Evaluation of Antimicrobial Potency of Disinfectants,Anti-Infective . (Phenol Coefficient Method,Tube dissolution method ,Agar Plate method)

1. Evaluation of Antimicrobial potency of
Disinfectants, Anti-infective and antibiotics(.Phenol
Coefficient Method,Tube dissolution Method,Agar
Plate Method )
PRESENTED BY AREEBA SHAFIQ
M.PHILL PHARMACEUTICS SCHOLAR (ISLAMIA UNIVERSITY
BAHAWALPUR,PAKISTAN)

2. Contents
 Disinfectants
 Evaluation of Antimicrobial Potency of Disinfectants
 Anti-Infective and Antibiotics
 Evaluation of Antimicrobial Potency of Anti-Infective and
Antibiotics .

3. Disinfectants
• A disinfectant is a chemical substance or compound used to
inactivate or destroy microorganisms on inert surfaces.
• Disinfection does not necessarily kill all microorganisms, especially
resistant bacterial spores; it is less effective than sterilization, which
is an extreme physical or chemical process that kills all types of
life.

4. Classification of Disinfectant
 Phenol: Chlorophenols.etc
 Alcohols:Ethanol.etc
 Aldehydes:Formaldehyde. etc
 Dyes:Triphenylmethane .etc.
 Surface Active Agents :Sodium Stearate etc
 Halogens:Chorine etc
 Metals:Copper etc .
 Guanidines &Amidines:Chlorhexidine.etc
 Furan Derivatives :Nitrofurazone .etc
 Quinolines and Isoquinolines :Hydroxyquinoline.etc.

5. Need for testing the disinfectants
A disinfectant must be tested
• To know the required effective dilution.
• To know the time taken for the onset of action.
• Periodical monitoring of its activity. As disinfectants are known to
loose their action on long standing & in the presence of organic
matter their efficacy must be tested periodically.

6. Evaluation of Antimicrobial potency of Disinfectant
Phenol Coefficient Method
• The measure of the disinfecting power of a substance, determined by dividing the figure
indicating the degree of dilution of the disinfectant that kills a microorganism within a
given time by that indicating the degree of dilution of phenol killing the microorganism
under similar conditions.
Two types of phenol coefficient tests are done:
1. Rideal Walker method
2. Chick Martin test

7. 1.Rideal Walker Method
Phenol coefficient test is suitable for testing disinfectants miscible
with water and which exert their antimicrobial action in manner
similar to that of phenol.
• Test Organism: Salmonella typhi
• Standard disinfectant: Phenol

8. • Different dilutions of the test disinfectants and phenol are prepared and 5ml of each
dilution is inoculated with 0.5 ml broth culture of the organisms for 24 hr.
• All tubes (disinfectants + organisms and phenol + organisms)are placed in 17.5 °C water
bath.
• Subcultures of each reaction mixture are taken and transferred to 5ml sterile broth after
2.5, 5, 7.5 and 10 min.
• The broth tubes are incubated at 37 °C for 48 to 72 hr and are examined for presence or
absence of growth.

9. • If a phenol coefficient or Rideal-Walker coefficient of a given test disinfectant
is 1, it means that disinfectant has same effectiveness as phenol.
• If a phenol coefficient or Rideal-Walker coefficient of a given test disinfectant
is less than 1, it means that disinfectant is less effective than phenol.
• If a phenol coefficient or Rideal-Walker coefficient of a given test disinfectant
is more than 1, it means that disinfectant is more effective than phenol.
• If the phenol coefficient of the test disinfectant is 20 it means that the
disinfectant is 20 times more active than phenol.

10. 2.Chick Martin
• Chick martin test incorporates the presence of organic matter as the test is not carried
out in the water but yeast suspension or 4% dried human feces.
• The total time of the test is 30 minutes.
• Both S. typhi and S. aureus cultures are used to test the efficacy of disinfectants.
• The calculation method is the same as that Rideal Walker test.

