Disinfection and tests for disinfection(Microbiology)

2. DEFINITION - DISINFECTION
 Process of elimination of most pathogenic
organisms excluding bacterial spores on
inanimate objects.
 Sterilization and Disinfection are NOT
synonymous.

3.  Wide spectrum of activity.  Active at any pH
 Stable  High penetrating power
 Long shelf life  Able to destroy microbes
 No bad odour within a particular time
 Speedy in action  Efficacy shouldn’t be
 Non-toxic/ Non-allergic/ lost on reasonable
Non-corrosive/Non-irritant dilution
 Be active in the presence of
organic matter

4. Considerations of Disinfection
Factors influencing the effectiveness of a disinfectant:
a)Nature of the item to be disinfected
b)Number and resilience of the contaminants
c)Amount of organic material present
d)Type and concentration of disinfectant
e)Duration and temperature of exposure

6. 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.

7. Various methods are employed to test the
efficacy of a disinfectant:
I. Koch's method
II. Minimum Inhibition Concentration
III. Rideal Walker Method
IV. Chick Martin test & Garrod’s test
V. Capacity use dilution test (Kelsey-Sykes test)
VI. Stability test
VII. In-use test

8. BASIC PRINCIPLE
 All these tests use the same principle in evaluating
the efficacy of the test disinfectant:
 After the indicator bacterium have been exposed to
contact with the disinfectant, their viability is tested by
sub-culturing them on media prepared without
disinfectant
 The disinfectant carried over to the sub-cultures should
therefore neutralized either by diluting them to sub-
inhibitory conc. Or by adding a substance that inactivates
it.
 1/10 dilution for phenols, aldehydes
 0.5% Na.thiosulphate for chlorine comp’s, iodophores
 1% Na. bisulphate for HCHO and glutaraldehude

9. KOCH'S METHOD
 Spores of Bacillus anthraces were dried on
silk thread and were subjected to action of
disinfectants.
 Later, it was washed and transferred to solid
medium.

10. Measures the lowest concentration
of the disinfectant that inhibits the growth of
known strain of organism.
Disadvantages:
i. No. of organisms considered-too low
ii. Time of exposure- too long
iii. No sufficient organic matter

11. Method for calculating MIC
Indicator bacteria + sterile DW Prepare suspensions till it appears faintly turbid
Incorporate different known concentrations of
disinfectant in plates of Nutrient Agar
Inoculate streaks of different bacterial suspensions onto each plate of NA.
Incubate for 37 C-48hrs
The lowest concentration that entirely prevents the growth of the indicator
bacteria is the MIC of that disinfectant in that particular medium.

12. RIDEAL WALKER METHOD
 PHENOL-COEFFICIENT TEST.
 Bactericidal potency is assessed by measuring the rate of
killing a selected range of bacteria under specified
conditions.
 B.cidal power of a particular disinfectant as compared
with that of pure PHENOL.
¥ Minimum time of 10min.
¥ Usually employed cultures are S.typhi& St.aureus.
¥ Phenol coefficient is considered to be 1.

13. Determine MIC of disinfectant for S.typhi Determine MIC of phenol for S.typhi
Prepare 5 graded concentrations in DW Prepare dilutions of 1/95, 1/100,
lowest conc.<MIC 1/105, 1/110, 1/115
Take 5ml into sterile test tube
Add 0.2ml of 24hr broth of S.typhi
Shake well
At regular intervals (say 5min) remove a large loopful from each mixture& transfer
to a tube of 5ml broth
Inoculate broth for 48hrs
Note tubes in which turbidity(bacterial growth) appears
Calculate phenol coefficient by dividing the figures indicating the dilution of test that
shows growth after exposure for 5min to that of no growth after long exposure-10min

15. Interpretations of Rideal Walker Method:
A. Higher the phenolic coefficient, more is the
effectiveness.
B. If the value is >1, test disinfectant is more potent
than phenol
Disadvantages:
1. Ineffective in the presence of organic
matter.
2. Test is not reproducible
3. Can be done only for phenolic disinfectants
4. S. typhi may not be appropriate
5. The time allowed for disinfection is short

16. CHICK-MARTIN TEST
☻Modification of Rideal Walker Method
☻Is done conditions mimicking naturality i.e., in the
presence of organic matter.
☻3% dried human feces is added
☻Contact time is increased to 30min
☻Both S.typhi & St.aureus are used for testing efficacy

17. GARROD’S TEST
♥ Modification of Chick-Martin Test.
♥ Contains yeast instead of feces.
♥ 5g of dry yeast is mixed with 100ml DW.
♥ 48ml suspension is added to 2ml broth of
S.typhi

19. IN-USE TEST/ TEST OF MAURER
 Intended to estimate the number of living organism in a vessel of
disinfectant in actual use.
 The disinfectant that is already in use is diluted 1/10 by mixing 1 ml
of the disinfectant with 9 ml of sterile nutrient broth.
 Ten drops of the diluted disinfectant (each 0.02 ml) is placed on
two nutrient agar plates
 One plate is incubated at 37oC for 3 days while the other is held at
room temperature for 7 days
 The number of drops that yielded growth is counted after
incubation
 If there growth in more than five drops on either plate, it
represents failure of disinfectant.

20. 9ml of quarter strength Ringer soln
1ml of test disinfectant 9ml isotonic soln that inactivates it
Prepare a suspension
Transfer 10 small drops onto separate areas of
the surface of each of two dried agar plates
Incubate at 37 C-48hr Incubate at room temp
Examine the plates
Score the growth from each drop
Growth from >5drops = failure

21. CAPACITY USE DILUTION TEST /KELSEY-SYKES TEST
 Kelsey-Sykes test is a triple challenge test, designed
to determine concentrations of disinfectant that will
be effective in clean and dirty conditions.
 Method:
 Effective in clean and dirty conditions.
 The dilutions of the disinfectant are made in hard water for clean
conditions and in yeast suspension for dirty conditions
 The contact time of disinfectant and test organism is 8 min.
 Test organism alone or with yeast is added at 0, 10 and 20
minutes interval
 Depending on the type of disinfectant, a single test organism is
selected from S. aureus, P. aeruginosa, P. vulgaris and E. coli.

22. Test disinfectant + standard hard water solution
Add suspensions of test
bacterium
After each addition of organism, portion of
the mixture are removed
Cultured in a solid media.
Observe for any positive growth

23. ∆ The disinfectant is evaluated on its ability to kill
microorganisms or lack of it and the result is reported
as a pass or a fail and not as a coefficient.
Disadvantage:
Rather complicated.

24. STABILITY TESTS
 It measures the stability & long-term
effectiveness of the diluted disinfectant in
clean and dirty medium.
 Used to supplement the information
obtained from capacity use dilution tests.

25. There is no single reliable test to
determine the efficacy of a disinfectant.
This is due to the fact that many
parameters play a role in influencing the
action of disinfectant

26. THANK YOU…!!!