DEFINITION - DISINFECTION Process of elimination of most pathogenic organisms excluding bacterial spores on inanimate objects. Sterilization and Disinfection are NOT synonymous.
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
Considerations of DisinfectionFactors influencing the effectiveness of a disinfectant:a)Nature of the item to be disinfectedb)Number and resilience of the contaminantsc)Amount of organic material presentd)Type and concentration of disinfectante)Duration and temperature of exposure
Need for testing the disinfectantsA 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.
Various methods are employed to test theefficacy of a disinfectant: I. Kochs 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
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
KOCHS 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.
Measures the lowest concentrationof the disinfectant that inhibits the growth ofknown strain of organism.Disadvantages: i. No. of organisms considered-too low ii. Time of exposure- too long iii. No sufficient organic matter
Method for calculating MICIndicator 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.
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.
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 wellAt regular intervals (say 5min) remove a large loopful from each mixture& transferto 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
Interpretations of Rideal Walker Method: A. Higher the phenolic coefficient, more is theeffectiveness. B. If the value is >1, test disinfectant is more potentthan phenolDisadvantages: 1. Ineffective in the presence of organicmatter. 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
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
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
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.
9ml of quarter strength Ringer soln1ml 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
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.
Test disinfectant + standard hard water solution Add suspensions of test bacteriumAfter each addition of organism, portion ofthe mixture are removed Cultured in a solid media. Observe for any positive growth
∆ 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.
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.
There is no single reliable test todetermine the efficacy of a disinfectant. This is due to the fact that many parameters play a role in influencing the action of disinfectant