EVALUATION OF DISINFECTANTS

REQUIRED CONCENTRATIONS AND TIMES FOR CHEMICAL DESTRUCTION OF MICROORGANISMS

Disinfectants	Microorganisms	Disinfectant concentration	Time
Chlorine	Mycobacterium tuberculosis	50 ppm	50 sec
	Entamoeba cyst	0.1 ppm	150 min
	Hepatitis A Virus	3 ppm	30 min
Ethyl alcohol	Staphylococcus aureus	70 %	10 min
	Escherichia coli	70 %	2 min
	Poliovirus	70 %	10 min
Hydrogen peroxide	Staphylococcus aureus	3 %	12.5 sec
	Neisseria gonorrhoea	3 %	0.3 sec
	Herpes simplex virus	3 %	12.8 sec
Quaternary Ammonium compounds	Staphylococcus aureus	450 ppm	10 min
	Salmonella typhi	300 ppm	10 min
Ethylene oxide gas	Streptococcus faecalis	500 mg/L	2 – 4 min
	Influenza virus	10,000 mg/L	25 hrs

EVALUATION OF DISINFECTANTS

(i) Phenol coefficient test

Phenol

·      Phenol (Carbolic acid) is recognized as first chemical agent with excellent Antimicrobial properties used by Joseph Lister in 1867.

·       At concentrations of 0.1 % to 1 %, it is Bacteriostatic (i.e. stops the reproduction of bacteria). At higher concentrations (from 1 % to 2 %), Phenol is Fungicidal and Bactericidal (i.e. destroys the fungus or bacteria). Phenol can kill Anthrax spores at 5 % concentration within 48 hours.

·    Although phenol has excellent antiseptic properties, it is not used as a common antiseptic due to its systemic toxicity on the skin.

Testing of Phenol coefficient

·     Since Lister introduced Phenol as a first Disinfectant, it has been the Standard disinfectant to which other disinfectants are compared under the same conditions.

·       Phenol coefficient is a number obtained by dividing Dilution ratio of Test Disinfectant with the Dilution ratio of Phenol.

·       In 1903, British Chemists Samuel Rideal and J. T. Ainslie Walker established a protocol to compare the effectiveness of a variety of chemicals with that of Phenol, using as their test organisms Staphylococcus aureus & Salmonella typhi.

·     They exposed the test bacteria to the antimicrobial chemical solutions diluted in water for 7.5 minutes. They then calculated a Phenol coefficient for each chemical for each of the two bacteria tested.

·     A Phenol coefficient of 1.0 means that the chemical agent has about the same level of effectiveness as Phenol.

·     A chemical agent with a Phenol coefficient of less than 1.0 is less effective than Phenol. An example is Formalin, with Phenol coefficients of 0.3 (S. aureus) and 0.7 (Salmonella typhi).

·     A chemical agent with a Phenol coefficient greater than 1.0 is more effective than Phenol. Lysol has a coefficient of 5.0 against Staphylococcus aureus but only 3.2 when used on Salmonella typhi, whereas Ethyl alcohol has a value of 6.3 against both.  Chloramine has a Phenol coefficients of 133 and 100 respectively.

Phenol coefficient of various Chemical Disinfectants

Factors affecting Phenol coefficient test

·       Four major factors affect the disinfectants and thus produce wrong results for Phenol coefficient tests.

·       These factors are

a)     Temperature - An increase in temperature has shown increased disinfectant properties.

b)     pH - Optimal growth is achieved at pH between 6 to 8; thus, the recommended pH for the tests is 7.5.

c)     Surface activity - The Surface active compounds in low concentrations may increase the disinfectant power.

d)    Presence of Interfering substances - Interfering substances such as certain Salts may hinder disinfectant activity.

Limitations of Phenol coefficient test

·       The Phenol coefficient tests are designed specifically for determining the Disinfection power of Phenol-like disinfectants. However, some chemicals whose structure and properties are completely different from phenol but are Germicides (such as chlorine, picric acid, hydrogen peroxide, formalin, iodine, etc.) have been compared with Phenol.

