Chemical antimicrobial agents or Disinfectants used in hospitals and laboratories must be periodically tested to determine its efficacy.
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. Testing of antimicrobial agents is regulated by two different federal agencies. The U.S. Environmental Protection Agency regulates disinfectants, whereas agents used on humans and animals are under the control of the Food and Drug Administration.
There are several methods of testing disinfectants, and each method have advantages and disadvantages. These tests are divided into the following disinfectant tests: carrier test, suspension test, capacity test, practical test, field test or in-use test.
Carrier tests
These tests are the oldest tests and was described by Robert Koch in 1881. In this test, a carrier such as a silk or catgut thread or a penicylinder (a little stick) is used. This carrier is contaminated by submersion into a liquid culture of the test organism. The test organism used by Robert Koch was liquid culture of Bacillus anthracis and he used a silk thread. The carrier will be then dried and brought in contact with the disinfectant to be tested for a given exposure time. After the exposure, the carrier is cultured in a nutrient broth; no growth indicates activity of the disinfectant tested whereas growth indicates that it is not effective as a disinfectant. Example of a carrier test is the use-dilution test.
Limitation of the carrier tests are:
a) the number of bacteria dried on a carrier cannot be standardized
b) the survival of the bacteria on the carrier during drying is never constant.
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, they are the qualitative suspension tests, quantitative suspension tests and the phenol coefficient test.
In a qualitative suspension test, a loopful of bacterial suspension will be brought into contact with the disinfectant and then a loopful of this mixture will be cultured for surviving organisms. Results were expressed as ‘growth’ or ‘no growth’.
In quantitative suspension test, the same above procedure will be carried out, but here the number of surviving organisms are counted and compared to the original inoculum size.
Phenol coefficient test: Phenol coefficient of a disinfectant is calculated by dividing the dilution of test disinfectant by the dilution of phenol that disinfects under predetermined conditions.
1. Rideal Walker method: Phenol will be diluted to different concentrations and the test disinfectant also will be diluted to different concentrations. Their bactericidal activity will be determined against Salmonella typhi suspension. Subcultures are performed from both the test and phenol at various time 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. Suppose that the phenol dilution was 1/90 and maximum effective dilution for disinfectant was 1/450. The phenol coefficient of disinfectant would be 5.
Disadvantages of the Rideal-Walker test are: No organic matter is included; the microorganism Salmonella typhi may not be appropriate; the time allowed for disinfection is short; it should be used to evaluate phenolic type disinfectants only.
2. Chick Martin test: 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 3% dried human feces to simulate the presence or 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.
3. Garrod’s Test: This test is a modified Chick Martin Test, where the organic material added is 5% of yeast suspension.
Capacity tests
The ability to retain activity in the presence of an increasing load of dirt and bacteria 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. The best known capacity test is the Kelsey-Sykes test.
Kelsey-Sykes test: This is a triple challenge test, and is done to evaluate effectiveness of disinfectant both at clean and dirty conditions. The dilutions of the disinfectant are made in hard water for testing under clean conditions and in yeast suspension for testing under dirty conditions. Test organism may be Staphylococcus aureus, Pseudomonas aeruginosa, Proteus vulgaris or E coli. There will be three successive additions of the test bacteria, at 0, 10 and 20 minutes’ interval. The disinfectant will be evaluated for its ability to kill the bacteria 8 minute after each challenge or addition of test bacteria by inoculating into a nutrient media and bacterial growth if any will be monitored after incubation. The disinfectant passes at the dilution tested if negative results (no bacterial growth) are obtained after first and second challenge. The result will be reported as a Pass or Fail, not as a coefficient.
Stability Test: if the disinfectant found to be effective after capacity test, its stability upon storage should be determined. So if the prepared disinfectant solutions are to be kept for more than 24 hours, then its stability is measured by using the supplementary test. Pseudomonas aeruginosa is used. For this test the disinfectant solution is prepared in two sets
1. First set will be inoculated with the test organism and will be incubated for 7 days and will be tested for bacterial growth after 7 days.
2. The second set will be kept aside at room temperature for 7 days and then inoculated with the test organism and will be tested for bacterial growth.
If bacteria is found to survive the particular dilution of the disinfectant tested, a higher concentration of the disinfectant must be tested.
Practical tests
The practical tests under real-life conditions are performed after measuring the time-concentration relationship of the disinfectant in a quantitative suspension test. The objective is to verify whether the proposed use dilution is still adequate in the conditions under which it would be used. The best known practical tests are the surface disinfection tests.
Surface disinfection tests assess the effectiveness of the selected sanitizer against surface-adhered microorganisms. The test surface (a small tile, a microscopic slide, a piece of PVC, a stainless steel disc, etc.) is contaminated with a standardized inoculum of the test bacteria and dried: then a definite volume of the disinfectant solution is distributed over the carrier; after the given exposure time the number of survivors is determined.
There is an essential difference between a carrier test and a surface disinfectant test. In the carrier test, the carrier is submerged in the disinfectant solution during the whole exposure time, whereas in the surface disinfectant test, the disinfectant is applied on the carrier for the application time and then the carrier continues to dry during the exposure time. Surface tests can reflect in-use conditions like contact times, temperatures, use-dilutions, and surface properties.
Surface Time kill Test: A 24-hour culture in nutrient broth culture is prepared and spread onto the center of a number of sterile test surfaces. The inoculated test surfaces are treated with the disinfectant, each for different durations of time. After the treatment times, the test surfaces are placed into a solution that neutralizes the disinfecting action, and microorganisms surviving are cultured and enumerated. Untreated, inoculated test surfaces are kept as control.
In-use test
This was described by Maurer in 1985 to detect contamination of disinfectants. A 1 ml sample of the disinfectant is added to 9 ml diluent that contains an inactivator. Ten drops of this diluted disinfectant are placed on two nutrient agar plates. One is incubated at 37oC for three days and the other at room temperature for seven days. Five or more colonies on either plate indicate contamination of the disinfectant.
Testing scheme
The antimicrobial efficiency of a disinfectant is examined at three stages of testing. First phase involves laboratory tests which verify whether the chemical compound possesses antimicrobial activity, generally by quantitative suspension tests are considered. Second stage is carried out in laboratory using conditions simulating real-life conditions. Here disinfection procedures are examined by performing practical tests. Third phase involves field tests and in-use tests.
The antimicrobial activity is assessed in terms of activity towards vegetative bacteria (bactericidal tests), against fungi and yeasts (fungicidal tests), against mycobacteria (tuberculocidal tests), against viruses (virucidal tests) and against spores of bacteria (sporicidal tests).
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