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Microbiology

Disinfectant Validation Procedure

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Pharma Editor
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July 18, 2017
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For Disinfectant validation following materials are required:-

Staphylococcus aureus
B.Subtilis
E.coli.
Candida albicans
Apergillus niger.
Micrococcus species Environmental Isolate
Disinfectants for Validation.
Sterile distilled water.
Sterile Molten Soyabean Caesin Digest Agar
Sterile Molten Potato Dextrose Agar
Poured SCDA plates.
Poured PDA plates
Sterile forceps
Sterile membrane filtration units.
Sterile membranes
Vortex Mixer.

Preparation of Spore forming Culture:

1. Prepare SCDA slants.

2. Incubate slant for 48 hrs at 30 35 C as preincubation to cross check the cont
3. from the working culture streak a loopful of Bacillus subtilis culture on to the n
4. Incubate the slants at 30 35 C for 48 hrs.
 5. After 48 hrs of incubation cross check the culture for any cross contamination b
6. During Gram Staining also check for the presence of vegetative or spore cells.
7. After Gram Staining preserve the cultures for further 7 days.
8. After 7 days cross check the culture for any spore formation by simple Negative

Negative staining:

Prepare a thin smear on a clean slide.

Place the slide on a staining rack above boiling water.
over the smear with small pieces of tissue paper.
Pour malachite green and continue heating till boiling for 5 minutes.
Do not allow the stain to dry.
Gently wash the slide with water.

Preparation of Challenge Inoculum:

Prepare fresh SCDA /PDA slants as per the SOP 12043.

Incubate those slants at 30 35 C for 48 hrs to cross check any type of contamin
From the working culture streak a loopful of culture into the freshly prepared slan
Incubate those inoculated slants at 30 35 C for 48 hrs for bacterial cultures an
Add 5.0 ml of sterile saline into the slants aseptically taking care not to contamina
With the help of a sterile spatula streak the lawn of organisms.
Take 1.0 ml of the culture suspension and make serial dilutions ranging from 1:10

Plate the serial dilutions from 1:10 to 1:100 taking 1.0 ml of the culture in sterile pet

Pour sterile molten SCDA and PDA on to the inoculated plates.

Incubate the plates at the required specified temperature.

After plating the required dilutions do not discard the dilutions and preserve the sam

After the incubation count the number of colonies .

Select the dilution which is having 10000 to 100000 cells / ml for validation study .

Preserve the culture suspension.

Record the data in respective record

Preparation of Disinfectant solution:

Take the concentrated solution as received from the supplier and dilute the disinf
This will be the used dilution.

Determining the initial microbial count:

With the help of an calibrated micropipette pipette out 1.0 ml of any of the cultu
Vortex it for 5.0 minutes.
Filter the sample through a 0.45 m membrane.
Give three washings of 100 ml each with .1 % sterile peptone water.
After filtration , with the help of a sterile forcep take the membrane and place the
After incubation count the number of colonies present on the membrane.
Note down the number of colonies.
This will be the initial counts of the culture.

In the same manner proceed for all the other cultures which are going to be tested.

Record the initial counts in the report as Counts / 0.1ml

Determining the Efficacy of the Disinfectant by Use Dilution Method:

Prepare test tubes having 9.0 ml of sterile distilled water.

Add 1.0 ml of the use dilution for one disinfectant.
Vortex the tube for 5.0 minutes.
Add 0.1 ml of any one culture into the test sample.
Make the sample dilution in such a way that each contact time have two sets of s
Give a contact time of 0 hrs, 5.0 min , 10 min .
After the specified contact time, filter the samples through a 0.45 m membrane.
Give three washings of 100 ml each with .1 % sterile peptone water.
After filtration with the help of a sterile forcep take the membrane and place it on
Incubate the plates .
After incubation count the number of colonies present on the membrane.
Note down the number of colonies.
This will be the final counts of the exposed culture .
Select the plates which have least to Nil counts.
 Proceed in the same manner taking all the cultures to be tested .
Contact time for the usage of the disinfectant will be set on the basis of the resul

