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TABLE OF CONTENTS

APPROVAL SIGNATURE

OVERVIEW

• Objective
• Purpose & Scope
• Responsibility

EXECUTION TEAM
TRAINING RECORD

REQUIREMENTS FOR QUALIFICATION

SYSTEM/ EQUIPMENT DESCRIPTION

• Preparation of Bacillus subtilis spore culture

• Preparation of Challenge Inoculum
• Validation by Filtration Method
• Validation by Surface Method.

QUALIFICATION PROCEDURE OR METHODOLOGY

• General Recording Instructions

• General Safety Instruction for Execution
• Preparation of Bacillus subtilis spore culture

1. Take a preincubated sterile SCDA slant and a working culture of Bacillus

subtilis
2. Pick up a loopful of Bacillus subtilis culture and streak on surface of sterile
SCDA slant
3. Incubate the slant at 30 – 35 ° C for 7 days
4. Perform Spore staining for this incubated culture and observe for its purity
and spore formation
5. If the culture show spores (discard if contaminated), preserve this culture at 2-
8 °C and use this spore for the preparation of Inoculum of Bacillus subtilis
6. Record the Result as per Annexure-02

• Preparation of Challenge Inoculum

1. Purpose
2. Requirement
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3. Test Procedure

• Disinfectant Validation by Use Dilution Method

• Disinfectant Validation by Surface Method

ACCEPTANCE CRITERIA

• The reduction of Challenge Inoculum count should be more than 3 log for
bacterial cultures

OBSERVED DEVIATION (IF ANY)

QUALIFICATION REPORT

ABBREVIATIONS

LIST OF ANNEXURES

REFERENCE DOCUMENT

APPROVAL SIGNATURE

• This is specific protocol for Disinfectant Efficacy validation which is being used
in the plant for Cleaning and Fumigation purpose. The Author signature
indicates that this document has been prepared in accordance with existing
standards and adequately reflects the tasks and deliverables necessary for
disinfectant efficacy test.

• The reviewer’s signature indicates that, this document has been reviewed and
it accurately and completely reflects the tasks and deliverables necessary for
process.

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• The approver’s signature indicates that the documentation and information

contained herein complies with applicable regulatory, corporate, divisional /
departmental requirements, and current Good Manufacturing Practices.

OVERVIEW

Objective

To establish methodology for Disinfectant efficacy Validation used in Pharmasky Ltd.

Purpose & Scope

The purpose is to provide an outline for the efficacy of the Disinfectants used in the
plant for the disinfection of surfaces, air and disinfection of hands etc.

Responsibility

To conduct the qualification study, a team shall be formed. The team shall contain
the members from the, Quality Control and Quality Assurance departments. The
Validation team is described through the following responsibility.

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EXECUTION TEAM

Following personnel shall be responsible for the execution of qualification study;

1. Executive – QC (Microbiology) : To conduct the qualification study as per

protocol.
2. Executive - Formulations : To provide material for testing.
3. Executive Quality Assurance : To review & monitor the qualification study.

TRAINING RECORD

Purpose

The purpose of the training is to familiarize the trainees with the requirement of
Disinfectant Efficacy Validation.

Scope

This Training is applicable to the all concerned persons which are involved in this
activity.

Topics

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1. The following topics shall be covered during training:

2. Purpose & procedure of Disinfectant Efficacy Validation.
3. Identifying the responsibility of involved person.
4. Documentation practices to be followed.
5. General precautions / guidelines to be followed during qualification.
6. Attach training record with the report as Annexure – 01.

