VALIDATION PROTOCOL Navana Pharmaceuticals Ltd.

Rupshi, Narayanganj, Bangladesh

TITLE: VALIDATION PROTOCOL OF MICROBIAL LIMIT TEST BY POUR PLATE METHOD

Document No. AMVP/MICROBIAL LIMIT TEST Issue Date January 02, 2019 Revision No. 01
Copy No.
Supersedes 00,February 14,2016 Effective Date
Page 1 of 6
Department Quality Control Review Date January 01, 2022

1. INTRODUCTION
This test is designed to perform quantitative estimation of mesophilic bacteria and fungi that may
grow under aerobic condition as well as to determine whether a substance or preparation complies
with an established specification for microbiological quality.

2. AIM
To establish that the pour-plate enumeration method has the ability to ensure that the product has no
microbial inhibitory properties which could produce false negative and also has the ability to detect
microorganisms in the presence of the product to be tested.

3. SCOPE
This procedure is applicable for each batch of a substance or preparation required to meet
microbiological quality in the Navana Pharmaceuticals Ltd.

4. REFERENCE
4.1.1. USP 41, Chapter (61 & 62).
4.1.2. BP 2016, Appendix XVI B. Tests for Microbial Contamination
4.1.3. ISO 9001: 2008, Clause- 4.2.3 & 4.2.4.

5. EQUIPMENT & APPARATUS

5.1.1. Micro pipette and sterile tips
5.1.2. Vortex mixture
5.1.3. Inoculating loop
5.1.4. Burner etc

6. REAGENT/MEDIA
6.1.1. Tryptone Soya Broth
6.1.2. Tryptone Soya Agar
6.1.3. Sabouraud Dextrose Agar

7. CHALLENGE MICROORGANISMS
7.1.1. Staphylococcus aureus ATCC 6538
7.1.2. Pseudomonas aeruginosa ATCC 9027
7.1.3. Aspergillus brasiliensis ATCC 1 6404

Prepared by: Checked by: Approved by:

Name Md. Rasel Bhuiyan Shahana Shilpi Deb Narayan Biswas
Designatio Executive, Sr. Manager, QC DGM, QA
n Microbiology
Sign. &
Date
 VALIDATION PROTOCOL Navana Pharmaceuticals Ltd.
Rupshi, Narayanganj, Bangladesh

TITLE: VALIDATION PROTOCOL OF MICROBIAL LIMIT TEST BY POUR PLATE METHOD

Document No. AMVP/MICROBIAL LIMIT TEST Issue Date January 02, 2019 Revision No. 01
Copy No.
Supersedes 00,February 14,2016 Effective Date
Page 2 of 6
Department Quality Control Review Date January 01, 2022

7.1.4. Candida albicans ATCC 10231

7.1.5. Bacillus subtilis ATCC 6633

8. PROCEDURE
8.1.1. Growth promotion of the media
8.1.1.1. Follow the instruction of the growth promotion test SOP (SOP/NP/MB/003).

8.1.2. Suitability of the counting method in the presence of product

8.1.2.1. Preparation of the sample
The Methods for sample preparation depends upon the physical characteristics of the
material/product to be tested. Sample preparation can be carried out as follows:
Water-soluble product: Dissolve or dilute (usually 1 in 10 dilution prepared) the product
to be examined in buffered sodium chloride peptone solution pH 7.0 or phosphate buffer
solution pH 7.2 or Soyabean casein digest broth. If necessary adjust to pH 6-8. Further
dilutions where necessary are prepared with the same diluents.
Non-fatty products insoluble in water: Dissolve or dilute (usually 1 in 10 dilution is
prepared) the product to be examined in buffered sodium chloride peptone solution pH
7.0, or phosphate buffer solution pH 7.2 or soyabean casein digest broth. A surface-
active agent such as 1 g/l of Polysorbate 80 may be added to assist the suspension of
poorly wettable substances. If necessary adjust to pH 6-8. Further dilutions where
necessary are prepared with the same diluents.
Fatty Products: Dissolve in Isopropyl Myristate sterilized by filtration, or mixed the
product to be examined with the minimum necessary quantity of sterile Polysorbate 80
or another noninhibitory sterile surface active reagent heated, if necessary, to not more
than 40°C or in exceptional cases not more than 45°C. Mix carefully and if necessary
maintain the temperature in a water bath. Add a sufficient amount of prewarmed
chosen diluents to make a 1 in 10 dilution of the original product. Mix carefully while
maintaining the temperature for the shortest time necessary for the formation of an
emulsion. Further serial 10 fold dilution may be prepared using the chosen diluents
containing a suitable concentration of sterile Polysorbate 80 or another noninhibitory
sterile surface active reagent.
8.1.2.2. Dilution of the prepared sample:
Dilute sample prepared as directed above by buffered sodium chloride peptone solution
Prepared by: Checked by: Approved by:
Name Md. Rasel Bhuiyan Shahana Shilpi Deb Narayan Biswas
Designatio Executive, Sr. Manager, QC DGM, QA
n Microbiology
Sign. &
Date
 VALIDATION PROTOCOL Navana Pharmaceuticals Ltd.
Rupshi, Narayanganj, Bangladesh

