1.0 Objective:
The objective of this protocol is to provide sufficient documented
evidence in order to prove that the disinfectants employed for cleaning and
sanitization are effective against wide range of microorganisms by employing
Membrane Filtration method to establish the effective working concentration and
contact time.
2.0 Scope:
The scope of this Validation protocol is to
assure the efficacy of different disinfectant used for cleaning as well as
sanitizing the production manufacturing, microbiology testing areas and rest of
the company premises (general areas) which could be capable enough to kill wide
range of microbial flora generally prevailing in those areas.
3.0 Pre –Validation Requirement:
3.1 Selection of Microorganisms:
The microorganisms
selected are based on different classifications. The selected microorganisms
include:
ü Gram positive bacteria (Staphylococcus
aureus ATCC No. 6538 or equivalent)
ü Gram positive spore
forming bacteria (Bacillus subtilis
ATCC No. 6633 or equivalent)
ü Gram negative bacteria (Pseudomonas
aeruginosa ATCC No. 9027 or equivalent)
ü Yeast (Candida
albicans ATCC No. 10231 or equivalent)
ü Mold
(Aspergillus brasiliensis ATCC No.16404 or equivalent)
ü Environment Isolate if
available
3.2 Selection of
Disinfectants:
Disinfectants should be selected based on the following characteristics
as mentioned below.
ü
Should be able to kill a wide range of microbial flora including
pathogenic microbial strains.
ü
Should be non-toxic to humans on general application.
ü
Should not corrode the equipment surfaces on application.
ü Should not possess any
bad odour or residues on application.
3.3 Required Media, Diluents and other Accessories:
ü
Sterile molten Soyabean Casein Digest Agar (SCDA)
ü Sterile molten Sabouraud
dextrose agar (SDA)
ü Sterile Normal saline (0.9
% of sodium chloride solution)
ü Sterile Purified Water
or water for injection
ü Sterile Membrane
Filtration assembly with vacuum pump
ü Sterile Forceps
ü Vortex Mixer.
ü Sterilized graduated 1ml
and 10ml glass pipette.
ü Calibrated micropipettes
and sterile micro tips
ü Sterile 0.1% w/v peptone
water.
ü
Measuring cylinder.
ü
Disinfectant of different concentration.
ü
Sterile 0.9 % w/v normal saline tubes (10 ml)
ü
Sterile cotton swabs.
ü
Contact Plates.
ü
Cyclomixer
ü Poured sterile SCDA
plates
4.0 Selection of Different Surfaces:
Different surface are selected for the validation
of Disinfectants to provide the documented evidence that all the disinfectants
with required concentration and contact time are effective on all the surfaces
which we have included as per company premises product manufacturing and
testing areas.
4.1 List of different
surfaces included for disinfectant validation studies are given below:
ü
Stainless Steel surface
ü
Glass surface
ü
GI doors surface
ü
Panel surface
ü
Granite surface
ü
Cota stone Surface
ü
PU coated surface
ü
Paint coated surface
ü
Epoxy coated surface
ü
Glove Surface
5.0 Method for Disinfectant Validation:
Membrane Filtration technique is one of the
simplest and effective analytical method for evaluation of
sanitizer/disinfectant efficacy. This method involves the direct mixing of
microbial cells or spores with the sanitizer/disinfectant and subsequent
filtering of the solution at various intervals of contact time between
sanitizer/disinfectant solution and microbial cultures. This method completely
removes any the chances of bacteriostasis or Fungistasis.
6.0 Process for Validation
of Efficacy of Disinfectant will be performed under
following subheadings:
6.1 Preparation of challenge inoculums:
ü
Take required type of working culture (Spore culture in case of Bacillus subtilis).
ü
Take loopful of each working culture (Spore culture in case of Bacillus
subtilis) and inoculate in to different tubes containing 10 ml Sterile saline
and dilute the suspension serially using 10 fold dilution method from 10-1 to
10-8.
ü
Pipette out 0.1 ml of 10-1 diluted culture on to each of
two Sterile Petri dishes.
ü
Repeat this step for 10-2, 10-3, 10-4,
10-5, 10-6, 10-7, and 10-8. Repeat
the above 4 steps for all other cultures.
ü
Add 20 ml of SCDA medium for bacteria, SDA for Fungi that is melted and
cooled at approximately 450 C.
ü
Cover the Petri dishes and mix the sample with Agar.
ü
Allow to solidify at room temperature. Invert the Petri dishes and
incubate bacteria at 30-350 C for 48 hours, Candida albicans and Aspergillus brasiliensis at 20-250 C
for 5 days.
ü
Preserve all the dilutions at 2-80 C. Examine the plate count
as the average of two plates in terms of CFU’s/0.1 ml. Select the dilutions
containing 10,000 to 100000 CFU’s/0.1 ml, store at 2-80 C and
discard all other dilutions.
