Non-Tariff Barriers

Non-Tariff Measure

NTM classification

B82: Testing requirement 

Date when the measure came into force

14 July 1999 

Publication where the measure is specified

Government Notice R529 (Government Gazette 19999) Of 14 May 1999 

Regulation where the measure is specified

Compulsory Specification For Disinfectants And Detergent-Disinfectants 

Country/Region applying the measure

South Africa 

The rationale of the measure

This specification covers requirements for disinfectants and detergent-disinfectants intended for use on inanimate surfaces. 

Coded list of objectives

X: For purposes n.e.s. 

Description of the measure

5 Methods of microbiological testing
5.1 General
The tests shall be undertaken by persons experienced in microbiological techniques, using aseptic techniques.
NOTES
1 In order to ensure accuracy of these tests, it is recommended that each test be repeated.
2 Before these tests are carried out, the efficacy of the Inactivating system should be checked to ensure that it adequately inactivates the disinfectants or detergent-disinfectants to be tested.
3 For the purpose of checking the resistance of the test organisms and the other test conditions, it is advisable to include a reference standard. It is essential that it be a disinfectant or detergent-disinfectant based on the relevant active ingredient,
but, because a universal standard is difficult to select, each laboratory should make its own choice of material.
5.2 Laboratory ware
Ensure that all glassware is resistant to repeated heat sterilization and that the glass is free from inhibitory substances such as heavy metals and free alkalis. Borosilicate glass with an expansion coefficient of less than 6 x 10^(-6%)k^(-1) is recommended.
5.2.1 Universal container culture bottles
Bottles made of glass, fitted with standard screwed metal caps with rubber liners and of nominal capacity:
a) 30ml, and
b) 110 ml.
Do not use plastics containers or glass containers fitted with plastics tops.
5.2.2 Culture tubes
Rimless cylindrical tubes that have hemispherical ends and a nominal wall thickness of 1,5 mm and that are of the following sizes:
a) diameter and length 16 mm x 160 mm; and
b) diameter and length 20 mm x 200 mm.
Plug these tubes with cotton wool plugs or with plugs of a foam rubber suitable for autoclaving or use screw-capped tubes of similar dimensions.
5.2.3 Graduated pipettes
Total delivery pipettes for bacteriological purposes only, that have an outflow opening of diameter 2 mm to 3 mm, are graduated in units of 0,1 ml and are of sizes to deliver 1 ,0 ml 5,0 ml and 10,0 ml.
5.2.4 Volumetric cylinders

Graduated measuring cylinders with or without stoppers and of capacities 5 ml. 1 ml, 1 00 md, 500 ml and 1 000 ml.
5.2.5 Culture flasks
Culture flasks of capacities 250 m«, 500 ml and 1l
5.2.6 Erienmeyer flasks
Erienmeyer flasks of capacities 250 ml, 2 l and 3 l.
5.2.7 Petri dishes
Petri dishes of diameter and height 100 mm x 20 mm and made of glass or of wettable polystyrene.
5.2.8 Reagent bottles
Bottles of capacities 50 ml and 1 00 m« and that have polypropylene or other plastics stoppers of such design that they can be used to deliver drops of the reagent.

5.2.9 Inoculating loop
A length of platinum or platinum-iridium alloy wire of diameter 0.376 mm, mounted in a holder that consists of a thin metal rod or tube. The end of the wire is formed into a loop of diameter 4 mm at a distance of 38 mm from the holder. The loop is at such an angle to the axis of the wire that it can be kept in the horizontal plane while being lifted vertically off the surface of the liquid.
5.2.10 Droplet pipette
A droplet pipette that delivers 0,2 mi in about five droplets.
5.2.11 Micropipetter
A micropipetter, high precision, adjusted to dispense 20 \ii accurately, and sterile tips suitable for use with this micropipetter.
5.3 Equipment
5.3.1 Autoclave
A pressure vessel capable of producing steam or connected to a central steam source and capable of withstanding a pressure of 300 kPa. The autoclave is capable of attaining a temperature of 121 °C within 10 min of the beginning of the sterilization cycle.
5.3.2 Incubators and water-baths
Incubators and water-baths that have thermostatically controlled heating and cooling devices and that are so fitted with means of circulation that the temperature of the total enclosed space is maintained to within 2 °C of the thermostat setting.
5.3.3 Hot air oven (for sterilization by means of dry heat)
A thermostatically controlled oven heated by electricity or gas and so fitted with means of circulation that the temperature of the total enclosed space is maintained at 1 70 °C ± 5 °C, the heat supply being such that the working temperature is regained within 10 min of the momentary opening and closing of the oven door.
5.3.4 Stop-watch
A stop-watch accurate to within 1 s per hour.
5.4 Media and reagents
5.4.1 General
5.4.1.1 Water
Use only glass-distilled water or demineralized water of equivalent purity, that is clear, colourless and free from visible suspended matter and of which the pH value, measured at 25 °C, is in the range 5,0 to 7,5.
5.4.1.2 Quality of ingredients
In the preparation of the media and reagents, use only ingredients of quality acceptable for microbio- logical purposes. Use anhydrous salts unless otherwise specified.
5.4.1.3 Accuracy
Except where otherwise specified, allow the following tolerances:
a) on temperatures ………..± 2 °C
b) on masses ………………± 1 ,0 %
c) on volumes………………….. ± 1 ,0 %
d) on pH value………………………………….. ±0,1
5.4.1.4 Dehydrated media
Many of the media required are obtainable in dehydrated form and, for uniformity of results, the use of such media is recommended. If these are used, follow the manufacturer's instructions strictly with regard to reconstitution and sterilization.
5.4.1.5 Filtration Of media
Whenever it is necessary to filter a medium in the course of its preparation, proceed as follows:
a) filter a medium that does not contain a solidifying agent, i.e. a liquid medium or broth, through a medium-speed filter paper; or
b) if the medium contains a solidifying agent (for example, agar) filter it through al0mm to15mm thick layer of pre-wetted absorbent cotton wool. To prevent solidification of the medium during filtration, use a steam-jacketed funnel. Alternatively, carry out the filtration in a steam chamber.
5.4.1 .6 Adjustment of the pH value of media
Where the final pH value of a medium or reagent is specified, so adjust the pH value, if necessary, during preparation and, in the case of media, before sterilization, that, after preparation, the required pH value measured at 25 °C is obtained. Unless otherwise specified, use a solution of hydrochloric acid (c(HCI)
= 1 mol/l) or sodium hydroxide (cNaOH) = 1 mol/l), as appropriate, to adjust the pH values.

5.4.1.7 Dispensing
Where specified quantities of media are to be dispensed into bottles, use 30 mi universal bottles (see 5 2.1(a)). Where bulk sterilizing is required, use any suitable glass container of the required quality or suitably stoppered culture tubes (see 5.2.2(a)). Dispense reagents into reagent bottles (see 5.2.8). Stir media constantly while dispensing. Whenever the preparation of slopes for surface cultivation is required,
dispense the medium in 10 m« volumes and sterilize as specified. Immediately after sterilization, place the bottles or, when relevant, the culture tubes, on a 1 -in-4 sloped surface and allow the agar to solidify.
5.4.1.8 Sterilization
When sterilization by autoclaving is specified and unless otherwise directed, autoclave the medium at 121 °C for 15min.
5.4.1.9 Control of prepared media
Ensure, by suitable incubation tests, that prepared media are sterile and are capable of supporting the growth of the relevant organisms under the stated conditions of incubation.
5.4.1.10 Storage Of media
Ensure that prepared media are carefully protected from exposure to heat and sunlight and have not evaporated or changed in concentration or in pH value, and that, unless otherwise specified, they are used within three months of preparation.
5.4.2 Bottom layer agar
5.4.2.1 Ingredients
Tryptone ………………13.0g
Agar………………… 11,0g
Sodium chloride…………….. 8,0g
Glucose…………… 1,0g
Water………………………. 1 000 ml

5.4.2.2 Preparation
Dissolve the ingredients in the water by warming. Cool to 45 °C to 50 °C and dispense into Petri dishes of nominal diameter 90 mm, ensuring that the depth of the agar in each plate is at least 3 mm.
5.4.3 Bovine albumin solution
5.4.3.1 Ingredients