Determining the Efficacy of the Disinfectant by Surface Method:

To get a practical approach for the efficacy apply the disinfectants on to any of th
Take S.S strips and Epoxy coated material having a surface area of 25 cm 2.
Wrap the strips and the epoxy coated surface with a aluminium foil and sterilize i
From the previously determined suspension having 10000 100000 cells per ml i
With the help of a sterile spatula spread the culture on the surface.
Keep it on the LAF bench for 30 minutes for drying.
After the exposed duration for drying spray the disinfectant on to any one surface
Allow the surface to be with the sanitiser for 0 hrs, 5.0 min , 10 min.
With the help of a sterile moistened swab , swab the surface gently covering all th
Use different swabs for all the strips.
Place the two swab sticks in a test tube having sterile solution of fluid caesin dige
Vortex the test tube gently for 5. 0 minutes .
Aseptically filter the samples through a 0.45 m membrane.
Give three washings of 100 ml each with .1 % sterile peptone water.
After the filtration with the help of a sterile forcep take the membrane 5.52 and p
Incubate the plates .
After incubation count the number of colonies present on the membrane.
Note down the number of colonies in both the test sample and that with the une
Proceed in the same manner taking all the cultures to be tested and the various s

Evaluation of Results:

The decrease in the bacterial load to the exposed disinfectant indicates that the disi

Schedule Revalidation:

Revalidation shall be carried out in case of

A new disinfectant is received .

If the manufacturer revise the concentration of ingredients.
If a new microorganism is isolated from that particular area.
Documentation:

Validation Report contains the following documents.

Approved Validation protocol

Media Preparation SOP
Gram Staining GTP
Test report for different disinfectants
List of Approved Disinfectants
Summary Conclusion

Conclusion:

Summary report will contain discussion and conclusion which clearly state the succe
Procedure

cubation to cross check the contamination.

acillus subtilis culture on to the newly prepared slant.
ure for any cross contamination by simple gram staining technique.
nce of vegetative or spore cells.
urther 7 days.
ore formation by simple Negative Staining technique.

oiling for 5 minutes.

cross check any type of contamination.

ure into the freshly prepared slants as per the SOP.
r 48 hrs for bacterial cultures and 20 25 C for 5 days for fungal cultures.
ally taking care not to contaminate the slants .
of organisms.
erial dilutions ranging from 1:10 to 1: 100 .

.0 ml of the culture in sterile petridishes.

he dilutions and preserve the samples at a temperature of 2 8 C till the incubation period .

cells / ml for validation study .

the supplier and dilute the disinfectant to prepare a stock solution, which is twice that of the recommended concentration.

tte out 1.0 ml of any of the culture and inoculate into 9.0 ml of the 0.9 % sterile saline solution

rile peptone water.

ake the membrane and place the membrane on a SCDA or m (HPC) agar plate.Incubate the plate .
esent on the membrane.

es which are going to be tested.

Dilution Method:

h contact time have two sets of samples.

es through a 0.45 m membrane.

rile peptone water.
ke the membrane and place it on a SCDA or m (HPC) agar.

esent on the membrane.

es to be tested .
l be set on the basis of the results which will have least counts.

ace Method:

the disinfectants on to any of the surface which is present in that particular area which will be decontaminated by spraying the disinfectan
a surface area of 25 cm 2.
h a aluminium foil and sterilize it in an Dry heat Steriliser at 200 degree C for 2 hrs.
ng 10000 100000 cells per ml inoculate one culture on to three different S. S surface and Epoxy coated surface.
ure on the surface.

isinfectant on to any one surface of the recommended used ilution .

s, 5.0 min , 10 min.
the surface gently covering all the area of the surface.

erile solution of fluid caesin digest Soya broth .

rile peptone water.

p take the membrane 5.52 and place it on a SCDA or m (HPC) agar.

esent on the membrane.

est sample and that with the unexposed strip.
es to be tested and the various sanitisers..

sinfectant indicates that the disinfectant is capable of reducing the contaminant when used in the area.
ion which clearly state the successful achievement of objective of validation studies.
d concentration.
ated by spraying the disinfectant.