ICS 71.100.35  
 to come on the last page  
Reference number  
DRS 457 : 2021  
© RSB 2021  
  
 RWANDA 
STANDARD  DRS  
457 
 
First edition  
Approved by RSB 2021 -mm-dd 
 
Determination of bactericidal efficacy of 
disinfectants  
 
   
DRS 457 : 2021  
i                ©RSB 2021  - All rights reserved  
 In order to match with technological development and to keep continuous progress in industries, 
standards are subject to periodic review. Users shall ascertain that they are in possession of the latest 
edition 
 
 
 
 
 
 
 
 
© RSB 2020  
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized 
in any form or by any means, electronic or mechanical, including photocopying and microfilm, without 
prior written permission  from R SB. 
Requests for permission to reproduce this document should be addressed to : 
Rwanda Standards  Board  
P.O Box 7099 Kigali -Rwanda  
KK 15 Rd, 49  
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Toll Free: 3250  
E-mail: info@rsb.gov.rw  
Website: www.rsb.gov.rw  
ePortal: www.portal.rsb.gov.rw   DRS 457 : 2021  
©RSB 2021  - All rights reserved                  ii 
 Contents  Page 
1 Scope  ................................ ................................ ................................ ................................ ............  1 
2 Normative references  ................................ ................................ ................................ ..................  1 
3 Terms and definitions  ................................ ................................ ................................ .................  1 
4 Methods of microbiological testing  ................................ ................................ ...........................  2 
4.1 General  ................................ ................................ ................................ ................................ ..........  2 
4.2 Accuracy  ................................ ................................ ................................ ................................ ....... 2 
4.3 Laboratory ware  ................................ ................................ ................................ ...........................  2 
4.4 Test media  ................................ ................................ ................................ ................................ .... 3 
4.4.1  Reconstitution of media  ................................ ................................ ................................ ..............  3 
4.4.2  Standard hard water  ................................ ................................ ................................ ....................  3 
4.4.3  Nutrient agar  ................................ ................................ ................................ ................................ . 3 
4.4.4  Nutrient broth  ................................ ................................ ................................ ...............................  4 
4.4.5  Reculture medium ................................ ................................ ................................ ........................  4 
4.4.6  Wright and Mundy medium (synthetic broth AOAC)  ................................ ...............................  5 
4.4.7  Yeast suspension  ................................ ................................ ................................ ........................  6 
4.5 Test organisms  ................................ ................................ ................................ ............................  7 
4.6 Maintenance of test organisms  ................................ ................................ ................................ .. 7 
4.7 Preparation of cultures for test suspensions  ................................ ................................ ...........  8 
4.8 Minimum inhibitory concentration test  ................................ ................................ .....................  8 
4.8.1  Preparation of the test suspensions ................................ ................................ ..........................  8 
4.8.2  Preparation of the test sample dilutions  ................................ ................................ ...................  8 
4.8.3  Test procedure  ................................ ................................ ................................ .............................  8 
4.8.4  Interpretation of results  ................................ ................................ ................................ ..............  9 
4.9 Kelsey Sykes test ................................ ................................ ................................ .........................  9 
4.9.1  Test organism  ................................ ................................ ................................ ..............................  9 
4.9.2  Preparat ion of culture for test organism suspensions  ................................ ............................  9 
4.9.3  Preparation of test organism suspension for the test under “clean” conditions  .................  9 
4.9.4  Preparation of test organism suspension for the test under “dirty” conditions  ..................  9 
4.9.5  Estimation of the number of viable organisms in the test organism suspension  ................  9 
4.9.6  Preparation of test solutions  ................................ ................................ ................................ .... 10 
4.9.7  Test procedure under “clean” conditions  ................................ ................................ ...............  10 
4.9.8  Test procedure under “dirty” conditions  ................................ ................................ ................  11 
4.9.9  Interpretation of results  ................................ ................................ ................................ ............  12 
 