REQUIREMENTS FOR QUALIFICATION

Following items shall be qualified before execution of Disinfectant Efficacy validation:

• Microbial Culture

1. Staphylococcus aureus
2. Bacillus subtilis
3. Pseudomonas aeruginosa
4. Clostridium sporogenes
5. Candida albicans
6. Aspergillus niger
7. E. coli
8. S. abony
9. Environment isolate

• Media

1. Sterile Molten Soybean Casein Digest Agar

2. Sterile Molten Sabouraud Chloramphenicol Agar
3. Reinforced Clostridial Agar
4. Sterile Peptone water
5. Sterile Letheen broth
6. Preincubated sterile SCDA, SCA plates

• Sterile forceps and scissors

• Sterilized filtration unit and Membrane filters
• Manifold
• Vortex mixer
• Forceps
• Micropipette
• Biosafety Cabinet
• Disinfectants to be validated

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SYSTEM/ EQUIPMENT DESCRIPTION

The validation will be performed under the following subheadings:

• Preparation of Bacillus subtilis spore culture.

• Preparation of Challenge Inoculum.
• Validation by Filtration Method.
• Validation by Surface Method.

QUALIFICATION PROCEDURE OR METHODOLOGY

General Recording Instructions

• Read the contents of the document thoroughly before proceeding for

Execution of the activity (in case of doubts / contradictions / contact the
approvers of the document for clarifications).
• Recording of all the observations and data shall be as per good documentation
practices SOP.
• Recording will be done on controlled copy of Disinfectant efficacy validation
annexure.
• In each of the tests/process outlined in the protocol the test parameter,
specifications, acceptance criteria and any other requirement are pre-defined.
Other columns viz. observations, verified by and other columns are to be filled
in by the member of execution team manually.
• Follow the specific instructions as mentioned in the particular test for verifying
the parameters & recording the observations.
• Recording the requested information in a particular test indicates that it has
been inspected / verified according to specifications by visual examination,
product literature, using a measuring device etc.
• All the deviations / discrepancies observed during the activity are to be
explained & justified if acceptable.
• Record all deviations / discrepancies observed during the execution in the
Observed deviations format of protocol.

General Safety Instruction for Execution

Safety will be one of the key considerations during the execution of this document.
The following guidelines must be observed during the execution stage.

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• All personnel involved with the execution shall identify hazards associated
with performance of testing and precautions to be taken.
• All personnel involved with the execution shall check that utilities are safely
isolated when energizing or de-energizing.
• All personnel involved in the execution shall inform the company management
any hazard, to themselves or others, associated with the materials,
equipment, method of working and the precautions to be taken.

Preparation of Bacillus subtilis spore culture

• Take a preincubated sterile SCDA slant and a working culture of Bacillus

subtilis.
• Pick up a loopful of Bacillus subtilis culture and streak on surface of sterile
SCDA slant.
• Incubate the slant at 30 – 35°C for 7 days.
• Perform Spore staining for this incubated culture and observe for its purity
and spore formation.
• If the culture show spores (discard if contaminated), preserve this culture at 2-
8°C and use this spore for the preparation of Inoculum of Bacillus subtilis.
• Record the Result as per Annexure-02.

Preparation of Challenge Inoculum

Purpose

The purpose of the test is to prepare the challenge Inoculum of selected culture used
for Disinfectant Efficacy Validation.

Requirement

Sterile Peptone water, Sterilized tips, Micropipette, culture & Biosafety cabinet etc.

Test Procedure

• Take required types of working cultures (Spore culture in the case of Bacillus
subtilis).
• Inoculate a small loop of each working culture (Spore culture in the case
of Bacillus subtilis) and inoculate in to different bottles containing 50 ml

