What are disinfectants?
As per the definition given by WHO ( World health organization ) : a disinfectant is a chemical agent, which destroys or inhibits growth of pathogenic microorganisms in the non-sporing or vegetative state.
Why Evaluation?
Evaluation of disinfectants is used to check the ability or efficacy of any disinfectant against specific microorganisms to establish its effectiveness.
Evaluation tests of bactericide.
1. RIDEAL WALKER TEST
This test is also known as the phenol coefficient test,in which any chemical is compared with phenol for its antimicrobial activity.
The result is shown in the form of phenol coefficient.
▪ If a phenol coefficient of a given test disinfectant is less than 1, it means that disinfectant is less effective than phenol.
▪ If a phenol coefficient of a given test disinfectant is more than 1, it means that disinfectant is more effective than phenol.
Procedure
1.1 Different dilutions of the test disinfectant and phenol are prepared and 5 ml of each dilution is inoculated with 0.5ml of the 24 hour growth culture of the organisms.
1.2 All tubes(Disinfectant + organisms & phenol + organisms) are placed in a water bath ( at 17.5° C)
1.3 Subcultures of each reaction mixture are taken and transferred to 5ml sterile broth at an interval of 2.5 minutes from zero to 10 mintues.
1.4 Broth tubes are incubated at 37° C for 2 to 3 days & examined for the presence or absence of the growth.
1.5 Then the Rideal Walker coefficient is calculated :
2. CHICK MARTIN TEST.
CHICK MARTIN test is performed in the much similar way as the RIDEAL Walker test but with a little variation.
Principle : This test is carried out in the presence of organic matter like 3% human feces or dried yeast.
Procedure
2.1 Serial dilutions of test solution and phenol is prepared in distilled water.
2.2 To this 3% yeast suspension is also added.
2.3 To this solution the S. typhi is added
2.4 After contact time of 30 mins the above mixture is transferred to the freshly prepared 10 ml of broth.
2.5 The test tubes are incubated at 37°C for 48 hours.
2.6 Presence or absence of the growth is calculated.
Evaluation tests of Bacteriostatic.
1. Tube dilution & Agar plate Method
1.1 The chemical agent is incorporated into nutrient broth or agar medium and inoculated with test micro-organisms.
1.2 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.
1.3 The results are recorded and the activity of the given disinfectant is compared.
2. Cup plate method
2.1 Agar is melted and cooled at 45° Celsius.
2.2 Then inoculated with test micro-organisms and poured into a sterile petri plate.
2.3 In the cup plate method, when the inoculated agar has solidified, holes around 8mm in diameter are cut in the medium with a steel cork borer.
2.4 Now the antimicrobial agents are directly placed in the holes.
2. Bactericidal & Bacteriostatic
• Bactericidal (bac-teri-cidal )
• Word origin: bacterio- + -cide
• Bactericidal refers to antibiotics that kill
the bacteria.
• Bacteriostatic (Bacterio - static)
• Word origin: bacterio- & stasis
• Bacteriostatic refers to antibiotics that
prevent the growth of bacteria.
4. A. Evaluation of Bactericidal activity
1) also known as the phenol coefficient test.
2) Three microorganisms are used for this test.
Salmonella typhi
Staphylococcus aureus
Pseudomonas aerugenosa
3) The result is shown in the form of phenol coefficient.
If phenol coefficient<1 , disinfectant is less effective than phenol.
If phenol coefficient>1 , disinfectant is more effective than phenol.
1. Rideal-Walker (RW) test
5. Procedure
• Different dilutions of the test disinfectant and phenol are prepared and
5 ml of each dilution is inoculated with 0.5ml of the 24 hour growth
culture of the organisms.
• All tubes (Disinfectant + organisms & phenol + organisms) are placed
in a water bath ( at 17.5° C)
• Subcultures of each reaction mixture are taken and transferred to 5ml
sterile broth after 2.5,5,7.5,10 minutes.
• Broth tubes are incubated at 37° C for 48 to 72 hours & examined for
the presence or absence of the growth.
• Then the Rideal Walker coefficient is calculated :
6. Rideal Walker Co-efficient =
Dilution of test disinfectant killing in 7.5 min not in 5 min
Dilution of the standard phenol killing in 7.5 min not in 5 min
7. 2. Chick Martin Test
Procedure
Serial dilutions of test solution and phenol is prepared in distilled water.
To this 3% yeast suspension is also added.
To this solution the S. typhi is added
After contact time of 30 mins the above mixture is transferred to the freshly prepared
10 ml of broth.
The test tubes are incubated at 37°C for 48 hours.
Presence or absence of the growth is calculated.
Principle: This test is carried in the presence of organic matter like 3% human feces or
dried yeast.
8. Chick Martin Co-efficient=
Mean of highest phenol Conc. inhibiting and lowest permitting growth
Mean of highest disinfectant Conc. inhibiting and lowest permitting growth
9. B. Evaluation of Bacteriostatic activity
1) The chemical agent is incorporated into nutrient broth or agar
medium and inoculated with test micro-organisms.
2) These tubes are incubated at 30°C to 35°C for 2 to 3 days and then the
results in the form of turbidity or colonies are observed.
3) The results are recorded and the activity of the given disinfectant is
compared.
1. Tube dilution & Agar plate Method
10.
11. 2. Cup plate method
• Agar is melted and cooled at 45° Celsius.
• Then inoculated with test micro-organisms and poured into a sterile
petri plate.
• In the cup plate method, when the inoculated agar has solidified, holes
around 8mm in diameter are cut in the medium with a steel cork borer.
• Now the antimicrobial agents are directly placed in the holes.
• In all cases zones of inhibition may be observed, the diameter of the
zones gives an indication of the relative activity of antimicrobial
substance.
14. 3. Ditch plate method
• The nutrient agar is melted, cooled suitably, and poured into Petri
dishes.
• The solidified media is cut with a sterile blade to make a ditch.
• A solution of the antimicrobial substances is carefully run into the
ditch and a loop of each microorganism is then streaked outwards from
the ditch on the agar surface.
• Microorganisms resistant to antimicrobial growth right up to the ditch,
whereas susceptible microorganisms show a zone of inhibition
adjacent to the ditch or centre of the plate.
• Width of the inhibition zone gives an indication of the relative activity
of the antimicrobial substance against the various test microorganisms.
15.
16. Reference
1) Dr Wani Imtiyaz. Pharmaceutical microbiology. 1st Edition . Punjab:
S.Vikas & Company;2018. pg 153-156
2) Jain N. K. Pharmaceutical microbiology. 3rd Edition . New Delhi:
Vallabh Prakashan;2019. pg 200-209