 DRS 457 : 2021  
iii                ©RSB 2021  - All rights reserved  
 Foreword  
Rwanda Standard s are prepared  by Technical Committees and approved by R wanda Standards Board (RSB) 
Board  of Directors in  accordance with the procedures of RSB, in compliance with Annex 3 of the World Trade 
Organisation/Technical Barrier to Trade ( WTO/TBT ) agreement on the pre paration, adoption and application of 
standards.  
The main task of technical committees is to prepare national standards.  Final Draft Rwanda S tandards adopted 
by Technical committ ees are ratified  by me mbers of RSB Board of Director s for publication and gaze ttment as 
Rwanda Standards.  
DRS 457  was prepared by Technical Committee RSB/TC 024, Organic and Inorganic Chemicals.  
In the preparation of this standard, reference was made to the following standard:  
US 1692 : Determination of bactericidal efficacy of disinfectants/sanitizers  
The assistance de rived from the above source is hereby acknowledged with thanks.  
Committee membership  
The following organizations were represented on the Technical Committee on Organic and Inorganic Chemicals 
(RSB/TC 024) in the pr eparation of this standard.  
Star Construction and Consultancy Ltd  
Rwanda Inspectorate, Competition and Consumer Protection Authority  
Rwanda Food and Drugs Authority  
Rwanda Investigation Bureau  
Rwanda Forensic Laboratory  
Rwanda Social Security Board  
Rwanda Environment Management Authority  
BARANYUZWE Cosmetics Ltd  
SULFO Rwanda Industries Ltd  
UBURANGA Natural Products  
Divine Hope Company Ltd  DRS 457 : 2021  
©RSB 2021  - All rights reserved                  iv 
 University of Rwanda /College of Medicine and Health Sciences  
University of Rwanda /College of Sciences and Technology  
University of Rwanda /College of Education  
Rwanda Polytechnic – IPRC Kigali  
Standards for Sustainability (SfS)  
Rwanda  Standards Board  (RSB) – Secretariat  
  DRS 457 : 2021  
1                ©RSB 2020  - All rights reserved  
 
 Determination of bactericidal efficacy of disinfectants  
1 Scope  
This Working Draft prescribes a method to determine the bactericidal efficacy of disinfectants using Kelsey 
Sykes test (modified).  
This method is also applicable  to detergent -disinfectants.  
2 Normative references  
The following documents are referred to in the text in such a way that some or all of  their content constitutes 
requirements of this document.  For dated references, only the edition  cited applies. For undated references, the 
latest edition of the referenced document (including any amendments) applies.  
EAS 384 , Disinfectants  — Glossary of terms  
ISO 6888 -1, Microbiology of food and animal feeding stuffs — Horizontal method for the enumeration of 
coagulase -positive staphylococci (Staphylococcus aureus and other species — Part 1: Technique using Baird -
Parker agar medium  
ISO 7218, Microbiology of food and anima  feeding stuffs — General requirements and guidance for 
microbiological examinations  
ISO 7251, Microbiology of food and animal feeding stuffs — Horizontal method for the detection and 
enumeration of presumptive Escherichia coli — Most probable number techn ique 
ISO 16266, Water quality — Detection and enumeration of Pseudomonas aeruginosa — Method by membrane 
filtration   
3 Terms and definitions  
For the purposes of this standard, the terms and definitions given in EAS 384 and the following apply.  
3.1 
bacterici dal efficacy  
ability of a disinfectant/sanitizer product to kill or prevent the growth of microorganisms, particularly bacteria, as 
required  
  