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sterile saline and dilute the suspension serially using 10 fold dilution method
from 10-1 to 10-10.
• Pipette out 1.0 ml of 10-4 diluted culture onto each of two sterile petri dishes
• Repeat this step for 10-5, 10-6, 10-7, 10-8, 10-9& 10-10. Repeat the above 4
steps for all other cultures (as per point no. 5.1.1) also.
• Add 20-25 ml of Agar Medium (SCDA for Aerobic bacteria, RCA for Anaerobic
bacteria and SCA for Fungi) that is melted and cooled to approximately 45°C.
• Cover the petri dishes and mix the sample with agar by tilting or rotating
the petri dishes.
• Allow to solidify at room temperature. Invert the petri plates and
incubate. (Aerobic bacteria at 30° to 35° for 48 hrs. Anaerobic bacteria at 30°
to 35° for 48 hrs in an anaerobic jar, Candida albicans at 20° to 25° for 72 hrs.
and Aspergillus niger at 20° to 25° for 5 days.)
• Preserve all the dilutions at 2-8 ° C. Examine the plates, count the number
of colonies and express the average of 2 plates in terms cfu/plate. Select
the dilutions containing 100000 to 1000000 CFU’s / ml, preserve at 2-8°C
and discard all other dilutions
• Use the preserved dilute culture suspensions within 7 days.
• Record the data as per Annexure-03.

Disinfectant Validation by Use Dilution Method

Purpose

This test is performed so as to ensure the suitable concentration & contact time for
the disinfectant to achieve ≥ 3 Log reduction by filtration technique.

Requirements

Filtration Funnels, flasks, vacuum pump, sterile membrane filters, sterile peptone
water, medium etc.

Test Procedure

• Dilute disinfectant solution as per the recommendation of manufacturer

and also prepare 1/2 and 1/4 concentrations from the
manufacturers recommendation.
• Use sterile purified water or sterile water for injection as diluent.
• Arrange 3 sets (3 bottles in each set) of sterile empty 25 ml tubes (for
one culture).
• Add 10 ml of manufacturer’s recommended concentration of the
disinfectant solution in 1st tube of each set.
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• Add 10 ml of 50% strength of disinfectant solution as per

manufacturer recommended concentration of the disinfectant in 2nd tube of
each set.
• Add 10 ml of 25% strength of disinfectant solution as per
manufacturer recommended concentration of the disinfectant in 3rd tube of
each set.
• Arrange one sterile bottle containing 10 ml of sterile peptone water (for initial
count of the challenge Inoculum).
• Add 1 ml of prepared challenge inoculum of E.coli to this 100 ml of diluent.
• Dilute this solution using 10 fold dilution methods from 10-1 to 10-4.
• Arrange the sterile filter holders having 0.45 μm membrane filters on
the manifold and assemble the manifold to vacuum source.
• Add 1 ml of the Challenge Inoculum of E.coli to all the tubes of 3 sets.
• Mix and immediately filter the solutions of 1st set using individual filter holder
for each tube. This is ‘0 min’ contact sample.
• Filter the solution meant for initial count using another filter holder.
Rinse each membrane filter with 3X100 ml of sterile 0.1% peptone water.
• After rinsing, place each membrane filter on the surfaces of individual
agar medium plates.
• After 5 minutes, mix and filter the solutions of 2nd set using individual
filter holder for each tube. This is ‘5 min’ contact sample. Rinse each
membrane filter with 3X100 ml sterile 0.1% peptone water.
• After rinsing, place each membrane filter on the surfaces of individual
agar medium plates.
• After another 5 minutes, mix and filter the solutions of 3rd set using individual
filter holder for each tube. This is ‘10 min’ contact sample.
• Rinse each membrane filter with 3X100 ml sterile 0.1% peptone water.
• After rinsing, place each membrane filter on the surfaces of individual
agar medium plates.
• Repeat the whole procedure for each organism as per above point.
• Use same agar medium and incubation conditions as mentioned in point
no. ‘Preparation of Challenge Inoculum’.
• Examine the plates, count the number of colonies on each plate.
• Compare the ‘0 min’ ‘5 min’ ’10 min’ contact culture plates with ‘Initial count’
plates.
• Record the results as per Annexure-04.

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Disinfectant Validation by Surface Method

Purpose

To get a practical approach for the efficacy apply the disinfectants on to any of
the surface which is present in that particular area which will be decontaminated
by spraying the disinfectant.

Requirement

SS strip, Epoxy Coated strip, sterile swab.

