i. Carrier tests:

In carrier tests, the carrier such as a silk or catgut thread or a penicylinder (a little stick) is contaminated by submersion in a liquid culture of the test organism. The carrier is then dried and is brought in contact with the disinfectant for a given exposure time. After the exposure, it is cultured in a nutrient broth; no growth indicates activity of the disinfectant tested whereas growth indicates a failing. Example of a carrier test is the in use-dilution test of the American Association of Official Analytical Chemists (AOAC, 1990).

E.g. In Use-dilution test:

The AOAC Use-dilution test is a carrier-based test. The organisms used are Salmonella cholerasuis, S. aureus and P. aeruginosa. Carriers (stainless steel cylinders) are meticulously cleaned, sterilized by autoclaving in a solution of aspargine, cooled and inoculated with a test organism by immersing in one of the culture suspensions. The cylinders are drained on filter paper, dried at 37°C for 40 minutes, exposed to the use-dilution of the disinfectant for 10 minutes, and cultured to assess the survival of the bacteria

ii. Suspension tests:

In these tests, a sample of the 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. There are different kinds of suspension tests:

a. The qualitative suspension tests,

b. The quantitative suspension tests and

c. The test for the determination of the phenol coefficient (Rideal and Walker, 1903)

a. Qualitative test:

In this test, a loopful of bacterial suspension is brought into contact with the disinfectant and again a loopful of this mixture was cultured for surviving organisms. Results are expressed as ‘growth’ or ‘no growth’.

b. Quantitative methods:

In this method, the number of surviving organisms is counted and compared to the original inoculum size. By subtracting the logarithm of the former from the logarithm of the latter, the decimal log reduction or microbicidal effect (ME) is obtained. An ME of 1 equals to a killing of 90% of the initial number of bacteria, an ME of 2 means 99% killed. A generally accepted requirement is an ME that equals or is greater than 5: at least 99.999% of the germs are killed.

c. Phenol coefficient test:

Phenol coefficient may be defined as the killing power of germicide or an antimicrobial agent towards a test organism compared to that of phenol under identical conditions. It is the ratio of the higher dilution of disinfectant killing the test organism in 10min. but not in 5min, to the of the highest dilution of phenol showing the same result.

Procedure

— To a series of dilutions of disinfectant being tested (5 ml per tube), 0.5 ml of 24hrs broth culture of test organism (Staphylococcus aureus or Salmonella typhi) is added

— All tubes (disinfectant + organisms & phenol+ organisms) are placed in 20°C water bath.

— At intervals of 5, 10 and 15 min, subcultures are made with a loop into sterile media tubes and incubated for 48-72 hrs at 37°C and then observed for growth.

— The greatest dilution of disinfectant killing the test organism in 10 min but not in 5 min is divided by the greatest dilution of phenol showing the same result.

— The number obtained by this division is the phenol coefficient of the substance tested.

Table 1: Phenol coefficient method

Antimicrobial agents	Dilutions	5 min	10 min	15 min
Disinfectant (X)	1:100 1:125 1:150 1:175 1:200	0 + + + +	0 + 0 + +	0 0 0 0 +
Phenol	1:90 1:100	+ +	0 +	0 +

E.g. I: Rideal and Walker method;

It is developed by Rideal and Walker in 1903. In this technique, phenol is diluted from 1:400 to 1:800 and the test disinfectant is diluted from 1:95 to 1:115 and their bactericidal activity is determined against Salmonella typhi suspension. Subcultures are performed from both the test and phenol at intervals of 2.5, 5, 7.5 and 10 minutes. The plates are incubated for 48-72 hours at 37°C. That dilution of disinfectant which disinfects the suspension in a given time is divided by that dilution of phenol which disinfects the suspension in same time gives its phenol coefficient.

Table 2: Rideal and Walker method

Disinfectant	Dilution	2.5 min	5 min	7.5min	10 min
Test disinfectant	1:400 1:500 1:600 1:700 1:800	NG G G G G	NG NG G G G	NG NG NG G G	NG NG NG NG G
Phenol	1:95 1:100 1:105 1:110 1:115	G G G G G	NG G G G G	NG NG G G G	NG NG NG NG G

Here, after 7.5 minutes, the test organism was killed by the test disinfectant at a dilution of 1:600. In the same period the test organism was killed by phenol at a dilution of 1:100.