©RSB 2021  - All rights reserved                  2 
 
 4 Methods of microbiological testing  
4.1 General  
The test shall be undertaken by persons competent in microbiological te chniques, using aseptic techniques. 
Media, reagents and equipment shall comply with the requirements and guidelines given in ISO 7218.  
NOTE 1  In order to ensure accuracy of these tests, it is recommended that tests be repeated.  
NOTE 2  Before these tests ar e carried out; the efficacy of the re -culture medium should be checked to ensure that it 
adequately inactivates the disinfectant to be tested.  
NOTE 3  For the purpose of checking the resistance of the test organisms and other test conditions, it is advisabl e to 
include a reference standard. It is essential that it is a disinfectant based on the relative active ingredient, but, because  a 
universal standard is difficult to select, each laboratory should make its own choice of material.  
4.2 Accuracy  
Except where ot herwise specified, allow the following tolerances on:  
a) Temperature:  ± 2 °C; 
b) Masses:    ± 1.0%;  
c) Volumes:   ± 1.0%; and  
d) pH value:   ± 0.1 pH units.  
4.3 Laboratory ware  
4.3.1  Glassware , resistant to repeated heat sterilization with the glass free from inhibitory substances such 
as heavy metals and free alkali. Borosilicate glass with an expansion coefficient of less than 6 x 10-6 K-1 is 
recommended.  
4.3.2  Universal container culture bottles , made of glass, fitted with standard screwed metal caps with 
rubber liners  and that have nominal capacities of 30 ml and 100 ml. plastic containers or glass containers fitted 
with plastic tops shall not be used.  
4.3.3  Culture tubes , rimless and cylindrical with hemispherical ends and nominal wall thickness of 1.5 mm, 
of internal  diameter 16 mm and of length 160 mm.  
4.3.4  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.  
4.3.5  Measuring cylinders , graduated with or without stoppers and of capacities 5 ml, 10 ml, 100 ml, 500 
ml and 1 l.   
3                ©RSB 2021  - All rights reserved  
 4.3.6  Culture flasks , of capacities 250 ml, 500 ml and 1 l.  
4.3.7  Erlenmeyer flasks , of capacities 250 ml, 2 l and 3 l.  
4.3.8  Petri dishes , of diameter 90 mm and of height 20 mm and made of glass or of wettable polystyrene.  
4.3.9  Reagent bottles , of capacities 50 ml and 100 ml that have polypropylene or other plastic stoppers of 
such design that the can be used to deliver drops of t he reagent.  
NOTE  The analyst may use other volumes of laboratory ware, different from those stated above, as appropriate. 
However, the principle of the test method should be adhered to.  
4.4 Test media  
4.4.1  Reconstitution of media  
Many of the media required are obta inable commercially 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 regarding 
the reco nstitution and sterili zation. The water used in the preparation  of media shall be distilled sterile water . 
Where sterilization by autoclaving is specified, autoclave the medium at 121 °C ± 3 °C for 15 min, unless 
otherwise stated. Unless otherwise specified, use a 0.1 M solution of hydrochloric acid or sodium hydroxid e for 
pH adjustment as is relevant.  
4.4.2  Standard hard water  
4.4.2.1  Ingredients  
Calcium chloride (CaCl 2):     0.304 g  
Magnesium chloride (MgCl 2.6H 2O):   0.139 g  
Water:          1000 ml  
4.4.2.2  Preparation  
Dissolve the ingredients in the water. Dispense 100 ml volumes into suitable containers and sterilize by 
autoclaving.  
4.4.3  Nutrient agar  
4.4.3.1  Ingredients  
Agar:         15.0 g  
Peptone:        5.0 g  
Sodium chloride:       5.0 g   
©RSB 2021  - All rights reserved                  4 
 
 Yeast extract:      2.0 g  
Beef extract:       1.0 g  
Water:        1000 ml  
4.4.3.2  Preparation  
Dissolve the ingredients in the water and adjust the pH value to 7.1. Dispense in 10 ml and 15 ml volumes into 
suitable bottles and sterilize by autoclaving. Allow only the 10 ml volumes to solidify in a sloped position.  
4.4.4  Nutrient broth  
4.4.4.1  Ingredients  
Peptone:        5.0 g  
Sodium chloride:       5.0 g  
Yeast extract:       2.0 g  
Beef extract:        1.0 g  
Water:         1000 ml  
4.4.4.2  Preparation  
Dissolve the ingredients in the water and adjust the pH value to 7.1. Dispense in 10 ml volumes into suitable 
bottles and sterilize by autoclaving.  
4.4.5  Reculture medium  
4.4.5.1  Ingredients  
Beef extract:        10 g 
Peptone:        10 g 
Sodium chloride:       5 g 
Polyoxyethylene sorbitan mono -oleate:  30 g 
Water:         1000 ml  
4.4.5.2  Preparation   
5                ©RSB 2021  - All rights reserved  
 Dissolve t he ingredients in the water and adjust the pH to 7.5. Dispense in 1o ml volumes into tubes and sterilize 
by autoclaving.  
4.4.6  Wright and Mundy medium (synthetic broth AOAC)  
4.4.6.1  Part A  
4.4.6.1.1  Ingredients  
/-cystine:          0.05 g  
d/-methionine:         0.37 g  
/-arginine hydrochloride:       0.4 g  
/-histidine hydrochloride:       0.3 g  
/-lysine hydrochloride:       0.85 g  
/-tyrosine:          0.21 g  
d/-threonine:          0.5 g  
d/-isoleucine:          0.44 g  
glycine:           0.06 g  
d/-serine:          0.61 g  
d/-alanine:          0.43 g  
/-glutamic acid hydrochloride:      1.3 g  
/-aspartic acid:         0.45 g  
d/-phenylalanine:         0.26 g  
d/-tryptophan:         0.05 g  
/-proline:           0.05 g  
Water:           500 ml.  
4.4.6.1.2  Preparation   
©RSB 2021  - All rights reserved                  6 
 