ü Record the data in
Attachment No-II.
6.2 Preparation of Bacillus spore culture:
ü
Take a pre-incubated sterile SCDA slant and a working culture of
Bacillus.
ü
Pick a loopful of Bacillus culture and streak on surface of sterile SCDA
slant.
ü
Incubate the slant at 30-35 0C for 7 days.
ü
Perform spore staining of this incubated culture and observe for its
purity and spore formation.
üIf the culture shows spore, preserve this culture at 2-80C
and use this spore culture for the preparation of inoculum of Bacillus as
mentioned above.
ü
Record the data in Attachment No-I.
ü
Use the preserved dilution culture suspension within 7 days.
7.0 Disinfectants
and Concentrations:
Prepare the disinfectants in sterile purified water
or sterile water for injection and filter the disinfectants with 0.22 micron
filters. The concentration and contact time of disinfectants should be based on
the manufacturer recommendation. Prepare three different concentration of one
disinfectants in which select one concentration which is recommended by manufacturer,
one lower concentration and one higher concentration than the recommended
concentration. Same with the contact time, use one contact time which is recommended
by manufacturer, then choose one lesser contact time and one higher contact
time. For example if recommended concentration by manufacturer is 1.0% for any
disinfectant with 10 minutes of contact time then we can select 0.5% which is
lower concentration with contact time of 5 minutes which is again less contact
time than the recommended contact time. Select one higher
concentration of 2.0% which is higher than the recommended and higher contact
time of 15 minutes. By preparing three different concentrations and different contact time
perform the validation study.
7.1 Use
Dilution Method:
ü
Dilute disinfectant solution as per the selected concentration.
ü
Use sterile purified water or sterile water for injection as diluent.
ü
Arrange required number of sterile empty test tubes in a test tube rack.
ü
Use calibrated micropipettes or graduated pipettes for dispensing the
inoculums and disinfectant solution.
ü
Add 10 ml of manufacturer recommendation concentration of disinfectant
solution in a test tube.
ü
Add 10 ml of lower and higher concentrations of disinfectant in other
test tubes.
ü
Arrange one sterile tube containing 10 ml of diluent (for initial count
of challenge inoculum).
ü
Add 0.1 ml of prepared challenge inoculum to 10 ml of diluent.
ü
Dilute the solution using 10 fold dilution from 10-1 to
10-8 and plate from 10-1 Dilution.
ü
Incubate these plates for initial count determination at appropriate
temperatures.
ü
Arrange the sterile filter holder having 0.45µm membrane filter on
manifold and assemble the manifold to vacuum source.
ü
Add 0.1 ml of challenge inoculum to all the tubes which contains
disinfectant solution.
ü Contact time of all concentrations
(0.5%, 1.0%, and 2.0%) should be done at room temperature first for 5 minutes.
For positive controls, inoculation done in duplicate for each challenge
microbial culture strain and Negative control should be kept un-inoculated.
After the completion of ‘5’ minutes contact time, the solutions should be filtered
using separate filter holder. Each membrane filter was rinsed with 3 X 100 ml
sterile 0.1% peptone water to remove the traces of disinfectants.
ü
Neutralizers could be used if required to neutralize any traces of
disinfectant on the filter or we may use media containing neutralizers.
ü
After rinsing, place each membrane filter on the surface of individual
pre-incubated agar medium plates.
ü
Similarly challenge all the prepared concentration for contact time of 10
and 15 minutes intervals individually, mix and filter the solution of each tube
using individual filter holder for each time. Rinse each membrane filter
with 3 X 100 ml sterile 0.1% peptone water.
ü
After rinsing place each membrane filter on the surface of individual
pre-incubated agar medium plates.
ü
Incubate all plates at 30oC – 35oC for 48 hours for bacteria and the plates with the challenge inoculums of Candida albicans and Aspergillus niger at 20oC – 25oC
for 5 days. Examine all plates each day for the presence of any microbial
growth and compare with initial count.
ü
Similarly Challenge different concentrations of other disinfectants with
0.1 ml of the inoculums used in the previous test at different contact time
individually and proceed as given above employing membrane filtration.
ü Record the data in
Attachment No-III.
7.2 Surface Challenge Test:
This method involves application of disinfectants
to the different surfaces, challenged with standard test organisms, at the
selected use concentration with a specified contact time and determining the
log reduction of the challenge microorganisms.
7.3 Determination of efficacy of disinfectant:
ü Perform all the
activities under Laminar Air Flow.
ü Prepare plates of
Soyabean casein digest Agar (SCDA) and Sabouraud Dextrose Agar (SDA).
ü Use pre-incubated plates
for validation study.
ü Use the dilutions of the
culture suspension and challenge the surfaces of the material to be evaluated
with 0.1 ml of the inoculums.
