B.1. Fertilities Test
Media TSA
Date/
Analyst
Microorganism Blank Media + microorganism Remark

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(cfu/plate)
Media SDA
Date/
Analyst
Microorganism Blank
Media + microorganism
(cfu/plate)
Remark

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B.2. Effectiveness Test and ID Test
Media TSA
Date/
Analyst
Product
Name
Lot No. Microorganism
TSA +
microorganism
(cfu/plate)
TSA + Product +
microorganism
(cfu/plate)
%
Recovery
ID Test

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Media SDA
Date/
Analyst
Product
Name
Lot No. Microorganism
TSA +
microorganism
(cfu/plate)
TSA + Product +
microorganism
(cfu/plate)
%
Recovery
ID Test