PHARMACEUTICAL MICROBIOLOGY (BP303T)Unit-III Part-5 Evaluation of Bactericidal and Bacteriostatic (Disinfectant). The common methods used for evaluation of a disinfectant are as follows, Tube Dilution Method. Agar Plate Method. Filter Paper & Cup Plate Method. Ditch-Plate Method. Phenol Coefficient Method. The official phenol coefficient tests include, Rideal-Walker Test (RW Test). Chick-Martin Test. United States FDA Test for Phenol Coefficient. (FDA Test) The US Association of Official Agricultural Chemists Test (FDA Test) A. Rideal-Walker Test: Kelsey Sykes Method

1. PHARMACEUTICAL MICROBIOLOGY (BP303T)
Unit-III
Part-5
Evaluation of Bactericidal and Bacteriostatic (Disinfectant).
Name: Ms. Pooja Deepak Bhandare
Assistant Professor
G H RAISONI UNIVERSITY
SCHOOL OF PHARMACY

2. Evaluation of Disinfectants
• The common methods used for evaluation of a disinfectant are as
follows,
1. Tube Dilution Method.
2. Agar Plate Method.
3. Filter Paper & Cup Plate Method.
4. Ditch-Plate Method.
5. Phenol Coefficient Method.
6. Kelsey Sykes Method.

3. 1. Tube Dilution Method
• Most commonly used method.
• In this method, a series of tubes is set up, each containing the
same quantity of a standard growth liquid medium
(Commonly Mueller-Hinton broth) and a gradually increasing
concentration of the disinfectant to be tested.
• The tubes are then inoculated with the same quantity of cell
suspension of the test microorganism and incubated for 2-3 days
@ 30-35℃.
• The first tube in the series where there is complete absence of
growth of the test microorganism denotes the minimal inhibitory
concentration (MIC) of the disinfectant.

5. 2. Agar Plate Method
•It is very similar to the tube dilution method.
•In this method, Petri plates containing a standard growth
agar medium (usually Mueller-Hinton agar) are taken at the
place of lubes containing liquid medium.
•Disinfectants of various concentrations are inoculated on the
agar surface of plates already inoculated with the same
quantity of the test microorganism.

6. •Plates are incubated and then examined for growth.
•The first plate in the series where there is complete
absence of growth of the test microorganism denotes
the minimal inhibitory concentration (MIC) of the
disinfectant

7. 3. Filter Paper & Cup Plate Method:
•These methods are known as “Agar Diffusion Tests”.
•The agar is melted and cooled @ 45℃, inoculated with test
microorganisms and poured in a sterile petri plate.
•In case of “Cup Plate Method” when the agar is solidified
the holes of 9 mm diameter were made by using sterile
cork borer and the disinfectant is directly placed in it.
•In case of the Filter Paper method the filter paper disks
soaked in disinfectant solution are placed in the holes.

8. • The zone of inhibition is observed after incubation @ 30-35 ℃.
• During incubation, the disinfectant diffuses into the agar.
• The diameter of the zone of inhibition gives indication of activity of the
disinfectant, larger the diameter greater is the efficiency of the disinfectant.
• A zone of Inhibition is proportional to the amount of the antimicrobial
agent added to the filter paper disc, the solubility of the agent, the diffusion
coefficient, and the overall effectiveness of the agent.

9. 3. Ditch-Plate Method:
• Agar is melted and then solidified in a petri plate.
• A ditch is made in the petri plate by cutting the soliodified agar.
• The disinfectant solution is made to run through the ditch carefully.
• The test organisms are streaked outwards from the ditch.
• The petriplate is incubated at desired temp. And time period.
• The microorganisms which are resistant to the disinfectant grow even
near the start at the ditch itself.
• The sensitive organisms show a zone of inhibition near the ditch or at
center of the petri plate.
• The width of the zone of inhibition is an indication of activity against
the test organism.

11. 4. Phenol Coefficient Method:
• In this method the efficacy of the disinfectant in use is rated by
comparing it with the activity of Phenol taking as a standard.
• The test is carried out by adding an increasing amount of phenol and
disinfectant in test tubes containing microorganisms.
• In UK the test organism used is Salmonella typhi while the USA uses
Salmonella typhi, Staphylococcus aureus and Pseudomonas
aeruginosa.
• The official phenol coefficient tests include,
• Rideal-Walker Test (RW Test).
• Chick-Martin Test.
• United States FDA Test for Phenol Coefficient. (FDA Test)
• The US Association of Official Agricultural Chemists Test (FDA Test)

12. A. Rideal-Walker Test:
• Introduced by Rideal and Walker the British chemists in 1903 are still in
use.
• The test uses Rideal, Walker Broth and Salmonella typhi as a test
organism.
• Different dilutions of the phenol and test disinfectants are made and 5
ml of each is inoculated with the 0.5 ml of the 24 hr broth culture of
the test microorganism.
• All the test tubes are placed in a water bath at 17.5 ℃.
• Subcultures from each test tube are taken and transferred to 5 ml
sterile broth after 2.5, 5, 7.5 and 10 minutes.

13. • The broth tubes are incubated at 37 ℃ for 2 - 3 days and are examined for
the presence or absence of the growth and the Phenol coefficient is calculated
using the formula,
• The Phenol Coefficient for phenol is considered as “1” any value for
disinfectant coming below one is considered as less while above it is more.

14. • Advantages of Phenol Coefficient Test:
• Quick.
• Inexpensive.
• Reproducible results.
• Useful to eliminate useless products.
• Sets standard for the preparations.
• Disadvantages of Phenol Coefficient Test:
• Most tests use only one test organism i.e. Salmonella typhi which represents
inadequate information.
• Compares the activity of disinfectants at only one concentration at fixed
conditions.
• Presence of organic matter at action time is not considered.
• Does not give information about tissue toxicity of the disinfectant.
• Sampling errors are large.

15. 5. Kelsey-Skyes Mehod:
• Developed in 1969, this test overcomes many drawbacks of the RW Test.
• The test uses several test organisms viz. Staphylococcus aureus, Proteus vulgaris,
Eschericia coli and Pseudomonas aeruginosa.
• Tests can be carried out in a clean as well as dirty environment.
• In both clean and dirty cases the final bacterial concentration should be about
109/ml.
• Clean conditions are simulated by using a clean broth while dirty conditions are
simulated by using a yeast suspension or inactivated horse serum.
• The samples are taken at 8, 18 and 28 minutes are then incubated at 30 to 32 ℃
and observations of growth of microorganisms in test tubes are recorded.
• Result interpretation: A disinfectant is considered satisfactory if,
• No growth in 2 or 5 tubes of 18 min sample or,
• No more than 5 colonies from 5 drops on the agar plate.

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