Procedure

• Take 3 sets (3 strips in each set) each of S.S and Epoxy coated strips having
a surface area of 25 cm2.
• Take one more strip each of SS and Epoxy coated.
• Wrap all the strips with aluminum foil and Depyrogenate it in hot air oven
at 250°C for 1 hrs.
• Prepare different concentrations of the disinfectant solution as mentioned
in the ‘Use dilution method’.
• Arrange the strips in the LAF.
• Add 1 ml of Challenge Inoculum to each Epoxy coated and SS strips.
• Allow the culture to dry on the surface of strips for 30 minutes or until they dry.
• Spray the manufacturer recommended concentration of disinfectant
on surfaces of 1st strip of each set, 50% of manufacturer
recommended concentration of disinfectant on surfaces of 2nd strip of each
set and 25% dilution of manufacturer recommended concentration of
disinfectant on surfaces of 3rd strip of each set.
• Do not spray the disinfectant on 10th strip, which will be used to determine the
‘Initial count’.
• With the help of different, individual sterile swabs, swab the
disinfectant exposed culture surfaces of strips of 1st set. Add each swab to
individual test tube containing 10 ml of sterile Letheen broth.
• Mix, filter and follow further process as mentioned for ‘0 min’ contact sample
of ‘Use dilution Method’.
• Swab the culture surfaces (Not exposed to disinfectant) of SS and Epoxy strip
surfaces and add each swab to individual test tube containing 10 ml of sterile
Letheen broth. Dilute this solution up to 10-4
• Mix, filter and follow further process as mentioned for ‘Initial count sample’ of
‘Use dilution Method’.

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• After 5 minutes, swab the disinfectant exposed culture surfaces of all the strips
of 2nd set with the help of different, individual sterile swabs and add each
swab to individual test tube containing 10 ml of sterile Letheen broth.
• Follow the filtration, plating and incubation procedure as mentioned in ‘5 min’
contact sample of ‘Use dilution Method'.
• After further 5 minutes, swab the disinfectant exposed culture surfaces of
the strips of 3rd set with the help of different, individual sterile swabs and
add each swab to individual test tube containing 10 ml of sterile Letheen
broth.
• Follow the filtration, plating and incubation procedure as mentioned in
‘10 min’ contact sample of ‘Use dilution Method'.
• Repeat the whole procedure for each Challenge inoculum as mentioned in
above point.

• Examine the plates; count the number of colonies on each plate.

• Compare the ‘0 min’ ‘5 min’ ’10 min’ contact culture plates with ‘Initial count’
plates.
• Record the results as per Annexure-05.

ACCEPTANCE CRITERIA

The reduction of Challenge Inoculum count should be more than 3 log for bacterial
cultures.

OBSERVED DEVIATION (IF ANY)

• Allot a sequential number starting from 01 for each deviation / discrepancy

observed during the execution of the activity. Record the details of the test /
parameter during which the deviation / discrepancy was observed, along with
a brief description of the deviation / discrepancy.
• The deviation / discrepancy shall be addressed as per the SOP “Deviation
Process”.

QUALIFICATION REPORT

• The qualification report shall consist of a summary document, in narrative

form, which shall briefly describe the activity performed along with the
observations recorded in relevant exhibits.
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• This report shall also include the related documents and attachments / exhibit
which were completed at the time of qualification activity.

ABBREVIATIONS

SCDA : Soyabean Casein Digest Agar

RCA : Reinforced Clostridial Agar

LAF : Laminar Air Flow

SCA : Sabouraud Chloramphenicol Agar

CFU : Colony Forming Unit

ANNEXURES

01 - Training Record

02 - Report for Bacillus subtilis Culture

03 - Report for Preparation of Challenge inoculum

04 - Report for Disinfectant Efficacy test by Use Dilution method.

05 - Report for Disinfectant Efficacy test by surface method.

REFERENCE DOCUMENT

Nil

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