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Disinfection & testing for Disinfection- Microbiology


By: rajeshmoola (71 month(s) ago)

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

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

Considerations of Disinfection:

Considerations of Disinfection Factors influencing the effectiveness of a disinfectant: Na ture of the item to be disinfected Number and resilience of the contaminants Amount of organic material present Type and concentration of disinfectant Duration and temperature of exposure

Need for testing the disinfectants:

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.

Methods of testing disinfectants:

Methods of testing disinfectants Various methods are employed to test the efficacy of a disinfectant: Koch's method Minimum Inhibition Concentration Rideal Walker Method Chick Martin test & Garrod’s test Capacity use dilution test (Kelsey-Sykes test) Stability test 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


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.

Minimum Inhibitory concentration :

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

Method for calculating MIC :

Method for calculating MIC 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. Indicator bacteria + sterile DW Prepare suspensions till it appears faintly turbid


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.

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Determine MIC of disinfectant for S.typhi Determine MIC of phenol for S.typhi Prepare 5 graded concentrations in DW lowest conc.<MIC Prepare dilutions of 1/95, 1/100, 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

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 :

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

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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 CHICK-MARTIN TEST

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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 GA RROD’S TEST


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.

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1ml of test disinfectant 9ml of quarter strength Ringer soln 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 .

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

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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.

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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

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