Albumin (bovine) ………………………….1 5,0 g
Water……………………………. 1 000 ml
5.4.3.2 Preparation
Dissolve the albumin in the water. Sterilize by passing through a filter with maximum effective pore size of 0,45 µm. Dispense 10 ml volumes into bottles (see 5.2.1(a)).
5.4.4 Cetrimide inactivator
5.4.4.1 Ingredients
Polyoxyethylene sorbitan mono-oleate…………….. 8,0 g
Sodium taurocholate…………… 8,0 g
Sodium thiosulfate…………..1.5 g
Potassium phosphate, monobasic………………. 0,5 g
Sodium citrate…………… 0,5 g
Water…………………. 1 000 ml
5.4.4.2 Preparation
Dissolve the ingredients in the water by heating. Dispense 20 ml volumes into bottles (see 5.2.1 (a)) and sterilize by autoclaving.
5.4.5 Diluent used in the "5,5,5" test
5.4.5.1 Ingredients
Albumin (bovine)………………………. 0,3 g
Sodium chloride………………………………………. 9,0 g
Water ……………………………..1 000 ml
5.4.5.2 Preparation
Dissolve the ingredients in the water. Sterilize by passing through a filter with maximum effective pore size of 0,45 µm. Dispense 10 ml volumes into bottles (see 5.2.1(a)).
5.4.6 Hard water used in preparing the yeast suspension
5.4.6.1 Ingredients
Calcium chloride (CaCl2) ……………..1 ,0 g
Magnesium chloride (MgCl2-6H2O)………………………… 8,5 g
Water …………………………………..1 000 ml



5.4.6.2 Preparation
Dissolve the ingredients in the water. Dispense 1 00 ml volumes into bottles {see 5.2.1 (b)) and sterilize by autoclaving.

5.4.7 Inactivator media
5.4.7.1 Inactivator medium No. 1
5.4.7.1.1 Ingredients
Monopotassium phosphate (KH2PO4) …………………0,5 g
Sodium citrate (Na3C6H5O7.3H2O)……………………………. 0,5 g
Sodium taurocholate……………………………….. 8,0 g
Sodium thiosulfate (Na2S2O3.5H2O)……………………… 1 .5 g
Polyoxyethylene sorbitan mono-oleate ………………………….8,0 g
Water……………. 1 000 ml
5.4.7.1.2 Preparation
Dissolve the ingredients in the water by heating. Dispense 20 mi volumes into bottles (see 5.2.1 (a)).
Sterilize by autoclaving.
NOTE - This inactivator has been found suitable for disinfectants and detergent-disinfectants based on iodopliors, organic halogen compounds {other than iodine compounds) and most quaternary ammonium compounds.
5.4.7.2 Inactivator medium No. 2
5.4.7.2.1 Ingredients
Polyoxyethylene sorbitan mono-oleate ……………30,0 g
Beef extract…………………. 20,0 g
Peptone………………………. 20,0 g
Sodium chloride……………………………..1 0,0 g
Water……………………….. 1 000 ml
5.4.7.2.2 Preparation
Dissolve the ingredients in the water and adjust the pH value to 7.1 . Dispense 9 md and 10 md volumes into bottles (see 5.2.1(a)) and sterilize by autoclaving.
NOTE -This inactivator has been found suitable for disinfectants and detergent-disinfectants based on glutaraldehyde.
5.4.7.3 Inactivator medium for the "5,5,5" test

5.4.7.3.1 Ingredients
Lecithin (made from soya, purified)………………………… 3,0 g
l-histidine………………………………1,0g
Phosphate buffer 0,25N (see 5.4.12)……………………… 10,0 ml
Sodium thiosulfate (Na2S2O3.5H2O)………………… 5.0 g
Polyoxyethylene sorbitan mono-oleate…………………………….. 30.0 ml
Water………………………………………….. 1 000 ml
5.4.7.3.2 Preparation
Dissolve the ingredients in the water by heating. Dispense 20 me volumes into bottles (see 5.2.1 (a)).
Sterilize by autoclaving.
5.4.7.4 Neutralizer broth medium

Use TSB (see 5.4.22) but to each 100 ml of broth, add 3 g of soy lecithin (azolectin) and 20 g of polyoxyethylene sorbitan mono-oleate. When testing an antiseptic that contains cetrimide, also add 20 ml of cetrimide inactivator (see 5.4.4). Adjust the pH value to between 7,0 and 7,4. Dispense 9 ml volumes into bottles (see 5.2.1 (a)) and sterilize by autoclaving for 20 min.
NOTES
1 In order to dissolve the azoiectin and polyoxyethylene sorbitan mono-oleate in the TSB, use half the volume of TSB in a large container and add the azoiectin and polyoxyethylene sorbitan mono-oleate while stin-ing. Boll for 30 min to 60 min with constant stirring until all the azoiectin granules are dissolved. Allow to cool before diluting to the final volume with TSB.
Adjust the pH value and dispense as required. Do not fill the bottles to more than half their volume before sterilizing, to prevent boiling over of the contents, which will alter the azolectin/polyoxyethylene sorbitan mono-oleate ratio.
2 Sometimes the azolectin/polyoxyethylene sorbitan mono-oleate emulsion settles to the bottom of the container. If this
happens, storage of approximately one week at room temperature usually allows the solids to redissolve. If the neutralizer is required sooner, healing in a water-bath at 100 °C followed by occasional swirling during cooling will redissolve the solids.
3 This inactivator has been found suitable for antiseptics based on chlorhexidine gluconate and for some disinfectants and detergent-disinfectants based on quaternary ammonium compounds and glutaraldehyde.
5.4.8 Mait extract agar

5.4.8.1 Ingredients
Malt extract………………………….. 30,0 g
Agar……………………………………. 15,0 g
Soya peptone……………………………….. 5,0 g
Water …………………………….1 000 m
5.4.8.2 Preparation
Dissolve the ingredients in the water. Dispense 10ml and 1 5 ml volumes into bottles (see 5.2.1 (a)) and sterilize by autoclaving. Allow only the 10 ml volumes to solidify in a sloped position.
5.4.9 Nutrient agar
5.4.9.1 Ingredients
Agar…………………………….. 15,0 g
Peptone……………………………….. 5,0 g
Sodium chloride …………………………………5,0 g
Yeast extract …………………………….2,0 g
Beef extract……………….. 1,0 g
Water …………………………1 000 ml

5.4.9.2 Preparation
Dissolve the ingredients in the water and adjust the pH value to 7,1 . Dispense 1 m{ and 15 ml volumes into bottles (see 5.2.1 (a)) and sterilize by autoclaving. Allow only the 1 ml volumes to solidify in a sloped position.
5.4.10 Nutrient broth No. 2 (double strength)
5.4.10.1 Ingredients
Peptone………….. 10,0 g
Sodium chloride …………………………5,0 g
Refined meat extract ……………….10,0 g^1
Water ………………………………..1 000 ml

5.4.10.2 Preparation
Dissolve the ingredients in water, adjust the pH value to 7,1 and dilute the solution to 1 t Dispense 5 ml volumes into culture tubes (see 5.2.2(a)) and sterilize by autoclaving.

5.4.11 Nutrient medium
5.4.11.1 Ingredients
Peptone…………………….. 5,0 g
Sodium chloride…………………………….. 5,0 g
Yeast extract ………………………………2,0 g
Beef extract ………………………………1 ,0 g
Water…………………………. 1 000 ml

5.4.11.2 Preparation

Dissolve the ingredients in the water and adjust the pH value to 7,1 , Dispense 10 mi and 50 mi volumes into bottles (see 5.2.1(a) and 5.2.1(b), respectively) and sterilize by autoclaving.

5.4.12 Phosphate buffer 0,25N
5.4.12.1 Ingredients
Monopotassium phosphate (KH2PO4)………………………. 34,0 g
Water ……………………………………….500 ml
5.4.12.2 Preparation
Dissolve the monopotassium phosphate in the water. Adjust the pH value to 7,2 with 1 N NaOH. Dispense into 30 m{ bottles (see 5.2.1(a)) and sterilize by autoclaving.
5.4.13 Physiological saline solution
5.4.13.1 Ingredients

Sodium chloride……………………………… 9.0 g
Water…………………. 1 000 ml

5.4.13.2 Preparation

Dissolve the sodium chloride in the water. Dispense into 250 mi culture flasks (see 5.2.5) and sterilize by autoclaving.
1) Meat extract made from specially selected raw materials of a light colour adjusted to neutrality and dried to a fine powder.