TITLE: VALIDATION PROTOCOL OF MICROBIAL LIMIT TEST BY POUR PLATE METHOD

Document No. AMVP/MICROBIAL LIMIT TEST Issue Date January 02, 2019 Revision No. 01
Copy No.
Supersedes 00,February 14,2016 Effective Date
Page 3 of 6
Department Quality Control Review Date January 01, 2022

pH 7.0, or phosphate buffer solution pH 7.2 or soyabean casein digest broth further 1 in
100 and 1 in 1000.
8.1.2.3. Inoculation of organisms in to the diluted sample( positive product control)
Add a sufficient volume of each of the organisms listed in 7.0 separately to the all
dilution of the prepared to obtain of inoculums of not more than 100 cfu and the
volume of suspension of the inoculum should not exceed 1% of the volume of diluted
sample and follow the table1 and 2.
8.1.2.4. Inoculation of organisms in to the control (positive control)
Add a sufficient volume of each of the organisms listed in the point 7.0 separately to
dilution medium to obtain of inoculums of not more than 100 cfu and the volume of
suspension of the inoculum should not exceed 1% of the volume of dilution medium
and follow the table1 and 2.
8.1.2.5. Negative control
Verify the testing condition by performing a negative control, using the chosen diluent
in place of the test preparation and follow the table 1 and 2.
8.1.2.6. Enumeration of Total Aerobic Microbial Count (Bacillus subtilis, Pseudomonas
aeruginosa, Staphyloccus aureus)
Table 1
StepNo. Negative control Positive control Product control
1 Aseptically pipette out 1 ml Aseptically pipette out 1 ml Aseptically pipette out 1 ml
into duplicate sterile into duplicate sterile into duplicate sterile
petridishes. petridishes. petridishes from each diluted
product
2 To each of the petridishes To each of the petridishes To each of the petridishes add
add about 15-20 ml of add about 15-20 ml of about 15-20 ml of Tryptone
Tryptone Soya agar, mix and Tryptone Soya agar, mix Soya agar, mix and allow
allow solidifying. and allow solidifying. solidifying.
3 Invert the petridishes and Invert the petridishes and Invert the petridishes and
incubate at 30-35°C for five incubate at 30-35°C for five incubate at 30-35°C for five
days. days. days.
4 Count the number of Colony Count the number of Count the number of Colony
Forming Units developed at Colony Forming Units Forming Units developed at
the end of the incubation developed at the end of the end of the incubation and
and record the results. the incubation and take take arithmetic average of

Prepared by: Checked by: Approved by:

Name Md. Rasel Bhuiyan Shahana Shilpi Deb Narayan Biswas
Designatio Executive, Sr. Manager, QC DGM, QA
n Microbiology
Sign. &
Date
 VALIDATION PROTOCOL Navana Pharmaceuticals Ltd.
Rupshi, Narayanganj, Bangladesh

TITLE: VALIDATION PROTOCOL OF MICROBIAL LIMIT TEST BY POUR PLATE METHOD

Document No. AMVP/MICROBIAL LIMIT TEST Issue Date January 02, 2019 Revision No. 01
Copy No.
Supersedes 00,February 14,2016 Effective Date
Page 4 of 6
Department Quality Control Review Date January 01, 2022

arithmetic average of counts .

counts.

Results and interpretation:

The product under test is considered non inhibitory to microorganism under the defined test if the
following conditions are not met at the lowest possible dilution factor such as ( 1 in 10, 1 in 100, 1 in
1000):
 The test is valid, if negative control shows no growth.
 The recovery of organism in positive product control should do not differ by a factor greater than 2
from positive control.