ü Spread the cell
uniformly over the surface of 2 inch X 2 inch square with the help of sterile L
shaped spreader.
ü Allow the suspension to
dry.
ü After completely drying
spray the recommended concentration of the chosen disinfectant on the surface
where inoculums are spread and note the contact time.
ü Take required number of
readymade sterile swab for sampling and wet those swabs in sterile 0.9% saline
solution under LAF.
ü Take the swab from the
surface of 2 inch X 2 inch square by following horizontal strokes first
followed by vertical strokes.
ü Replace the swab in to
the tube with saline and mark the swab tube properly with date of sampling and
name of organisms.
ü Vortex the tube by
cyclomixer to make the suspension uniform.
ü Filter the content of
the swab by 0.45µm pore size filter paper.
ü Wash membrane with 3x100
ml of 0.1% sterile peptone water to remove residue of disinfectant.
ü Aseptically transfer the
membrane filter to the pre-incubated plate of Soyabean casein digest agar and Sabouraud dextrose agar.
Avoid air trapping between the filter and agar surface.
ü Incubate the plates of
bacteria at 30-35°C for 48 hours and for fungus 5 days.
ü
Observe the plates after completion of incubation; express the results
in cfu/plate.
ü Record the data in
Attachment No-IV.
ü Fill the validated
concentration and contact time for particular disinfectant as per Attachment
No.-V.
8.0 Acceptance Criteria:
ü
The concentration of disinfectant which shows required reduction in
microbial count at the least concentration and contact time shall be selected as effective
concentration.
ü
The disinfectant solution should bring a minimum of 3-log reduction for
vegetative cells and minimum 2 log reduction for spore cells.
9.0 Re-Validation:
The Disinfectant Efficacy validation shall be
re-validated in one or more of following cases:
ü
Replacement of any disinfectant.
ü
Change in the composition of disinfectant by manufacturer.
ü Change in cleaning
procedure
10.0 Change Control System:
Any changes in the proposed equipment/system/procedure shall
be carried out through the change control procedure.
11.0
Validation Report:
ü
The Validation report shall consist of a summary document, in narrative
form, which shall briefly describe the activity performed along with the
observations recorded.
ü
This report shall also include the related documents and
attachments/annexure which completed at the time of validation activity.
Preparation
of Bacillus subtilis Spores
Working
Culture No
|
|
MTCC
No.
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Equivalent
ATCC No.
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Incubation
Temperature
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Agar
medium used
|
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Incubation
Period
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Medium
Lot No.
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Incubator
ID No.
|
|
Staining Method used:
Result of Staining: ________________________________________________________________________________
________________________________________________________________________________
Done By/Sign: Checked By/Sign: Reviewed By/Sign:
Preparation
of Challenge inoculum
Name
of the Culture: Working
culture tube No.Used:
Strain
No:
Equivalent ATCC No.:
Name
of the Diluent used: Dilution made:
Agar
Medium used: Medium Lot No:
Incubation
period and Temperature:
Incubator I.D.No.:
Dilution
Plated
|
Count/0.1ml/plate
|
Plate-1
|
Plate-2
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Average
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Interpretation: As per
the above Observation it is estimated that the________ dilution tube contain
the culture concentration of ______________________ CFU’s/0.1 ml
Done By/Sign: Checked By/Sign: Reviewed By/Sign:
Use
Dilution Test
Name of Disinfectant:
MTCC No.:
Name of the Organism: Equivalent ATCC No.:
Date of test:
Date of observation:
Name
of the Organism
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|
Initial
Count
|
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Log
of Initial Count
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Concentration
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Contact
Time _______________
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Contact
Time _______________
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Contact
Time _______________
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Log Reduction Calculation:
|
Concentration and Contact time
|
Log
of CFU’s recovered in initial count – Log of CFU’s recovered in Test
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Remarks: Minimum concentration
and Contact Time found to be effective_______________
Acceptance Criteria:The disinfectant should bring a minimum of 3-log
reduction or more.
Done By/Sign: Checked By/Sign: Reviewed By/Sign:
Surface
Challenge Test
Name of Disinfectant: Name
of Surface:
Recommended Concentration:
Date of test:
Date of observation:
Observations:
|
Cultures
|
Contact
Time
|
Initial count
|
Log of Initial
Count (a)
|
Count
Observed in
Test
|
Log of Observed Count (b)
|
Log
Reduction
(a-b)
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Staphylococcus
aureus
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Bacillus
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Pseudomonas
aeruginosa
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C.albicans
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Aspergillus brasiliensis
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Negative Control
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Remarks: The disinfectant is able/
not able to bring _____ log reduction for the
microorganism at _______% concentration and _______min and is suitable
/not suitable for use.
Done
by/Date: Checked
By/Date: Approved
by/Date:
Done by/Date: Checked By/Date: Approved by/Date:
Thanks and have a nice day!