5.4.14 Recuiture medium
5.4.14.1 Ingredients

Beef extract………………………. 10 g
Peptone………………………………… 1 g
Sodium……………………… chloride 5 g
Polyoxyethylene sorbitan mono-oleate………………………….. 30 g
Water…………………………… 1 000 ml

5.4.14.2 Preparation
Dissolve the ingredients in the water and adjust the pH value to 7,5. Dispense 1 mfi volumes into culture tubes (see 5.2.2(a)) and sterilize by autoclaving.

5.4.15.1 Sodium thiosulfate (20 g/H)
5.4.15.1 ingredients
Sodium thiosulfate…………………………… 20 g
Water……………………………… 1 000 ml

5.4.15.2 Preparation

Dissolve the sodium thiosulfate in the water, dispense 20 m? volumes into bottles (see 5.2.1(a)) and sterilize by autoclaving.
NOTE - This Inactivator has been found suitable for disinfectants and detergent-disinfectants based on stabilized inorganic chlorine compounds arid stabilized chlorine compounds.

5.4.16 Sodium thiosuifate (10 g/e)
5.4.16.1 Ingredients
Sodium thiosulfate………………….. 1 g
Water ………………………………..1 000 ml

5.4.16.2 Preparation
Dissolve the sodium thiosulfate in the water, dispense 20 ml volumes into bottles (see 5.2.1(a)) and sterilize by autoclaving.
5.4.17 Sporulation medium SM 1
5.4.17.1 Ingredients
Agar…………………… 12,0 g
Manganese sulfate (MnS04.4H20)…………………………… 0,03 g
Dipotassium phosphate…………………………. 4,0 g
Nutrient broth …………………………………3,1 25 g
Water…………………………… 1 000 ml

5.4.17.2 Preparation
Dissolve the ingredients in the water and adjust the pH value to 6,6. Dispense 20 mS. volumes into bottles (see 5.2.1(a)) and sterilize by autoclaving. Allow the medium to solidify in a sloped position.
5.4.18 Standard hard water
54.18.1 Standard hard water No. 1
5.4.18.1.1 Ingredients
Calcium chloride……………………….. 2,1 g
Water………………………………. 7 500 ml

5.4.18.1.2 Preparation

Dissolve the calcium chloride in the water. Dispense 97 ml volumes into 1 10 m« bottles (see 5.2.1(b)) and sterilize by autoclaving.

NOTE -This hard water has been found suitable tor the microbiological testing of disinfectants and detergent-disinfectants based on glutaraldehyde, organic halogen compounds (other than Iodine compounds), quaternary ammonium compounds, stabilized inorganic chlorine compounds and stabilized chlorine compounds.

54.18.2 Standard hard water used for the microbiological testing of disinfectants and detergent- disinfectants based on iodophors

5.4.18.2.1 Ingredients
Magnesium chloride………………………… 1 8,5 g
Calcium chloride……………………………. 7,9 g
Sodium bicarbonate…………………………… 22,4 g
Water……………………………. 2 500 ml

5.4.18.2.2 Preparation

Dissolve the magnesium chloride and calcium chloride in water, adjust the pH value to between 7,6 and 8,0 and dilute to 1 « with water. Sterilize by autoclaving (solution A). Dissolve the sodium bicarbonate in water, adjust the pH value to between 7,6 and 8,0 and dilute to 1 8 with water. Sterilize by filtration (solution B). Add 1 me of each of solutions A and B to 95 ml. of sterile water in an Erlenmeyer flask (see 5.2.6).

5.4.18.3 Standard hard water for use in the Kelsey-Sykes test
5.4.18.3.1 Ingredients

Calcium chloride (CaCl2)………………………… 0.304 g
Magnesium chloride (MgCl2.6H2O) ……………………..0,139 g
Water …………………………..1 000 ml

5.4.18.3.2 Preparation
Dissolve the ingredients in the water. Dispense 100 md volumes into 1 10 mi bottles (see 5.2.1 (b)) and sterilize by autoclaving.

5.4.18.4 Standard hard water used in the Rideal- Walker test
5.4.18.4.1 Ingredients
Calcium chloride ……………………..0,15 g
Magnesium sulfate…………………….. 0,15 g
Water……………. 1 000 ml

5.4.18.4.2 Preparation
Dissolve the ingredients in the water. Dispense 100 ml volumes into 110 ml bottles (see 5.2.1(b)) and sterilize by autoclaving.

5.4.19 Standard phenol solution (50 g/l)
Using pure phenol that has a crystallizing point not lower than 40,5 °C, prepare a 50 g/l stock solution in water and use this for making the control dilutions (see 5.10.2).
NOTE - It is important that pure phenol be used, because cresol has approximately three times the bactericidal efficacy of phenol, and error resulting from its presence (as indicated by reduction of the crystallizing point to below 40,5 °C) might be considerable.

5.4.20 Sterile skimmed milk
5.420.1 Blend 50,0 g of skimmed milk powder with 400 ml of water in a high-speed blender for 5 min to 7 min. Dilute to 500 ml with water.

5ASi02 Filter through a coarse filter paper that has been previously wetted, or centrifuge for 10 min at a resultant centrifugal force of 6 kN/kg. If there is a visible residue on the filter paper or in the centrifuge tube, repeat the procedure with a different batch of skimmed milk.

5.4.20.3 Dispense 15 ml to 25 ml volumes into bottles (see 5.2.1(a)) and sterilize by autoclaving for 10 min. Store in a refrigerator maintained at 4 °C.
5.4.20.4 Test for freedom from growth-Inhibiting factors
5.4.20.4.1 Using sterile water, prepare a suspension of Staphylococcus aureus (see 5.5.2.1). So standardize the suspension, by using a spectrophotometer in conjunction with a standard curve, a haemocytometer, Petroff-Hausser countina chamber or any other suitable means, that it contains approximately 100 million organisms (10*) per milliiitre. Use the suspension within three hours of preparation.
5.4.20.4.2 Mix 0,12 ml of this suspension with 15 ml of melted nutrient agar (see 5.4.9) that has been cooled to 45 °C and pour the mixture into a flat-bottomed Petri dish of diameter 100 mm (see 5.2.7), placed on a level surface. After the agar has set, place the Petri dish for at least 15 min in a refrigerator maintained at 2 °C to 5 °C. Put three sterile, polished, stainless steel cylinders (penicillin cups) of outer
diameter 8 mm ± 0,5 mm, inner diameter 6 mm ± 0,5 mm and length 10 mm ± 1 mm on the agar and fill each with the milk to be tested.
5.4.20.4.3 Leave the Petri dish in the refrigerator for 2 h and then incubate the Petri dish at 37 °C for 1 6 h to 20 h. Remove the cylinders from the Petri dish and examine the set agar plate for zones of growth inhibition.
5.4J20.4.4 Repeat the procedure given in 5.4.20.4.1 to 5.4.20.4.3 (inclusive), using:
a) Escherichia coli as the test organism , with:
1) a suspension containing 650 million (6,5 x 10®) organisms per milliiitre; and
2) an inoculum of 0,15 ml in 15 ml of melted nutrient agar; and
b) Pseudomonas aeruginosa as the test organism, with:
1) a suspension containing 500 million (5,0 x 10®) organisms per milliiitre; and
2) an inoculum of 0,15 ml in 15 ml of melted nutrient agar.

5.4.20.5 If no zones of growth inhibition are visible on any of the three sets of plates, the milk may be used for the test.
5.4.21 Top layer agar
5.4.21.1 Ingredients
Tryptone……………………………..1 0,0 g
Sodium chloride………………………… 8,0 g
Agar ………………………………………….6,0 g
Glucose………………………….. 3,0 g
Water ………………………..1 000 ml

5.4.21.2 Preparation

Dissolve the ingredients in the water by warming. Dispense 90 ml volumes into 110 ml
bottles (see 5.2.1 (b)) and sterilize by autoclaving.

