Rideal Walker Co-effiecient

Rideal Walker Co-effiecient

A test to determine the Germicidal value of a given substance.

In the year 1903 Rideal and Walker developed a test to determine the Germicidal value or measure the bactericidal activity of disinfecting agents and this process was later modified and accepted by the British Standards Institution.

The disinfectant value of Phenol (Carbolic Acid) is taken as 1 (unit) and thus disinfectant value of other products is relative or coefficient of phenol. Rideal Walker test measured the value of the agent for disinfecting pure cultures of Bacterium Typhosum. Later the Americans modified Rideal Walker test by changing the organisms and culture media depending on the type of disinfectant tested such as Staphylococcus Aureus. The test procedure mentioned here is what has been recommended in Schedule ‘O’ of Drugs Act and Bureau of Indian Standards IS 1061. Method of determination of Rideal Walker Coefficient (R.W.C.)

Preparation of sample : The Sample of disinfectant fluids to be tested should be mixed thoroughly taking care that no air is beaten into the fluid immediately before withdrawing any portion for testing. The rest portion should be withdrawn from the middle of the sample.

Apparatus : A loop, 4 mm in internal diameter is made at end of a 28 swg (0.376mm) Wire of platinum or platinum iridium alloy, 38 mm long from the loop to the holder. The loop is bent at such an angle to the length of the wire as will facilitate in removal vertically from the surface of the liquid while keeping the place of the loop horizontal.

Incubator : Set and maintained at 37 °C ± 1 °C

Pipettes : Standard graduated pipettes of capacity 10 ml, 5 ml and 1 ml.

Dropping Pipette : Made to deliver 0.2 ml.

Medication Tubes : 5 sterile plugged rimless test tubes 125 mm x 22 mm (5″ x 3/4 ”) made of hard neutral glass.

Broth Tubes : About 2 dozens of the same description as medication tubes.

Standard measuring cylinders stoppered and graduated: 500 ml graduated in 10 ml: one 100 ml graduated 1 ml: five. All apparatus must be scrupulously clean and sterile immediately before use.

Reagants :

(a) Broth : Prepare a mixture of the following ingredients :

Meat extract (Microbiological grade) 20 g. Peptone (Microbiological grade) 20 g. Sodium Chloride (Reagent Quality) 10 g. Distilled Water: 1000 ml.

Dissolve the solids in distilled water. Add sufficient sodium hydroxide to neutralise the solution ; then boil it to bring down phosphates and filter while hot. The broth thus prepared is then adjusted to pH 7.6 with normal Hydrochloric acid. The broth is then sterilised by autoclaving at 15 Ibs. Pressure for 20 minutes. It is then filtered and placed in 5 ml quantities in sterilised broth tubes. The tubes of media thus prepared are sterilised by autoclaving at 15 Ibs pressure for 10 minutes. The final pH of the medium should lie between 7.3 and 7.5 further resterilisation in bulk or in tubes is not permissible.

(b) Test Organism : The test organism used is salmonella typhi (NCTC 786) of which suitable culture shall be obtained from the Director, Central Drugs Laboratory, Calcutta. This culture is maintained by weekly sub-culture on a nutrient agar slope (made by dissolving 2.5 per cent Agar (Bacteriological grade) in the broth prepared as above), incubating the sub-culture for 24 hours at 37°C and then storing in refrigerator at a temperature below 22°C. For the purpose of the test a little of the growth from the most recent sub-culture in nutrient agar slope is placed in tube of R.W. broth and incubated for 23 hours at 38°C. A standard loopful is then transferred to a second tube and incubated as before. This is done for at least three times before a test is carried out. Sub-culturing in broth is limited to 14 days.

(c) Standard Phenol : 5 percent W/V solution is sterile distilled water of chemically pure phenol having a crystallising point of not less than 40.5°C is prepared. Test dilutions are prepared from this stock solution containing 1 g of phenol in each 95, 100, 105, 155 ml. of the solution made. These dilutions shall be used within a week of preparation.

(d) Test dilutions of Disinfectant (sample) : The sample is prepared as described under ‘Preparation of samples’. A test portion of 5 ml is withdrawn and discharged into about 480 ml of sterile distilled water in a 500 ml glass stoppered sterile measuring cylinder and the pipette is rinsed three times or more in the clear liquid. The whole is then made up to 500 ml with sterile distilled water, the cylinder is stoppered and the contents thoroughly mixed by inverting the cylinder several times. Suitable test dilutions in sterile distilled water are then immediately prepared from this stock solution.

Procedure : 5 ml of 4 chosen dilutions of the disinfectant are placed in 4 medication tubes which are then placed in a rack provided with water bath maintained at a constant temperature between 17°C and 19°C, with the strongest dilution on the left. The fifth medication tube containing 5 ml of the particular phenol dilution is placed on the right. When the content of the medication tubes and broth culture of the test organism have reached the temperature of the water bath, starting at Zero time, 0.2 ml of the culture is added to the left hand medication tube and the tube is shaken gently. After 30 seconds the next tube is inoculated similarly and the process if repeated with each successive tube at at intervals of 30 seconds until the phenol control has been inoculated. Thirty seconds after this last addition (that is 2½ minutes from zero) a loopful of the well shaken content of the tube at the extreme left is withdrawn and placed in tube containing 5 ml of broth medium. Thirty seconds after this, similar operation is performed on the second medication tube.

The procedure is repeated at an interval of 30 seconds with each of the 5 medication tubes working from left to right until 4 sets of cultures have been made i.e. at 2½, 5, 7½ and 10 minutes respectively after exposure. In each withdrawal care should be taken to ensure that the loop is removed vertically from the surface of the liquid with its plane horizontally and without touching the side of the test tubes. The loop shall be sterilised by flaming between each operation, care being taken that the loop is cooled before being again used. The inoculated broth tubes are incubated for not less than 48 hours and not more than 72 hours at 37°C, when the tubes showing growth of the test organisms will be recognised by turbidity of the broth.

Calculation of Coefficient : The R.W.Co-efficient is obtained by dividing that dilution of the disinfectant which shows life of test in 2½ and 5 minutes but no life thereafter by that dilution of the phenol which gives the same response. A typical set of sample is given below :