Evaluation of Disinfectants

Introduction

Disinfectants are applied on environments, especially surfaces of inanimate objects, where they eliminate a wide range of
contaminating organisms. Its evaluation is largely focused on its ability to kill a wide range of organisms. A single method that would accommodate the variety of environments it acts on is impractical. Its antimicrobial effect, in practice, depends on different factors.

Evaluation of Disinfectants

There are two categories of evaluation methods:

1. Methods used for routine tests of disinfectants
2. In-use evaluation of disinfectants in real practical conditions.

Routine tests include:

1. Phenol tests: Rideal-Walker test and Chick-Martin test
2. AOAC Available Chlorine test

Considerations for the Phenol coefficient test include:

• The Test culture
• The Medium
• Interpretation of result

Rideal-Walker Phenol

This was reported by S. Rideal and J.T.A Walker in 1903 and standardized by
the British Standards Institution in 1934.

• Culture medium: Standard nutrient broth with pH adjusted to 7.6.
• Test Organism: Salmonella typhi NCTC 786; only cultures 22-26 hours old and from 3rd to 14th subculture are used.
• Disinfectant dilutions: The standard phenol solutions contain 1g of Phenol in 95,100,105,110 and 115ml of solution made with sterile distilled water. For the disinfectants, the dilutions should vary by an arithmetic unit of 50 resulting in dilutions of 1:200, 1:250, 1:300 and 1:350.

PROCEDURE

1. A volume of 5ml of each dilution of test disinfectant and standard phenol is placed in separate sterile test tubes.
2. A 24 hour broth culture is prepared.
3. The dilutions and culture are placed in the water bath and maintained at
   17-18°C.
4. A volume of 0.2ml of the culture is placed in each dilution of phenol and
   disinfectant.
5. At 2.5, 5, 7.5 and 10mins, a loopful of the reaction mix is transferred to a
   recovery medium which is incubated at 37°C for at least 48 hours and at most 72 hours.

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Rideal Walker Phenol Coefficient is calculated by dividing the highest dilution of the standard phenol which allows growth at 2.5 and 5 mins but not at 7.5 and 10 mins with the highest dilution of the disinfectant with the same effect.

Chick-Martin Phenol Coefficient Test

Chick-Martin Phenol Coefficient Test was developed by H. Chick and C.J. Martins five years after Rideal-Walker test was first reported. It was designed as an improvement by considering the disinfection period and effect of organic matter on antimicrobial activity.

• Culture medium: Standard nutrient broth with pH adjusted to 7.6.
• Test Organism: “S” strain of Salmonella typhi; only cultures from 3rd to 14th subculture are used.
• Yeast Suspension: A 5% suspension of yeast is required.
• Serial dilutions of both Standard Phenol and test disinfectant should exhibit concentrations decreasing by 10%.

PROCEDURE

1. The test, standard phenol, a 50ml culture (containing 2ml of test
   culture and 48 ml of the yeast suspension) are placed in the water
   bath at 20°C.
2. 2.5ml of culture is placed in each tube to bring it up to 5ml.
3. At 30 mins, a loopful of each reaction mix is transferred in duplicate
   to 10ml of recovery medium.
4. The recovery media is incubated at 37°C for 48 hours

The Chick-Martin Phenol coefficient test is calculated as the mean of the highest concentration of phenol showing growth in duplicate tubes and the lowest concentration showing no growth in duplicate tubes divided by the corresponding mean concentration of the test disinfectant.

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LIMITATIONS

• Phenol used as a standard for other classes of disinfectants
• Consideration of organic matter
• Use of single organism
• Use of fixed temperatures for both tests
• Disinfection period.

IMPROVED KELSEY-SYKES TEST

This test takes into account that disinfectants can be used in either clean or dirty environment.
The test is performed as follows:

• The MIC is used to determine the most resistant organism amongst Pseudomonas aeruginosa, Proteus vulgaris, Escherichia coli and Staphylococcus aureus.
• A 24 hour culture of the selected organism is used for the test; the inoculum is suspended in hard water such that each inoculum would have 108 to 1010 cells/ml.
• The presence or absence of yeast is used to simulate clean and dirty environment.
• Tween 80 is added to the recovery tubes; these are arranged in 3 sets of 5 tubes for each concentration.
• Three different concentrations are used per test; the middle concentration being 50% less and more than the higher and lower concentrations respectively.
• The process requires consecutive addition of three 1ml inoculum of the selected organisms at 10 min intervals. Eight minutes after each addition, a drop of the reaction mix is transfer to a set of five recovery tubes.
• These tubes are incubated at 32+1°C for 48 hours.
• The effective concentration of the disinfectant is the lowest concentration producing no growth in at least two of the five tubes of the first two additions of the inoculum.
• For the result to be satisfactory, each recommended concentration must as well pass the test in three other repeat runs using freshly prepared inoculum and disinfectant concentrations.