5.4.22 Tryptone soy broth (TSB)
5.4.22.1 Ingredients
Tryptone …………………………….17,0 g
Sodium chloride………………………………….. 5,0 g
Soy peptone……………………………………………… 3,0 g
Potassium hydrogen dibasic-phosphate……………………………………… 2,5 g
Dextrose…………………………………. 2,5 g
Water ……………………………1 000 ml

5.4.22.2 Preparation
Dissolve the ingredients in the water, heating if necessary. Adjust the pH value to 7,3, dispense 10 ml volumes into bottles (see 5.2.1(a)) and sterilize by autoclaving.

5.4.23 Wright and Mundy medium (synthetic broth AOAC)
5.4.23.1 Part A
5.4.23.1.1 Ingredients
l-cystine………………… 0-05 g
dl-methionine……………………………….. 0,37 g
l-arginine hydrochloride………………………… 0,4 g
dl-histidine hydrochloride………………………. 0,3 g
l-lysine hydrochloride…………………… 0,85 g
l-tyrosine………………………………… 0,21 g
dl-threonine………………………………… 0,5 g
dl-valine…………….. 1,0 g
l-leucine ……………………………0,8 g

dl-isoleucine ……………………………….0.44 g
Glycine…………………………. 0,06 g
dl-serine…………………………………… 0,61 g
dl-alanine………………………. 0,43 g
l-glutamic acid hydrochloride ……………………………..1 .3 g
l-aspartic acid …………………………………………..0,45 g
d l-phenylalanine………………………………. 0,26 g
dl-tryptophan……………………………… 0,05 g
l-proline …………………………0,05 g
Water ……………………………….500 ml

5.4.23.1.2 Preparation
Dissolve the ingredients in the water and add 1 8 m{ of a sodium hydroxide (c(NaOH) = 1 mol/l) solution.
5.4.23.2 Parts
5.4.23.2.1 Ingredients
Sodium chloride ………………………………..3,0 g
Potassium chloride……………………………………… 0,2 g
Magnesium sulfate………………………………………….. 0,05 g
Potassium phosphate……………………………………… l,5 g
Disodium phosphate …………………………………4,0 g
Thiamine hydrochloride……………………… 0,01 g
Nicotinamide ………………………0,01 g
Water ……………………………..500 ml

5.4.23.2.2 Preparation

Dissolve the ingredients in the water. Mix Parts A and B. If necessary, adjust the pH value to 7,1 . Dispense half of the medium in 10 m{ ± 0,2 mi volumes and the other half in 6 m{ ± 0,2 mi volumes into bottles (see 5.2.1(a)) and sterilize by autoclaving.

Before use, add to each tube in the two sets of bottles 0,1 me and 0,06 me, respectively, of a sterile 100 g/l solution of glucose.

5.4.24 Yeast suspension
5.4.24.1 Preparation of 20 % (by mass) of moist yeast suspension

NOTES
1 Whenever possible, the 20 % (by mass) of moist yeast suspension should be sterilized on the day the yeast is received. If this is not feasible, the unopened yeast package should be stored at a temperature not higher than 5 °C for not longer
than 48 h before use.
Crumble approximately 500 g of baker's yeast by hand into a previously tared 1 l beaker and determine the mass of the moist yeast. Cream the yeast by adding a small volume of hard water (see 5.4.6) while stirring the mixture. Carefully transfer the creamed portion to an Erienmeyer flask of capacity 2 i (see 5.2.6) and add a further small volume of hard water to any lumpy residue remaining in the beaker.
Continue this process until all the yeast has been transferred from the beaker to the flask and the concentration of the yeast suspension in the flask has been reduced to approximately 40 % (by mass) of moist yeast. Shake the contents of the flask vigorously and remove large particles by passing the suspension through a sieve of aperture size 140 pm, supported in a funnel in an Erienmeyer flask of capacity 3 l (see 5.2.6). Add enough hard water to reduce the concentration of the yeast to approximately 20 % (by mass) of moist yeast. Shake thoroughly and, while agitating, dispense 100 ml
volumes into bottles (see 5.2.1 (b)). Sterilize by autoclaving and store at 4 °C until required for use.
5.4.24.2 Determination of moisture content

Pipette 25 ml of the sterilized yeast suspension (see 5.4.24.1 ) into a dry tared
dish and dry to constant mass in a hot air oven maintained at 100 °C. Use this mass to determine the additional volume of hard water that must be added to each bottle of sterilized yeast suspension to make a suspension that contains exactly 5 % (by mass) of dry yeast.
5.4.24.3 Adjustment of pH value

Using a sodium hydroxide (c(NaOH) = 1 mol/«) solution, adjust the pH value of 1 00 ml of the 20 % (by mass) of moist yeast suspension (see 5.4.24.1) to 7,0, and note the volume of the sodium hydroxide solution required.

5.4.24.4 Preparation of 5 % (by mass) of dry yeast suspension

Immediately before use add to 100 ml of 20 % (by mass) of moist yeast suspension (see 5.4.24.1), the volume of hard water (see 5.4.6) necessary to mal<e a suspension that contains exactly 5 % (by mass) of dry yeast and enough sodium hydroxide (c(NaOH) = 1 mol/fi) solution (see 5.4.24.3) to adjust the pH value to 7,0. Store the yeast suspension at 4 "C for not longer than 7 d before use.

5.5 Test organisms

Use the following test organisms:

a) Staphylococcus aureus SABS TCC Sta 53 and SABS TCC Sta 59;
b) Escherichia coli SABS TCC Esc 25;
c) Escherichia coli K-1 2 Hf r, NCTC1 2486 SABS TCC Esc 36;
d) Escherichia coli 36 with bacteriophage MS2 SABS TCC Phg-C1 in cases where virucidal
e) Escherichia coli ATCC 1 3706 SABS TCC Esc 37;
f) Escherichia coli 37 with bacteriophage <t)X1 74 SABS TCC Phg-C2 in cases where virucidal
g) Pseudomonas aeruginosa SABS TCC Pse 2 and SABS TCC Pse 16;
h). Salmonella typhi SABS TCC Sal 10 (NCTC 786. Lister strain)
i) Aspergillus r)iger SABS TCC 355 in cases where fungicidal
j) Bacillus subtilis var. globigii SABS TCC Bac 35 in cases where sporicidal
Organisms that have survived the action of a disinfectant or detergent-disinfectant shall under no circumstances be used in a test.

Notes
1 Additional organisms may be used if so desired.
2 The extreme importance of using the standard strain is emphasized.
5.5.1 Maintenance of test organisms

5.5.1.1 Escherichia coli. Salmonella typhi. Staphylococcus aureus and Pseudomonas aeruginosa
5.5.1.1.1 From a newly opened freeze-dried culture or recently received agar culture, subculture the test organisms into bottles of 10 ml nutrient medium (see 5.4.11).
(Use tryptone soy broth (see 5.4.22) in the case of disinfectants based on chlorhexidine gluconate.)
5.5.1.1.2 Incubate the bottles at 37 °C for 24 h. Make subcultures from the cultures in the bottles onto slopes of nutrient agar (see 5.4.9). Incubate the slopes at 37 °C for 24 h.
5.5.1.1.3 From each of these slope cultures, prepare four subcultures (stock cultures) of each test organism on 10ml nutrient agar slopes (see 5.4.9). Incubate the stock cultures at 37 °C for 24 h and then store them in a refrigerator maintained at 4 °C.

NOTE -Take not more than six serial subcultures from each stock culture before resorting to a new freeze-dried culture.
5.5.1.2 Aspergillus niger
Inoculate slopes of malt extract agar (see 5.4.8) with Aspergillus niger and incubate at 25 °C for 7 d.
5.5.1 .3 Bacillus subtilis var. globigii
5.5.1.3.1 Inoculate slopes of sporuiation medium Sr\/1 1 (see 5.4.17) with B. subtilis var. globigii and incubate at 30 °C for 7 d and then at ambient temperature until 80 % to 100 % spores are present (i.e. approximately 5 d).
5.5.1 .3.2 Determine the degree of sporuiation microscopically. When 80 % to 1 00 % spores are present, harvest the spores by adding sterile water and gently scraping the agar surface. Alternatively, use a sterile glass rod or sterile beads to rub the spores off.
5.5.1.3.3 Centrifuge the suspension for 20 min to 30 min. Decant the supernatant liquid. Wash the spores 3 times to 4 times with sterile water until the supernatant liquid is completely clear. Re-suspend the spores in sterile water and heat the suspension for 1 min at 80 °C. Cool the suspension rapidly and store at 4 °C for 7 d.
5.5.1.34 Finally, wash the spore suspension once more in sterile distilled water and so standardize the suspension, by using a spectrophotometer in conjunction with a standard curve, a haemocytometer, Petroff-Hausser counting chamber or any other suitable means, that it contains at least 10 million (10 ) spores per millilitre.

