Disinfection: Evaluation of disinfectants.

 

Introduction:

  • Disinfection is a process of removal or destruction of microorganisms and reducing their number to a non harmful level.

  • Disinfection usually kills the vegetative form of microorganisms and doesn't affect the endospores.

  • The chemical which is used for disinfection of the nonliving objects (Inanimate objects) is called a “Disinfectant”.

  • The chemical which is used for disinfection of the living objects is called an “Antiseptic”.

  • Mostly the disinfectants are “Bactericidal” while some may be “Bacteriostatic”.

  • Minimal inhibitory concentration (MIC) is the lowest concentration of a chemical agent or drug that inhibits growth of a particular microorganism.

  • The MIC is not a constant for a given chemical agent because it is affected by many factors like,

    • nature of the target microorganism.

    • inoculum size.

    • composition of the culture medium.

    • incubation time.

    • conditions of incubation such as temperature, pH and aeration.

Evaluation of Disinfectants:

  • The common methods used for evaluation of a disinfectant are as follows,

  1. Tube Dilution Method.

  2. Agar Plate Method.

  3. Filter Paper & Cup Plate Method.

  4. Ditch-Plate Method.

  5. Phenol Coefficient Method.

  6. Kelsey Sykes Method.


  1. Tube Dilution Method:

  • Most commonly used method.

  •  In this method, a series of tubes is set up, each containing the same quantity of a standard growth liquid medium (Commonly  Mueller-Hinton broth) and a gradually increasing concentration of the disinfectant to be tested.

  • The tubes are then inoculated with the same quantity of cell suspension of the test microorganism and incubated for 2-3 days @ 30-35℃.

  • The first tube in the series where there is complete absence of growth of the test microorganism denotes the minimal inhibitory concentration (MIC) of the disinfectant.

  1. Agar Plate Method:

  • It is very similar to the tube dilution method.

  • In this method, Petri plates containing a standard growth agar medium (usually Mueller-Hinton agar) are taken at the place of lubes containing liquid medium. 

  • Disinfectants of various concentrations are inoculated on the agar surface of plates already inoculated with the same quantity of the test microorganism.

  • Plates are incubated and then examined for growth. 

  • The first plate in the series where there is complete absence of growth of the test microorganism denotes the minimal inhibitory concentration (MIC) of the disinfectant.

  1. Filter Paper & Cup Plate Method:

  • These methods are known as “Agar Diffusion Tests”.

  • The agar is melted and cooled @ 45℃, inoculated with test microorganisms and poured in a sterile petri plate.

  • In case of “Cup Plate Method” when the agar is solidified the holes of 9 mm  diameter were made by using sterile cork borer and the disinfectant is directly placed in it.

  • In case of the Filter Paper method the filter paper disks soaked in disinfectant solution are placed in the holes.

  • The zone of inhibition is observed after incubation @ 30-35 ℃.

  •  During incubation, the disinfectant diffuses into the agar.

  • The diameter of the zone of inhibition gives indication of activity of the disinfectant, larger the diameter greater is the efficiency of the disinfectant.

  • A zone of Inhibition is proportional to the amount of the antimicrobial agent added to the filter paper disc, the solubility of the agent, the diffusion coefficient, and the overall effectiveness of the agent. 

  1. Ditch-Plate Method:

  • Agar is melted and then solidified in a petri plate.

  • A ditch is made in the petri plate by cutting the soliodified agar.

  • The disinfectant solution is made to run through the ditch carefully.

  • The test organisms are streaked outwards from the ditch.

  • The petriplate is incubated at desired temp. And time period.

  • The microorganisms which are resistant to the disinfectant grow even near the start at the ditch itself.

  • The sensitive organisms show a zone of inhibition near the ditch or at center of the petri plate.

  • The width of the zone of inhibition is an indication of activity against the test organism.

  1. Phenol Coefficient Method:

  • In this method the efficacy of the disinfectant in use is rated by comparing it with the activity of Phenol taking as a standard.

  • The test is carried out by adding an increasing amount of phenol and disinfectant in test tubes containing microorganisms.

  • In UK the test organism used is Salmonella typhi while the USA uses Salmonella typhi, Staphylococcus aureus and Pseudomonas aeruginosa.

  • The official phenol coefficient tests include,

    • Rideal-Walker Test (RW Test).

    • Chick-Martin Test.

    • United States FDA Test for Phenol Coefficient. (FDA Test)

    • The US Association of Official Agricultural Chemists Test (FDA Test)

  1. Rideal-Walker Test:

  • Introduced by Rideal and Walker the British chemists in 1903 are still in use.

  • The test uses Rideal, Walker Broth and Salmonella typhi as a test organism.

  • Different dilutions of the phenol and test disinfectants are made and 5 ml of each is inoculated with the 0.5 ml  of the 24 hr broth culture of the test microorganism.

  • All the test tubes are placed in a water bath at 17.5 ℃.

  • Subcultures from each test tube are taken and transferred to 5 ml sterile broth after 2.5, 5, 7.5 and 10 minutes.

  • The broth tubes are incubated at 37 ℃ for 2 - 3 days and are examined for the presence or absence of the growth and the Phenol coefficient is calculated using the formula, 

  • The Phenol Coefficient for phenol is considered as “1” any value for disinfectant coming below one is considered as less while above it is more.

Advantages of Phenol Coefficient Test:

  1. Quick.

  2. Inexpensive.

  3. Reproducible results.

  4. Useful to eliminate useless products.

  5. Sets standard for the preparations.

Disadvantages of Phenol Coefficient Test:

  1. Most tests use only one test organism i.e. Salmonella typhi which represents inadequate information.

  2. Compares the activity of disinfectants at only one concentration at fixed conditions.

  3. Presence of organic matter at action time is not considered.

  4. Does not give information about tissue toxicity of the disinfectant.

  5. Sampling errors are large.

5) Kelsey-Skyes Mehod:

  • Developed in 1969, this test overcomes many drawbacks of the RW Test.

  • The test uses several test organisms viz. Staphylococcus aureus, Proteus vulgaris, Eschericia coli and Pseudomonas aeruginosa.

  • Tests can be carried out in a clean as well as dirty environment.

  • In both clean and dirty cases the final bacterial concentration should be about 109/ml.

  • Clean conditions are simulated by using a clean broth while dirty conditions are simulated by using a yeast suspension or inactivated horse serum.

  • The samples are taken at 8, 18 and 28 minutes are then incubated at 30 to 32 ℃ and observations of growth of microorganisms in test tubes are recorded.

  • Result interpretation: A disinfectant is considered satisfactory if,

    • No growth in 2 or 5 tubes of 18 min sample or,

    • No more than 5 colonies from 5 drops on the agar plate.

Commonly Asked Questions:

  1. Define DDisinfection. Give ideal properties of Disinfectant.

  2. Write Mechanism of action and applications of ,

    1. Chlorine,

    2. Iodine,

    3. Heavy Metals.

    4. Formaldehyde.

    5. Cetrimide.

  3. Write a note on “Factors affecting Disinfection”.

  4. Give classification of disinfectants.

  5. List out different methods used for evaluation of Disinfectant. Write in detail about the Phenol Coefficient Method.

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