8.1.2.7 Enumeration of Total yeasts and molds Count (Aspergillus brasiliensis, Cndida albicans)
Table 2
Step Negative control Positive control Product control
No.
1 Aseptically pipette out 1 Aseptically pipette out 1 ml into Aseptically pipette out 1 ml into
ml into duplicate sterile duplicate sterile petridishes. duplicate sterile petridishes from
petridishes. each diluted product
2 To each of the To each of the petridishes add To each of the petridishes add about
petridishes add about about 15-20 ml of Sabouraud 15-20 ml of Sabouraud Dextrose
15-20 ml of Tryptone Dextrose Agar, mix and allow Agar, mix and allow solidifying.
Soya agar, mix and allow solidifying.
solidifying.
3 Invert the petridishes Invert the petridishes and incubate Invert the petridishes and incubate
and incubate at 30-35°C at 20-25°C for five days. at 20-25°C for five days.
for five days.
4 Count the number of Count the number of Colony Count the number of Colony
Colony Forming Units Forming Units developed at the end
Forming Units developed at the end
developed at the end of of the incubation and take
the incubation and of the incubation and take arithmetic average of counts and
record the results. subtract it from the count of positive
arithmetic average of counts.
product control in the same dilution
factor.

Results and interpretation:

The product under test is considered non inhibitory to microorganism under the defined test if the
following conditions are not met at the lowest possible dilution factor such as ( 1 in 10, 1 in 100, 1 in
Prepared by: Checked by: Approved by:
Name Md. Rasel Bhuiyan Shahana Shilpi Deb Narayan Biswas
Designatio Executive, Sr. Manager, QC DGM, QA
n Microbiology
Sign. &
Date
 VALIDATION PROTOCOL Navana Pharmaceuticals Ltd.
Rupshi, Narayanganj, Bangladesh

TITLE: VALIDATION PROTOCOL OF MICROBIAL LIMIT TEST BY POUR PLATE METHOD

Document No. AMVP/MICROBIAL LIMIT TEST Issue Date January 02, 2019 Revision No. 01
Copy No.
Supersedes 00,February 14,2016 Effective Date
Page 5 of 6
Department Quality Control Review Date January 01, 2022

1000) :
 The test is valid, if negative control shows no growth.
 The recovery of organism from positive product control does not differ by a factor greater than 2
from the positive control.
8.1.3 Neutralization or Removal of Antimicrobial Activity
If growth is inhibited, then modify the procedure for particular enumeration test to ensure the validity
of the results. Modification of the procedure may include, for example, a) An increase in the volume
of the diluents or culture medium; b) Incorporation of a specific or general neutralizing agents
(Table:3) into the diluents. If for a given product the antimicrobial activity with respect to a
microorganism for which testing is prescribed cannot be neutralized, then it is to be assumed that the
inhibited microorganism will not be present in the product.
Table 3
Interfering Substances Potential Neutralizing Agents/Method
Glutaraldehyde, mercurials Sodium hydrogen sulfite
Phenolics, alcohol, aldehydes, sorbate Dilution
Aldehydes Glycine
Quaternary ammonium compounds (QACs), parahydroxy Lecithin
benzoates (parabens), bis-biguanides
QACs, iodine, parabens Polysorbate
Mercurials Thioglycollate
Mercurials, halogens, aldehydes Thiosulfate
EDTA(edentate) Mg or Ca ions

1.0 CHANGE CONTROL

Head of the Department is only authorized to make any changes in this SOP through the change
control SOP
2.0 LIST OF DISTRIBUTION
Sl. Division/Department/Section Copy Sl. Division/Department/ Copy No.
No. No. No. Section
01 QUALITY ASSURANCE
(MASTER COPY)
02 QUALITY CONTROL(Central)
Prepared by: Checked by: Approved by:
Name Md. Rasel Bhuiyan Shahana Shilpi Deb Narayan Biswas
Designatio Executive, Sr. Manager, QC DGM, QA
n Microbiology
Sign. &
Date
 VALIDATION PROTOCOL Navana Pharmaceuticals Ltd.
Rupshi, Narayanganj, Bangladesh

TITLE: VALIDATION PROTOCOL OF MICROBIAL LIMIT TEST BY POUR PLATE METHOD

Document No. AMVP/MICROBIAL LIMIT TEST Issue Date January 02, 2019 Revision No. 01
Copy No.
Supersedes 00,February 14,2016 Effective Date
Page 6 of 6
Department Quality Control Review Date January 01, 2022

03 QUALITY CONTROL
(CEPHALOSPORIN PLANT)

3.0 CHANGE HISTORY

Sl. No. Name of Initiator Revision No. Effective date Reason for change
01 Farzana Afroze 00 01 FEB 2016 New
1. Logo Change
02 Md. Rasel Bhuiyan 01
2. Content Change

4.0 ANNEXURE
4.1 Annexure I: Validation Report of Microbial Limit Test by Pour Plate Method (F/NP/MB/024).

Prepared by: Checked by: Approved by:

Name Md. Rasel Bhuiyan Shahana Shilpi Deb Narayan Biswas
Designatio Executive, Sr. Manager, QC DGM, QA
n Microbiology
Sign. &
Date