5.5.1.3.5 Dispense into sterile bottles (see 5.2.1 (a)) and store the spore suspension at 4 °C until needed.
5.5.2 Preparation of cultures for test suspensions

5.5.2.1 Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa
5.52.1,1 For each of the test organisms, inoculate a nutrient agar slope (see 5.4.9) from a stock culture kept at 4 °C (see 5.5.1 .1 .3) and incubate at 37 °G for 24 h.
5.5.2.1.2 For the test, use a 24 h culture that has been subcultured for two successive days. After six subcultures, restart the process, using a fresh stock culture (see 5.5.1 .1 .3).
NOTE - The physiological condition of the test organisms is important and might influence inter-laboratory and intra- laboratory variations in test results.
5.5.2.1.3 Using 10 ml of sterile water, wash the bacterial growth resulting from 24 h incubation from the slope (see 5.5.2.1.1), scraping the agar surface if necessary. Carefully decant the suspended growth into a sterile Erienmeyer flask (see 5.2.6) and shake vigorously to suspend all growth in the water. So standardize the suspension, by using a spectrophotometer in conjunction with a standard curve, a haemocytometer, Petroff-HSusser counting chamber or any other suitable means, that it contains the
required number of organisms per millilitre as stipulated in the individual microbiological efficacy tests. Use the suspension within three hours of preparation.

5.5.2.2 Salmonella typhi
5.522.1 Transfer a small portion of growth from a stock culture (see 5.5.1 .1 .3) to 5 ml of Nutrient broth No. 2 (double strength) (see 5.4.10) and incubate at 37 °C for 24 h.
5.5.2.2.2 Continue subculturing into fresh tubes of Nutrient broth No. 2 (double strength) at daily intervals, always transferring one standard loopful of the culture. After two weeks of subculturing as described, begin the process again with a fresh stock culture.
SJ522.3 A subculture made on any day between the third day and the fifteenth day (inclusive) may be used for the test, subject to the following provisions:
a) omission of one daily subculturing operation requires no special reorganization of procedure but, if subculturing cannot take place on two successive days, three successive daily subculturing operations shall be carried out after the break before organisms suitable for the test are obtained; and
b) over week-ends, the following procedure may be adopted: on the Friday make a subculture at about 1 0:00 and incubate at 37 °C until 1 6:00. Transfer the subculture to an incubator maintained at 22 °C, and leave it there for the week-end. At 08:00 on the following Monday, transfer it back, for at least 2 h, to the incubator maintained at 37 °C. and then prepare the usual subculture for use on the Tuesday.
NOTE - Discard cultures that show signs of clumping or pellicle formation.
5.5.2.3 Aspergillus niger

Using 10 ml of a sterile 0,5 g/l solution of polyoxyethylene sorbitan mono-oleate, wash the growth resulting from 7 d incubation from the slope (see 5.5.1.2), scraping the agar surface if necessary.
Carefully decant the suspended growth into a sterile bottle (see 5.2.1(a)) and shake vigorously to suspend all growth in the water. So standardize the suspension, by using a haemocytometer, Petroff- Hausser counting chamber or any other suitable means, that it contains the required number of organisms per millilitre as stipulated in the individual microbiological efficacy tests. Use the suspension within three hours of preparation.

5.5.2.4 Bacillus subtilis var. globigii
Prepare the suspension in accordance with 5.5.1 .3.
5.5.2.5 Bacteriophage

NOTE - The preparation refers to bacteriophage MS2 strain (see 5.5(d)). The preparation of bacteriophage (PXI 74 strain is identical, except that a different bacterial culture, i.e. SABS TCC Esc 37, is used.
5.5.2.5.1 Preparation of tiie bacteriopiiage stock
Use either the plate method or the broth method.
5.5.2.5.1.1 Plate method
5.5.2.5.1.1.1 Inoculate a dilution of bacteriophage MS strain that produced a semi-confluent lysis on an agar plate with Escherichia coli SABS TCC Esc 36 into tubes containing top layer agar (see 5.4.21 ) . Pour the suspension onto bottom layer agar (see 5.4.2), and incubate at 37 "C for 18 h.

5.52.5.1.15 Dispense 2,5 ml of nutrient medium (see 5.4.1 1 ) aseptically onto each plate and remove the nutrient medium with top layer agar by scraping it from the bottom layer agar, using a sterile glass rod.
55.2.5.1.1.3 Homogenize the suspension by vigorous shaking and then centrifuge the suspension at 7 000r/min for 10 min.

5.5.2.5.1.1.4 Add 10 % (by volume) of chloroform to the bacteriophages in the supernatant and store this stock solution at 4 °C.

5.5.2.5.1.2 Broth method

5.5.2.5.1.2.1 Add the bacteriophage MS strain to a nutrient medium (see 5.4.11) culture of Escherichia Coll SABS TCC Esc 36 which is near the end of the log phase to ensure that virtually every bacterial cell is infected simultaneously.
5.5.2.5.1.2.2 Centrifuge the suspension at 7 000 r/min for 10 min after lysis has occurred (visible as a marked drop in turbidity).
5.5.2.5.1.2.3 Add 10 % (by volume) of chloroform to the bacteriophages in the supernatant and store this stock solution at 4 °C.
5.5.2.5.2 Titration of the bacteriophage suspension
5.5.2.5.2.1 Inoculate a nutrient agar slope from a stock culture of Escherichia coli SABS TCC Esc 36 (see 5.5.1 .1 .3) and incubate at 37 °C for 24 h.
5.5.2.5.2.2 Using 2 ml of nutrient medium (see 5.4.1 1), wash the bacterial growth from the 24 h culture from the slope, scraping the agar surface if necessary. Carefully decant the suspended growth into a sterile screw-top glass bottle and vortex to suspend all the growth in the medium.
5.5.2.52.3 Add 0,5 ml of the solution obtained in 5.5.2.5.2.2 to 50 m« of nutrient medium (see 5.4.11) and incubate for 2 h at 37 "C.
5.52.524 Verify that the absorbance measured at 620 nm ± 20 nm through cells of optical path 1 cm is between 0,05 and 0,10 (inclusive), representing a bacterial concentration of approximately 10 organisms per millilitre.
532J523 Prepare a tenfold dilution series of the bacteriophage suspension, using, for each step, 9 ml volumes of sterile distilled water and, to 0,1 ml of each of the dilutions obtained, add 0,9 ml of the 2 h £ CO// culture (see 5.5.2.5.2.4).
5.52.52.6 Place the suspensions in a water-bath or incubator at 37 °C for 15 min ± 1 min and then add 5,0 mi of top layer agar, which had been melted and kept in a water-bath at 44 "C to prevent solidification of the agar.
5.5.2.5.2.7 Vortex and immediately spread the mixture over the surface of the bottom layer agar in appropriately labelled Petri dishes, and swirl gently.
5.5.2.5.2.8 Allow the agar to solidify, invert and incubate the Petri dishes at 37 °C for 24 h.

5.5.2.5.2.9 After incubation, examine the plates for plaque formation. Count and record the plaques on each plate that contains between 10 and 100 plaques.
5.5.2.5.2.10 A bacteriophage titre of at least 10^7 per millilitre is recommended.
5.6 Disinfecting efficacy of disinfectants and detergent-disinfectants based on
ciilortiexidine gluconate
5.6.1 Inoculate a bottle of tryptone soy broth (see 5.4.22) from a daily subculture of each of the Staphylococcus aureus (SABS TCC Sta 59) and Pseudomonas aeruginosa (SABS TCC Pse 1 6) (see 5.5.2.1 .2) test organisms.