11. • To calculate for phenol coefficient, the number indicating the degree of dilution of
the disinfectant in which it kills the microorganism within a given time is divided
by the number indicating the degree of dilution of phenol in which the latter kills
the microorganism in the same period of time and under the same conditions.
• A phenol coefficient that is greater than 1 indicates that the disinfectant is more
effective than phenol. In contrast, a phenol coefficient that is lower than 1 means
the disinfectant is less effective than phenol.

12. Comparing Rideal Walker and Chick Martin test

13. Benefits
 Cheaply carried out and quickly Analyzable
 Effectiveness of the disinfectant can be expressed quantitatively.

14. Anti –Infective Agent
An agent that is capable of acting against infection, either by inhibiting the spread of an
infectious agent or by killing the infectious agent .
Antibiotics
Antibiotics are medicines that fight bacterial infections in people and animals. They
work by killing the bacteria or by making it hard for the bacteria to grow and multiply.
Evaluation of Antmicrobial Potency of Anti-
Infective Agent and Antibiotics

15. Need for testing Antibiotics
 Help find out which antibiotic will be most effective in treating
your infection.
 To obtain more realistic and precise measurement of potency to
overcome the antibiotic resistance problem.

16. 1.Agar Plate Method
1.Preparation of Inoculum
From each inoculated agar plate, a minimum of four colonies were touched with
a sterile loop and transferred into a tube containing normal saline (0.9%) or in
broth and density of each microbial suspension was adjusted equal to that of 108
cfu/ml (standardized by 0.5McFarland standard) and was used as the inoculum.
2.Inoculation of Test Plate
The test is performed by applying a bacterial inoculum of approximately 1–
2×108CFU/mL to the surface of a large (150 mm diameter) Mueller-Hinton agar
plate.

17. 3. Application of Disks
Then, filter paper discs (about 6 mm in diameter), containing the test
compound(Antibiotics ) at a desired concentration, are placed on the agar
surface with sterile Forceps or a suitable disc dispenser.
4.Incubation
Plates are incubated for 16–24 h at 35°C prior to determination of results.

18. 5.Zone of Inhibition
Generally, antimicrobial agent diffuses into the agar and inhibits
growth of the test microorganism . Measuring the diameter of zone of
Inhibition by using a ruler or Sliding Caliper.

19. Benefits
 Easy to perform
 Inexpensive
 Allow Visibility of growth
 Correct Inoculum

21. 2. Tube Dilution Method
• Used to determine minimal concentration of antibiotic to inhibit or kill
the microorganism.
• Achieved by dilution of antibiotic in either agar or broth media.
Minimum Inhibitory Concentration:
The lowest concentration of drug that inhibit the growth of the bacteria
isolated from the patient.

22.  In this method we use Sterile Muller Hinton Broth
 We make 2 folds dilution of Antibiotics in the broth i.e 2µg/ml,4µg/ml
,8µg/ml,16µg/ml and so on.
 Then we add broth culture (0.1ml) of test organism to the Prepared
dilutions.
 One tube without antibiotic served as the organism control.
 After adding test organism we incubate the tubes at 37ͦC for 24 hours .
 Examine tubes for visible signs of bacterial growth. The tube exhibiting no
visible growth and containing the least amount of antibiotic was
considered the minimal inhibitory concentration (MIC).

24. Benefits
 Standard method for determining level of Microbial Resistance to
an antimicrobial agent .
 The MIC provides the ability to precisely determine the
concentration of antibiotic required to inhibit growth of a pathogen.

25. References
 References
1.https://slidetodoc.com/antibiotic-susceptibility-testing-presented-by-dr-quratulain-antibiotic/.
2.https://www.slideshare.net/visprod/antibiotic-sensitivity-tests
3. https://www.rxlist.com/anti-infective/definition.htm.
4.https://www.biologyonline.com/dictionary/phenol-coefficient
5.1. Chitwood, L.A.J.A.M., Tube dilution antimicrobial susceptibility testing: efficacy of a microtechnique
applicable to diagnostic laboratories. 1969. 17(5): p. 707-709.
6.https://www.researchgate.net/figure/The-serial-two-fold-dilutions-of-a-given-antibiotic-were-made-
with-a-starting_fig15_237114428