Phenol coefficient = 600/100 = 6

This result indicates that the test disinfectant can be diluted six times as much as phenol and still possess equivalent killing power for the test organism

E.g. II: Chick Martin test

iii. In capacity tests:

This test also determines the phenol coefficient of the test disinfectant. Unlike in Rideal-Walker method where the test is carried out in water, the disinfectants are made to act in the presence of yeast suspension (or 3% dried human feces) to simulate the presence of organic matter. Time for subculture is fixed at 30 minutes and the organism used to test efficacy is S. typhi as well as S. aureus. In this method, a soiled instrument is placed into a container with disinfectant; a certain quantity of dirt and bacteria is added tothe solution. The ability to retain activity in the presence of an increasing load is the capacity of the disinfectant. In a capacity test, the disinfectant is challenged repeatedly by successive additions of bacterial suspension until its capacity to kill has been exhausted. Capacity tests simulate the practical situations of housekeeping and instrument disinfection. The Best known capacity test is the Kelsey-Sykes test (Kelsey and Sykes, 1969).

E.g. Kelsey and Sykes:

— It is a triple challenge test, designed to determine concentrations of disinfectant that will be effective in clean and dirty condition

— The disinfectant is challenged by three successive additions of a bacterial suspension during the course of the tests

— The concentration of the disinfectant is reduced by half by the addition of organic matter (autoclaved yeast cells), which builds up to a final concentration of 0.5%

— Test organism used includes selected S. aureus, P. aeruginosa, P. vulgaris and E. coli

— The method can be carried out under 'clean' or 'dirty‘conditions. The dilutions of the disinfectant are made in hard water for clean conditions and in yeast suspension for dirty conditions.

— Test organism alone or with yeast is added at 0, 10 and 20 minutes interval. The contact time of disinfectant and test organism is 8 min.

— The three sets of five replicate cultures corresponding to each challenge are incubated at 320C for 48 hours and growth is assessed by turbidity.

— 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.

— Sets that contain two or more negative cultures are recorded as a negative result.

— The disinfectant passes at the dilution tested if negative results are obtained after the first and second challenges.

— The third challenge is not included in the pass/fail criterion but positive cultures serve as inbuilt controls

Table 3: Kelsey and Sykes method

Inoculum count	8 minute incubation (1st set)	18 minute incubation (2nd set)	28 minute incubation (3rd set)	Result
2X109	+++++	+ + + + +	+ + + + +	Fail
2X109	- - - - +	- - + + +	+ + + + +	Pass
2X109	- - - - -	- - - - -	- - - - +	Pass

Figure 1: Kelsey and Sykes method

iv. In use test

A simple to use test was described by Maurer in 1985 that can be used in hospitals and laboratories to detect contamination of disinfectants. 1 ml sample of the disinfectant is added to 9 ml diluents which also contains an inactivator. Ten drops, each of 0.02 ml volume of the diluted sample are placed on each of two nutrient agar plates. One is incubated at 37°C for three days and the other at room temperature for seven days. Five or more colonies on either plate indicate contamination

v. Surface time kill test

In this test, A 24 hour culture in nutrient broth culture is prepared. A volume of microbial culture (usually

1.10 ml to 0.020 ml) is placed onto the center of each of a number of sterile test surfaces. This inoculum can be spread over the sterile test surface in a circular pattern to achieve a thin, uniform coverage with the test microorganism if desired. To measure initial microbial concentrations, one or more untreated, inoculated test surfaces are harvested and microorganisms are enumerated. The remaining inoculated test surfaces are treated with the disinfectant, each for a different length of time. Immediately after the treatment times have elapsed, the test surfaces are placed into a solution that neutralizes the disinfecting action of the product, and microorganisms surviving treatment with the disinfectant or sanitizer are cultured and enumerated. Results of the time kill study are tabulated and reported, usually by charting microbial concentrations on the test surfaces as a function of treatment time with the disinfectant or sanitizer.