5.6.2 Incubate the inoculated tryptone soy broth at 37 °C for 24 h and proceed as in 5.5.2.1.3, so standardizing the suspension that it contains at least 10 million (10^) but not more than 100 million (10 )
organisms per millilitre.
5.6.3 Test procedure
5.6.3.1 Using sterile distilled water, prepare a relevant dilution of the disinfectant or detergent- disinfectant (see 7.2(f)).
5.6.3.2 Dispense 9 mi volumes of this sample dilution aseptically into sterile test tubes and place for 10 min in a water-bath maintained at 37 "C. Repeat this procedure but use 9 ml of sterile distilled water as a control.

5.6.3.3 Melt the contents of a sufficient number of bottles (containing 15 ml volumes) of nutrient agar (see 5.4.9), cool to 45 °C and maintain them at this temperature.
5.6.3.4 Using a clean, sterile pipette, dispense 1 m« of the Staphylococcus aureus suspension (see 5.6.2) into one of the tubes of each disinfectant or detergent-disinfectant to be tested and to the control.
Mix well, using a vortex type mixer and maintain the suspension in the water-bath for the duration of the test.
5.6.3.5 Remove a 1 mi volume from each tube after 1 min and add each volume to separate 9 ml volumes of neutralizer broth medium (see 5.4.7.4) and stir well.
5.6.3.6 Prepare a tenfold dilution series of the sample, using for each step 9 ml volumes of sterile distilled water as diluent. Four to five serial dilutions are recommended for the control.
5.6.3.7 Using a clean, sterile pipette, dispense 1 ml of each dilution (see 5.6.3.5 and 5.6.3.6) onto two appropriately labelled, sterile plates (Petri dishes). Add 15 ml! of nutrient agar (see 5.6.3.3) to each of the plates and swirl gently to ensure an even distribution of colonies after incubation. Avoid spilling any of the contents of the plates during this process.
5.6.3.8 Allow the agar to solidify, invert the plates and incubate at 37 °C for 48 h.
5.6.3.9 After incubation, examine the plates for growth. Count and record the colonies on each plate (of the test solution and of the control) that contains between 30 and 300 colonies. If the least diluted sample (with the highest concentration) yields less than 30 colonies, count all the colonies. Ensure that the colonies that have been counted are derived from survivors of the test organisms and not from contamination.
5.6.3.10 Take the dilution factor (DF) for the test sample and for the control as the inverse of the dilution, for example if the sample or the control has been diluted to 1/1 000, take DF as 1 000.



5.6.3.12 Repeat the procedure described in 5.6.3.1 to 5.6.3.1 1 , using Pseudomonas aeruginosa as the test organism.

5.6.4 interpretation of results
Deem the disinfectant or detergent-disinfectant to comply with the requirements of 4.2 if, subject to the following conditions:
a) at a concentration of 2 % of chlorhexidine gluconate (in cases where the supplied disinfectant or detergent-disinfectant contains 5 % of chlorhexidine gluconate or more); or
b) at the prescribed concentration, i.e. in an undiluted form (see 7.2(f)) (in cases where the supplied disinfectant or detergent-disinfectant contains less than 5 % of chlorhexidine gluconate),
the disinfectant or detergent-disinfectant kills at least 99,9 % of each organism tested.
5.7 Disinfecting efficacy of disinfectants and detergent-disinfectants based on
glutaraldeliyde
5.7.1 Survivor count method
The following distinctions are made:
a) disinfectants or detergent-disinfectants intended for general use; and
b) disinfectants intended for use on medical instruments.
5.7.1 .1 Preparation of test organism suspensions
5.7.1.1.1 Disinfectants and detergent-disinfectants based on glutaraldeliyde, intended for general use
Prepare the test organism suspensions as described in 5.5.2.1 and 5.5.2.3 and ensure that each millilitre of growth medium contains:
a) at least 1 00 000 (1 0^5), but not more than 1 million (1 0^) Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa organisms; and
b) 1 00 000 (1 0^5) Aspergillus niger organisms, respectively.

5.7.1.1.2 Disinfectants based on glutaraldeliyde, intended for use on medical instruments
Prepare the test organism suspensions as described in 5.5.2.1 , 5.5.2.3 and 5.5.2.4, and ensure that each
millilitre of growth medium contains:

a) at least 1 million (1 0^7), but not more than 1 00 million (1 0^8) Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa organisms;
b) 1 million (10^7) Aspergillus n/ger organisms; and
c) at least 1 million (1 0^7) Bacillus subtilis var. globigii spores, respectively.
5.7.1.2 Preparation of control and test solutions
5.7.1.2.1 Control solutions
5.7.1.2.1.1 Disinfectants and detergent-disinfectants based on glutaraldehyde, intended for general use
Dispense 8,9 ml of standard hard water No. 1 (see 5.4.1 8.1 ) into each of three sterile glass bottles and to each add 0,1 ml of sterile skimmed milk (see 5.4.20).
5.7.1.2.1.2 bisinfectants based on glutaraldehyde, Intended for use on medical instruments
Dispense 9 ml of sterile distilled water into each of three sterile glass bottles.
5.7.1 .2.2 Test solution
5.7.1.2.2.1 Disinfectants and detergent-disinfectants based on glutaraldehyde, intended for general use
Prepare the prescribed concentration of the test sample as stated on the label (see 7.2(f)), using standard hard water No. 1 (see 5.4.18.1). Dispense 8,9 mfi of the test solution into each of three sterile glass bottles and to each add 0,1 ml of sterile skimmed milk (see 5.4.20). Use the test solution on the day of preparation.
5.7.1.2.2.2 Disinfectants based on glutaraldehyde, intended for use on medical Instruments
Prepare the relevant concentration of the test sample as stated on the label (see 7.2(f)), using sterile distilled water. Dispense 9 m{ of the test solution into each of three sterile glass bottles. Use the test solution on the day of preparation.
5.7.1.3 Temperature adjustment
Label the bottles that contain the control, the test solutions and the test organism suspensions and place them for at least 30 min in a water-bath maintained at 22 °C ± 1 °C.
5.7.1.4 Test procedure for disinfectants and detergent-disinfectants based on glutaraldehyde, intended for general use
NOTE - For the sake of brevity, the procedure for testing the disinfectant solution against one organism is given. For each organism, a control test Is carried out, using the relevant solution (see 5.7.1 .2.1 .1 and 5.7.1 .2.1 .2) instead of the disinfectant solution. Each control runs concurrently with its associated test (with a time lapse of about 30 s between associated actions). Use a stop-watch for this purpose.

5.7.14.1 Melt the contents of a sufficient number of bottles (containing 15 mi volumes) of nutrient agar (see 5.4.9), cool to 45 °C and maintain them at this temperature.

5.7.1.45 Using a clean, sterile pipette, dispense 1 m{ of the Pseudomonas aeruginosa test suspension (see 5.7.1 .1 .1(a)) into the test solution and to the control (without removing the bottles from the water-bath). Remove the bottles from the water-bath, vortex them to mix the contents thoroughly and immediately return them to the water-bath.
5.7.1.4.3 At the end of a 5 min exposure period (actual contact time), transfer 1 mH of the test solution containing the organism to 9 ml of inactivator medium No. 2 (see 5.4.7.2) and mix well into a uniform suspension (first dilution). Within 30 s, repeat this procedure with the control.
5.7.1.4.4 Using a clean, sterile pipette, transfer 1 ml of the inactivator medium containing the organism (see 5.7.1 .4.3) to a Petri dish and then 1 m{ to the first of a series of bottles, each containing 9 ml of sterile distilled water. Mix well into a uniform suspension (second dilution).

5.7.1.4.5 Repeat the procedure in 5.7.1.4.4, but transfer the suspension obtained after the second dilution to the second of the series of bottles. Dilute further until an end dilution of 1 :1 000 of the test sample is obtained.

