TESTING OF DISINFECTANTS

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4/20/12

DISINFECTION

-a process that eliminates many or all pathogenic microorganisms, except bacterial spores, on inanimate objects. -disinfectants which kill spores with prolonged exposure times(3-12hr)--- chemical sterilants. eg:2.4%glutaraldehyde, 7.5% stbilized Hydrogen peroxide.
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Scottish surgeon who first introduced antisepsis using carbolic acid

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High level disinfectants: kill all microorganisms except bacterial spores. eg:peroxides,ozone and aldehydes. Intermediate level disinfectants: kill mycobacteria, vegetative bacteria,most viruses and fungi. eg: halogens,alcohol,phenol Low level disinfectants: kill most of the vegetative bacteria, some fungi and viruses. eg: aniline dyes,heavy metals
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Factors affecting efficacy of disinfectants

1. 2.

Organic & inorganic load present Type & level of microbial contamination Conc. & exposure time of the disinfectant Physical nature of the object Presence of biofilms Temp. & pH of the disinfection process
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3.

4. 5. 6.

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disinfectants

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TESTING OF DISINFECTANTS

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INTRODUCTION

Disinfectants used in hospitals and laboratories must be periodically tested as:

v v

They lose potency on standing Addition of organic matter will decrease their potency

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Disinfection process validation is defined as-

establishing documented evidence that a disinfection process will consistently remove or inactivate known or possible pathogens from inanimate objects.

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 Different

methods

-carrier tests -suspension tests -capacity tests -practical tests -field or in-use tests

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 Carrier

tests
-oldest tests -described by Robert Koch(1881)

-a carrier such as silk or catgut thread or a penicylinder is contaminated by submersion in a liquid culture of the test organism -carrier is dried and brought in contact with the disinfectant for a given exposure time. It is then cultured in nutrient broth - no growth indicates activity of the test disinfectant
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 Use-dilution

test

-is a carrier based test -organisms used are S.cholerasuis, S.aureus and P.aeruginosa. -stainless steel cylinders are sterilized by autoclaving in a solution of asparagine,cooled and inoculated with test organism. -cylinders are drained on filter paper,dried at 37C for 40min,exposed to the use-dilution of the disinfectant for
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AOAC Use-dilution test

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 Limitations

of carrier tests

-the no. of bacteria dried on a carrier is hard to standardize -the survival of the bacteria on the carrier during drying is not constant.

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 Suspension

tests

-a sample of bacterial culture is suspended into the disinfectant solution and after exposure it is verified by subculture whether this inoculum is killed or not. -suspension tests are preferred to carrier tests as the bacteria are uniformly exposed to the disinfectant.

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 Different

kinds of suspension tests are

-qualitative -quantitative -tests for determination of phenol coefficient: -rideal-walker method -chick martin test -garrods test
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Phenol coefficient expresses the disinfecting power of a particular substance as compared to that of pure phenol. dilution of disinfectant which sterilises the suspension in a given time dilution of phenol which sterilises the suspension within the same time
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 Phenol

coefficient greater than 1.0 indicates agent is more effective than phenol.

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 Rideal

walker method:

-phenol is diluted from 1:95 to 1:115 and the test disinfectant is diluted from 1:400 to 1:800 -0.2ml of 24hr broth culture of S.typhi is added to these dilution tubes -the dilution tubes in the rack are kept in a water bath at 18C -subcultures are made from both the test and phenol at intervals of 2.5,5,7.5 and 10min
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disinfectan dilution t 1:400 Test 1:500 disinfectan 1:600 t 1:700 1:95 1:800 Phenol 1:100 1:105 1:110 1:115

2.5min 5min NG G G G G G G G G G NG NG G G NG G G G G G
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7.5min NG NG NG NG NG NG G NG G NG G G G NG NG G NG NG NG G

10min

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 Garrods

modification:

this test is allied to Rideal walker test with the difference that yeast extract is added to broth culture and dilutions of the disinfectant and phenol are allowed to act on standardized cultures for 30min instead of varying periods
Chick

martin test:

disinfectants are made to act in the presence of 3% dried human faeces to simulate organic matter.
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Kelsey-Sykes Capacity tests:

q

It determines the dilution of a disinfectant to be used in a hospital. Simulates the natural conditions under which disinfectants are used in hospitals. The disinfectant is challenged repeatedly by successive additions of bacterial suspension until its capacity to kill has been exhausted. contd..
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Procedure :
v

Prepare the dilutions of the disinfectant in water of 300p.p.m. hardness. Bacterial suspension is made in clean and dirty conditions.

clean test organism is suspended in broth to a conc. Of 109 c.f.u./ml. dirty with 20%inactivated horse serum or 2%yeast suspension.
v

Distribute the dilutions of disinfectant in 3ml amounts in 2 rows of tubes 4/20/12

v v

To first row,add 1ml of clean inoculum. To 2nd row, add 1ml of dirty inoculum .

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 Capacity

tests can establish the minimum time required for disinfectant to be effective

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 Disinfectant

is satisfactory-no. of colonies from 5drops collected after the 2nd increment are not more than five. organisms used are S.aureus NCTC 4163 P.vulgaris NCTC 4635 P.aeruginosa NCTC 6749 E.coli NCTC 8196

Test

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 Practical

tests:

-these tests verify whether the proposed use dilution is still adequate in the conditions under which it would be used. ex: surface time kill test 0.02ml of culture is placed onto the center of each of a no. of sterile test surfaces. spread over the sterile test surface in a circular pattern to achieve thin uniform coverage
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contd..

 Initial

microbial concentrations are measured by harvesting one of the inoculated test surfaces. inoculated test surfaces are treated with the disinfectant,each for a different length of time.

Remaining

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 Immediately

after the treatment times have elapsed ,the test surfaces are placed into a neutralizing solution and surviving microorganisms are cultured and enumerated are tabulated and reported,usually by charting microbial conc. as a function of treatment time with the disinfectant or sanitizer.

Results

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 In-use

test(Kelsey and Maurer):

-monitors the efficacy of a disinfectant used at a recommended dilution procedure: -1ml of the disinfectant fluid in use is added to 9ml of the diluent. -diluents used are nutrient broth-for alcohols,aldehydes, hypochlorites and phenolics nutrient broth with 3%Tween 80-for
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 Place One

10 drops on the surface of two nutrient agar plates. plate is incubated for 72hr at 37c, the other at room temp. for 7days. of 5 or more colonies from one or both the plates indicates failure of disinfection at the particular conc. used.

Growth

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References
1)

Diagnostic and practical microbiologyDr.A.K.Gulati Day and Day-Text book of medical microbiology CDC guidelines for sterilisation and disinfection,2008 www.microrao.com

2)

3)

4)

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THANK YOU
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