·      In some cases, Water-insoluble compounds are compared with Phenol either in their pure form or diluted in other kinds of solvents. Such misuse of Phenol coefficient creates confusion.

·      It is also not recommended to use Phenol coefficient for testing Antiseptics because Antiseptics are not used to kill Bacillus typhosus usually used in Phenol tests.

·    Different antiseptics kill different bacteria with varying antiseptic power. One example is Tincture Iodine, which is 760 times more disinfecting compared to 5 % Phenol solution. However, in reality, Tincture Iodine is not 760 times more germicidal than 5 % Phenol in practical conditions.

(ii) Filter Paper Method (Disk – Diffusion Method)

·      The Filter paper method of evaluating a chemical agent is simpler than determining a Phenol coefficient. It uses small filter paper disks, each soaked with a different chemical agent.

·     A disk of filter paper is soaked with a chemical and placed on an Agar plate that has been previously inoculated and incubated with the test organism.

·       After incubation, if the chemical is effective, a clear zone representing inhibition of growth can be seen around the disk.

 

(iii) Use – Dilution Test

·    The Use-Dilution Test is commonly used to determine a chemical’s disinfection effectiveness on an inanimate surface.

·       For this test, a Cylinder (8 cm × 10 mm) of Stainless steel or Glass is dipped in a culture of the Targeted microorganism and then dried at 37 °C.

·     The cylinder is then dipped in solutions of Disinfectant at various concentrations for 10 minutes at 20 °C.

·    Finally, the cylinder is transferred to a new test tube containing fresh sterile medium that does not contain disinfectant, and this test tube is incubated for 48 hrs.

      Bacterial survival is demonstrated by the presence of turbidity in the medium, whereas killing of the target organism on the cylinder by the disinfectant will produce no turbidity.

(iv) In - Use Test or Kelsey–Maurer Test

       In-Use Test or Kelsey–Maurer Test can determine whether an actively used solution of Disinfectant is microbially contaminated.

·     One ml sample of the used Disinfectant is diluted into 9 ml of sterile Broth medium that also contains Disinfectant Inactivator (a compound to inactivate the Disinfectant).

·    Ten drops, totaling approximately 0.2 ml of this mixture, are then inoculated onto each of two Agar plates.

·       One plate is incubated at 37 °C for 3 days and the other is incubated at room temperature for 7 days.

·       The plates are monitored for growth of microbial colonies.

·   Growth of five or more colonies on either plate suggests that viable microbial cells existed in the Disinfectant solution and that it is contaminated.

(v) Kelsey-Sykes Capacity Test

·    Kelsey-Sykes Capacity Test is used to detect the efficiency of the Disinfectant under both Clean and Dirty conditions.

·       The method can be carried out under either 'Clean' or 'Dirty' conditions.

·     The dilutions of the Disinfectant are made in Hard water for Clean conditions and in Yeast suspension for Dirty conditions.

·     Depending on the type of Disinfectant, a single test organism is selected from Staphylococcus aureus, Pseudomonas aeruginosa, Proteus vulgaris and Escherichia coli.

·     Bacterial inoculum (1 ml) was added into the Disinfectants in Three Successive lots at 0 mins, 1 mins and 5 minutes.

·       The contact time of Disinfectant and Test bacteria is 8 mins.

·     The 3 sets of 5 replicate cultures corresponding to each challenge are incubated at 32 °C 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.

·       Negative results (presence of Turbidity) are those that have more than one Negative culture in a set.

·   If Negative results are found in the First challenge, the concentration of the disinfectant should be increased to 1.5 ml for Second challenge and 2 ml for further Third challenge.

·      The Third challenge is not included in the Pass/Fail criterion but Positive cultures (No bacterial growth or No turbidity) serve as inbuilt controls.

·       If there are no Positive cultures after the Third challenge, it shows that the Disinfectants are completely ineffective.