5.7.1.4.6 Consecutively prepare, as described above, a dilution series using the control.
5.7AA.7 From each dilution of the test sample, transfer 1 m{ to each of two appropriately labelled, sterile plates (Petri dishes). Do the same for each dilution of the control. Add 15 me of nutrient agar (see 5.7.1 .4.1) to each of the plates and swirl gently to ensure an even distribution of colonies after incubation.
Avoid spilling any of the contents of the plates during this process.
5.7.1.4.8 Allow the agar to solidify, invert the plates and incubate them at 37 °C for 48 h.
5.7.1.4.9 After incubation, examine the plates for growth. Count and record the colonies on each plate (of the test solution and of the control) that contains between 30 and 300 colonies. If the least diluted sample (with the highest concentration) yields less than 30 colonies, count all the colonies. Ensure that the colonies that have been counted are derived from survivors of the test organisms and not from contamination.
5.7.1.4.10 Take the dilution factor {DF) for the test sample and for the control as the inverse of the dilution, for example if the sample or the control has been diluted to 1/1 000, take DF as 1 000.
5.7.1.4.11 The percentage kill
5.7.1.4.12), Staphylococcus aureus after 5 min contact time (see 5.7.1.4.13) and Aspergillus mger after 15 min contact time (see 5.7.1.4.14).

5.7,1.4.12 Repeat the procedure described in 5.7.1 .4.1 to 5.7.1 .4.1 1 (inclusive) with the Escherichia coli suspension (see 5.5.2.1).

5.7.14.13 Repeat the procedure described in 5.7.1 .4.1 to 5.7.1 .4.1 1 (inclusive) with the Staphylococcus aureus suspension (see 5.5.2.1).
5.7.14.14 Repeat the procedure descritDed in 5.7.1 .4.1 to 5.7.1 .4.1 1 (inclusive) with the Aspergillus niger
test organism suspension (see 5.5.2.3), but
a) use an exposure period (actual contact time) of 1 5 min,
b) add malt extract agar (see 5.4.8) to the suspensions on the plates, and
c) incubate lor 7 d at 25 °C.
5.7.1.5 Test procedure for disinfectants based on glutaraidehyde. intended for use on medical instruments
5.7.1.5.1 Pseudomonas aeruginosa and Aspergillus nIger
Carry out the procedure in 5.7.1 .4.1 to 5.7.1 .4.1 1 (inclusive), using the Pseudomonas aeruginosa test suspension (see 5.7.1 .1 .1(a)) and the Aspergillus nigenesX suspension (see 5.5.2.3).
5.7.1.5.2 Bacillus subtilis var. globigii
Carry out the procedure in 5.7.1 .4.1 to 5.7.1 .4.1 1 (inclusive), using the Bacillus subtilis var. globigii test suspension (see 5.5.2.4), but:
a) use an exposure period (actual contact time) of 4 h; and
b) incubate at 37 °C for 48 h.
5.7.1.6 Interpretation of results
Deem the samples to comply with the requirements of 4.2 if. for each organism tested, a result of at least a 99,99 % kill was obtained.
5.7.2 Sporicidal activity (Kelsey-Sykes test modified)
5.7.2.1 Medium
Use the inactivator medium No. 2 described in 5.4.7.2.
5.7.2.2 Test organism suspension
Use the spore suspension of B. subtilis var. globigii (see 5.5.2.4).
5 7.2.3 Preparation of test solution

Prepare the prescribed concentration of the test solution as stated on the label (see 7.2(f)). using sterile distilled water. Use the test solution on the day of preparation.

5.7.2.4 Test procedure
5.7.24.1 Dispense 3 ml of the test solution into a sterile glass bottle.
5 7.24.2 Label the bottles containing the test solution and the test organism suspensions and place them for at least 30 min in a water-bath maintained at 22 °C ± 1°C.
5.7.24.3 Then, without removing the bottle containing the test solution from the water-bath and using a clean, sterile pipette, add 1 m{ of the test organism suspension to the test sample. Simultaneously start a stop-watch. Remove the bottle from the water-bath, mix the contents well and immediately return it to the water-bath.

5.7.2.4.4 After exactly 4 h, transfer 0,02 ml of the suspension obtained in 5.7.2.4.3 to each of five tubes containing 10 ml of inactivator medium No. 2 (see 5.4.7.2).
5.7.2.4.5 Incubate the inoculated tubes of inactivator medium at 30 °C for 48 h.
5.7.2.4.6 After incubation, examine the tubes of inactivator medium for growth and record the results.
5.7.2.5 Interpretation of results
Deem the sample to comply with the requirements of 4.2 if no growth of the test organism is detectable in any of the five tubes of inactivator medium.
5.7.3 Kelsey-Sykes test
5.7.3.1 Procedure
Follow the procedure in 5.8, but use only Pseudomonas aeruginosa as the test organism.
5.7.3.2 Interpretation of results
Deem the sample to comply with the requirements of 4.2 if the initial concentration of disinfectant (concentration B) (see 5.8.2.6(a)) shows no growth of the test organism in at least two of the five tubes of reculture medium in sets inoculated at:
a) the eighth minute after the addition of the initial inoculum; and
b) the eighteenth minute after the addition of the initial inoculum.
An example of a series of test results and their interpretation is given in table 2.
5.8 Kelsey-Sykes test for detergent-disinfectants based on plienolics
5.8.1 Minimum inhibitory concentration test

5.8.1.1 Preparation of the test suspensions
Prepare a 1 :10 dilution in Wright and Mundy medium (see 5.4.23) of a freshly grown subculture of each of the test organisms (see 5.5.2.1).
NOTE - Before diluting a Pseudomonas aeruginosa culture, filter it through a coarse filter paper.
5.8.1 .2 Preparation of the test sample dilutions
5.8.1.2.1 To 5 ml of test sample in a glass bottle of capacity 30 ml (see 5.2.1 (a)), add 5 ml of Wright and Mundy medium (see 5.4.23). Mix well and transfer 5 mi of this dilution of test sample to a further 5 mi of Wright and Mundy medium. Repeat the procedure until 10 doubling dilutions of the test sample (from 1 :2 to 1 :1 024) have been prepared. Discard 5 mi of the last dilution (so that each bottle will contain 5 m{ of a dilution of the test sample).
5.6.122 Repeat 5.8.1 .2.1 until three sets of 10 doubling dilutions of the test sample have been prepared.
5.8.1.3 Test procedure
5.8.1.3.1 To each of the 10 dilutions of the test sample (see 5.8.1.2.2), add 0,02 mi of the
Staphylococcus aureus test suspension (see 5.8.1 .1). Incubate the inoculated bottles at 30 °C for 72 h.
Examine the bottles for growth. The minimum Inhibitory concentration is the highest dilution (minimum concentration) not showing growth.
5.8.1.3.2 Repeat the procedure given In 5.8.1.3.1 but use, successively, the Escherichia coll and Pseudomonas aeruginosa (see 5.8.1 .1) test suspensions.
5.8.1.4 Interpretation of results
Determine which of the three test organisms is most resistant to the test sample, i.e. the organism for which minimum concentration Is the highest. Use this organism for the remainder of the test (see 5.8.2).

5.8.2 Remainder of test
5.8.2.1 Selection of the test organism for the determination using the minimum inhibitory concentration test (see 5.8.1 ). determine which of the test organisms is the most resistant, and use It as the test organism for the determination.
5.8.2.2 Preparation of culture for test organism suspensions
On the day before the test is due to be carried out, inoculate a tube containing 10 ml of Wright and Mundy medium (see 5.4.23) from a daily subculture of the appropriate test organism (see 5.5.2.1 ) and incubate the inoculated medium at 37 °C for 24 h.
5.8.2.3 Preparation of test organism suspension for the test under "clean" conditions

5.8.2.3. 1 After incubation (see 5.8.2.2). centrifuge the culture of the test organism for 15 min at a resultant centrifugal of 6 kN/kg. using a sterile Pasteur pipette, remove and discard the supernatant liquid and resuspend the organism In 1 mi! of hard water prepared for use in this test (see 5.4.1 8.3).
5.8.2.3.2 Transfer this suspension to a sterile glass bottle of capacity 30 ml (see 5.2.1 (a)).
5.8.2.3.3 Add a few sterile glass beads and shake for 1 min.
5.8.2.4 Preparation of test organism suspension for the test under "dirty" conditions
Obtain a suspension that contains 5 % (by mass) of dry yeast by adding 6 ml of the culture of the test organism (see 5.8.2.3) to 4 md of 5 % (by mass) of the dry yeast suspension (see 5.4.24.4) contained in a sterile glass bottle of capacity 30ml (see 5.2.1 (a)). Add a few sterile glass beads to the mixture and vortex for 1 min.

