EVALUATION OF

ANTIMICROBIAL
AGENTS AND
DISINFECTANTS
METHODS
 Tube dilution and agar
plate method
 Filter paper and cup-
plate method
 Ditch-plate method
 Phenol coefficient
method
 Kelsey-Sykes method
 Tube dilution and Agar plate
method
 The chemical agent is incorporated into the nutrient broth or agar
medium and inoculated with the test microorganisms.
 These tubes are incubated at 30 to 35 C for 2 to 3 days and then
the results in the form of turbidity or colonies are observed.
 Filter paper, Cup-plate or Cylinder
plate method
 The agar is melted, cooled at 45 C ,inoculated with the test microorganisms and
poured into a sterile petri plate.
 When the inoculated agar has solidified, holes about 9mm in diameter are cut in
the medium with a sterile cork borer.
 The antimicrobial agent is directly placed in the holes.
 The antimicrobial agent is applied to the surface of the solidified, inoculated agar
by using a filter paper disc and cylinder respectively.
 The zone of inhibition is observed after incubation at 30 to 35 C for 2 to 3 days.
 The diameter of the zone of inhibition gives an indication of the relative activities
of different antimicrobial substances against the test.
 Ditch-plate method or Giant colony
method
 A solution of the antimicrobial agent substance is carefully run into the
ditch which is prepare in an agar plate.
 A loopful of each test microorganism is then streaked outwards from the
ditch on the agar surface.
 Microorganisms resistant to the antimicrobial agent grow right upto the
ditch whereas susceptible microorganisms show a zone of inhibition
adjacent to the ditch or centre of plate.
 The width of the inhibition zone gives an indication of the relative activity
of the antimicrobial substance against the various test microorganisms.
 Phenol coefficient method
 A test chemical is rated for its microbicidal property with reference to
phenol under identical conditions.
 In this test similar quantities of microorganisms are added to rising
dilutions of phenol and of the disinfectant to be tested.
 In the UK the organism used is Salmonella typhi and the USA Salmonella
typhi, Staphylococcus aureus, Pseudomonas aeruginosa are used.
 The phenol coefficient test includes:
1. Rideal -Walker test (RW test)
2. Chick –Martin test (CM test)
3. United States Food and Drug Administration test ( FDA test)
4. The US Association of Official Agriculture Chemists test (AOAC test)
 Rideal–Walker Test
 The phenol coefficient of test disinfectants may be calculated by
Rideal – Walker test that use Rideal – Walker broth and Salmonella
typhi as the sensitive microorganism.
 Different dilutions of the test disinfectant and phenol are prepared
and 5ml of each of dilution is inoculated with 0.5ml of the 24
hours broth culture of the organisms.
 All tubes( Disinfectant + Organisms and phenol + Organisms) are
placed in a 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 minutes.
 The broth tubes are incubated at 37 C for 48 to 72 hours and are
examined for the presence or absence of growth.
 Chick–Martin Test
 This type of test is done in controlled amount of organic matter in
the form of standardization of yeast cells.
 In this test all conditions are like Rideal-Walker test but the
temperature is 20 C and the exposure time is 30 minutes.
 Chick-Martin coefficient is the mean of highest concentration of
phenol showing growth and lowest concentration preventing
growth divided by the same mean of disinfectants under this test.
 If greater than one, so more effective (Test disinfectants)than
phenol coefficients.
 The Rideal – Walker coefficient of the disinfectant is then
calculated as follows:
RW coefficient =Dilution of disinfectant killing in 7.5 but not in
5 min.
Dilution of phenol killing in 7.5 but not in 5
min.
 If a phenol coefficient or Rideal – Walker coefficient of the given
test disinfectant is 1,it means that the disinfectant has the same
effectiveness as phenol.
 Phenol coefficient of test disinfectant is less than 1 means it is less
effective and more than 1 means it is more effective as compared
to phenol.
 Advantages
 They are inexpensive and can be performed quickly.
 They give reproducible results in the hands of experienced
workers.
 They are valuable to eliminate useless products and supply
standards for crude preparations.
 Disadvantages
 Choice of the test organism: In most tests only one organism is used i.e.
Salmonella typhi.
 Phenol coefficient tests compare the activity of bacteriocides at only one
concentration with a fixed death time and reaction temperature.
 Most phenol coefficient tests give no indication of the activity of
disinfectants in the presence of organic matter.
 These tests do not give any information related to tissue toxicity.
 Sampling errors are large in phenol coefficient tests.
 These tests do not give any indication of the effects of dilution on the
activity of the disinfectant and are used to evaluate phenolic disinfectants
only.
 Kelsey-Sykes method
 It is a triple challenge test (Test disinfectant is checked 3 times with successive addition
of microbial suspension with the same concentration of disinfectants).
 In this method several test bacteria such as Staphylococcus aureus, Proteus vulgaris,
Escherichia coli and Pseudomonas aeruginosa are used.
 This test can be carried out in clean (No organic matter) or dirty (With organic matter)
conditions.
 In both cases the final concentration of bacterial cells should about 10 9 /ml.
 Clean conditions are stimulated by using broth as the suspending fluid and dirty
conditions by the use of a yeast suspension or inactivated horse serum as the
suspending fluid.
 The dilutions of the disinfectant are made in hard water.
 The samples taken at 8,18 and 28 are then incubated at 30 to 32 C
and the number of tubes showing growth or the number of
colonies/ drop from the surface plate culture is recorded.

Interpretation of result: A disinfectant is satisfactory for use at