5.8.2.5 Estimation of the number of viable organisms in the test organism suspension
So standardize the suspension by using a spectrophotometer in conjunction with a standard curve, a haemocytometer, Petroff-Hausser counting chamber or any other suitable means, that it contains at least 100 million (10^6) but not more that 10^10 organisms per milliliter. Use the suspension with three hours of prepearation.
5.8.2.6 Preparation of test solutions
Using the hard water prepared for this test (see 5.4.18.3) and glass bottles of capacity 30 ml (see 5.2.1 (a)), prepare three different concentrations A. B and C of the test sample that are such that.
a) concentration B is that which is expected or claimed to pass the test;
b) concentration A is half of concentration B ; and
c) concentration C is one-and-a-half-times concentration B.
For example, if a test sample is expected to pass the test at a concentration of 1 %, concentration A is a 0,5 % concentration, B is a 1 % concentration and C is a 1 ,5 % concentration.
5.8.2.7 Test procedure under "clean" conditions
5.8.2.7.1 Dispense 3 ml of each concentration of the test sample (see 5.8.2.6) into glass bottles of capacity 30 ml (see 5.2.1(a)), and label these bottles A, B and C, as relevant.

5.8.2.7.2 Place these bottles and the bottle containing the test organism suspension (see 5.8.2.3) for at least 30 min in a water-bath maintained at 22 °C ± 1 °C. To maintain the reproducibility of the test, adhere strictly to the temperature stated.
5.8.2.7.3 Then, without removing the bottle containing test concentration A from the water-bath, add 1 ml of the test suspension (see 5.8.2.3) and at the same time start a stop-watch (zero time). Remove the bottle from the water bath, mix well and immediately return it to the water-bath.

5.8.2.7.4 One minute after zero time, add, in the same way, 1 ml of the test suspension to the bottle containing test concentration B.
5.8.2.7.5 Five minutes after zero time, add, in the same way, 1 ml. of the test suspension to the bottle containing test concentration C.
5.8.2.7.6 Eight minutes after zero time, transfer 0,02 m{ from the bottle containing test concentration A to each of five tubes of reculture medium (see 5.4.1 4) each of which has been labelled A1 .
5.8.2.7.7 Ten minutes after zero time, add, in the same way as described in 5.8.2.7.3 above, a further 1 ml of test suspension (see 5.8.2.3) to the bottle containing test concentration A.
5.8.2.7.8 Eighteen minutes after zero time, transfer 0,02 me from the bottle containing test concentration A to each of five tubes of reculture medium each of which has been labelled A2.
5.8.2.7.9 Concurrently with 5.8.2.7.1 to 5.8.2.7.8. treat the test concentrations B and C in the same way, but base the time intervals on the times at which the first additions of the test suspensions were made, and label the sets of tubes of reculture medium B1 and B2, and C1 and C2 (respectively). Thus in the case of test concentration B, addition and transference will be made one minute later than the times given for test concentration A (i.e at 9 min , 11 min and 19 min after zero time).
Note – In order to obviate errors in transference, a copy of the test timetable (see table 2) should be used during each test. Each step of the test should be ticked off on the timetable as it is carried out.

5.8.2.7.10 Incubate all the inoculated tubes of reculture medium at 30 °C for 48 h.
5.82.7.11 After incubation, examine the tubes of reculture medium for growth and record the results as
shown in the example given in table 3.
5.8.2.7.12 If test concentration B passes the test (see 5.8.2.8), repeat steps 5.8.2.7.1 to 5.8.2.7.1 1 on two subsequent days.

5.8.2.8 Test procedure under "dirty" conditions
carry out the procedure described in 5.8.2.7 but use the test organism suspension described in 5.8.2.4.
5.9 Disinfecting efficacy of coal-tar type disinfectant liquids (black and white)
— Rideai-Wailcer coefficient test

5.9.1 Preparation of test organism suspension

Mix thoroughly a culture of Salmonella typhi (see 5.5.2.2) in nutrient broth No. 2 (double strength) (see 5.4.10) that has been incubated for 24 h at 37 °C and, before use, place the tube for 30 min ± 5 min in the water-bath (see 5.3.2) maintained at 22 °C.

5.9.2 Preparation of control and test solution
5.9.2.1 Standard phenol control solution
From the 50 g/l of standard phenol solution (see 5.4.1 9), make five phenol control dilutions that contain 1 g of pure phenol in each of 95 mi, 100 ml, 105 mi 1 10 ml and 1 15 m£ of solution.
NOTE - These dilutions may be stored in the dark for not more than one week before use.
5.9.2.2 Stock solution of the test sample (1 :100)
5.9.2.2.1 Immediately before withdrawing any portion for testing, thoroughly mix a composite sample of the disinfectant to be tested, ensuring that air is not beaten into or shaken into the sample.
5.9.2.2.2 Withdraw the test portion from the middle of the sample by means of a 5 ml capacity pipette (see 5.2.3). Fill the pipette to just above the mark, wipe it clean on the outside with sterile cotton wool and then adjust the contents to the mark. Allow the contents of the pipette to discharge below the surface of about 480 ml of water at a temperature of 1 8 °C, contained in a measuring cylinder.
5.9.2.2.3 Rinse the pipette out three times (or more in the case of viscous liquids) by drawing up and returning some of the dilution.
5.9.2.2.4 Make the solution up to 500 ml with water, stopper the cylinder and thoroughly mix the contents by inverting, with a corkscrew motion, 50 times.
5.9.2.3 Test solutions
From the stock solution (see 5.9.2.2), prepare five suitable test solutions (see tables 4 and 6) based on the nominal RW coefficient stated on the label (see 7.2(m)). Place 5 me of each of the five chosen solutions in sterile culture tubes (see 5.2.2(a)). Mark and place these tubes in sequence in a rack in the water-bath (see 5.3.2), with the strongest disinfectant solution on the left.
5.9.2.4 Phenol solutions
Prepare five culture tubes (see 5.2.2(a)), each containing 5 m{ of a different phenol control solution (see 5.9.2.1), and mark and arrange them in the same way in the water-bath as the solutions of the disinfectant (see 5.9.2.3).
5.9.2.5 Culture medium

5.9.2.5.1 Mix thoroughly a culture of Salmonella typhi (see 5.5.2.2) in Nutrient broth No. 2 (double strength (see 5.4.10) that has been incubated at 37 °C for 24 h and, before use, place the tube for 30 min ± 5 min in a water-bath maintained at 22 "C.
5.9.2.5.2 Place two sets of 15 tubes (marked sequentially "1 " to "30"), each containing 5 mi of Nutrient broth No. 2 (double strength) (see 5.4.10), in the water-bath.
5.9.3 Test procedure
5.9.3.1 Starting exactly at zero time (use the stop-watch (see 5.3.4) for timing all operations), add 0,2 mlof the test organism suspension (see 5.9.1 ) from the droplet pipette (see 5.2.1 0) to the leftmost test tube containing the disinfectant solution (see 5.9.2.3). Ensure that all of the culture added is pipetted straight into the disinfectant solution and not onto the wall of the test tube. Shake the tube; 30 s after this, inoculate the next tube to the right with 0,2 ml of culture in a similar manner. Inoculate each successive tube, at intervals of 30 s, until the fifth tube has been inoculated.

5.9.3.2 Thirty seconds after this last addition, i.e. 2,5 min after zero time, withdraw a representative loopful (see 5.2.9) of the well-shaken contents of the tube on the extreme left and add this to the tube marked "1" and containing 5 ml of the nutrient broth No. 2 (double strength) (see 5.4.10). Immediately after inoculation, shake the tube containing the inoculated broth. Thirty seconds after this loopful has
been withdrawn, transfer, in the same way. a loopful of the contents of the second test tube to the tube of broth marked "2". Repeat this procedure at intervals of 30 s, working from left to right, until all five test tubes have been so treated.
5 9.3.3 Starting again in each case with the left hand tube, perform two further cycles of withdrawals until three sets of cultures (15 tubes) have been made, i.e. at 2.5 min, 5 min and 7,5 mm intervals, respectively, after exposure.

5.9.34 Repeat the procedure given in 5.9.3.1 to 5 

Reference of the measure

Regulation 5 and 6 

Measure also domestic

Yes 

Products affected by the measure.

Description

Disinfectants and detergent-disinfectants intended for use on inanimate surfaces. 

Countries/Regions affected by the measure.

Description

All countries