 Dissolve the ingredients  in the water and add 18 ml of a sodium hydroxide [c(Na OH) = 1 mol/l] solution.  
4.4.6.2  Part B  
4.4.6.2.1  Ingredients  
Sodium chloride:           3.0 g  
Potassium chloride:          0.2 g  
Magnesium sulphate:          0.05 g  
Potassium phosphate:          1.5 g  
Disodium phosphate:          4.0 g  
Thiamine hydrochloride:         0.01 g  
Nicotinamide:           0.01 g  
Water:             500 ml  
4.4.6.2.2  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 ml ± 0.2 ml volumes, and the other half in 6 ml ± 0.2 ml volumes into tubes, and sterilize by 
autoclaving. Before use, add to each tube in the two sets of tubes 0.1 ml and 0.06 ml (respectively), of a sterile 
100 g/l (10%)  solution of glucose.  
4.4.7  Yeast suspension  
4.4.7.1  Preparation of 20% (by mass) of moist yeast suspension  
NOTE  Whenever possible, the 20% (by mass) of moist yeast suspension should be sterilize on the day the yeast is 
received. If this is not feasible, the uno pened yeast package should be stored at a temperature not higher than 5 °C for not 
longer than 48 h before use.  
4.4.7.1.1  Crumble approximately 500 g of baker’s yeast by hand into a previously tarred 1 -l beaker and 
determine the mass of the moist yeast. Cr eam the yeast, adding a small volume of hard water (see 4.4.2) while 
stirring the mixture.  
4.4.7.1.2  Carefully transfer the creamed portion to an Erlenmeyer flask of capacity 2 l and add a further small 
volume of hard water to any lumpy residue remaining i n 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.   
7                ©RSB 2021  - All rights reserved  
 4.4.7.1.3  Shake the flask v igorously and remove large particles by passing the suspension through a sieve of 
aperture size 140 µm, supported in a funnel in an Erlenmeyer flask of capacity 3 l. Add enough hard water to 
reduce the concentration of the yeast to approximately 20 % (by m ass) of moist yeast.  
4.4.7.1.4  Shake thoroughly and while agitating, dispense 50ml volumes into bottles fitted with metal screw caps. 
Sterilize by autoclaving and store at 4 °C until required for use.  
4.4.7.2  Determination of moisture content  
Pipette 25 ml  of the sterilized yeast suspension (see 4.4.7.1) into a dry tarred 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 shall be 
added to each bottle of sterilized yeast suspe nsion to make a suspension that contains exactly 5% (by mass) 
of dry yeast.  
4.4.7.3  Adjustment of pH  value  
Using a sodium hydroxide [c(Na OH) = 1 mol/l] solution, adjust the pH value of 50 ml of the 20% (by mass) of 
moist yeast suspension (see 4.4.7.1) to 7.0 and the volume of the sodium hydroxide solution required.  
4.4.7.4  Preparation of 5% (by mass) of dry yeast suspension  
Immediately before use, add to 50 ml of 20 % (by mass) moist yeast suspension the volume (see 4.4.7.2) of 
hard water necessary to make  a suspension that contains exactly 5% (by mass) of dry yeast and enough sodium 
hydroxide [ c(Na OH) = 1 mol/l] solution (see 4.4.7.3) to adjust the pH value to 7.0. Store the yeast suspension 
at 4 °C for not longer than seven days before use.  
4.5 Test organism s 
Use the following test organisms:  
a) Pseudomonas aeruginosa:    RS ISO 16266  
b) Escherchia coli:      RS ISO 7251; and  
c) Staphylococcus aureus:    RS ISO 6888 -1. 
Organisms that have survived the action of an antiseptic shall in no circumstances be used in a test.  
NOTE 1  Additional organisms may be used if so desired.  
NOTE 2  The extreme importance of using the standard strain is emphasized.  
4.6 Maintenance of test organisms   
4.6.1  From a newly opened freeze -dried culture or recently received agar culture, subculture the test 
organisms into bottles of 10 ml nutrients broth (see 4.4.4).   
©RSB 2021  - All rights reserved                  8 
 
 4.6.2  Incubate the bottles at 37 °C for 24 h. prepare subcultures from the cultures in the bottles onto slopes 
of nutrient agar (see 4.4.3). incubate the slopes at 37 °C for 24 h.  
4.6.3  From each of these slope cultures, prepare four subcultures (stock cultures) of each test organism 
onto 10 ml nutrient agar slopes. Incubate the stock cultures at 37 °C for 24 h and then store in a refrigerator 
maintained at 4 °C, except for Pseudomonas aeruginosa , which is stored at room temperature.  
4.6.4  Use the stock cultures to prepare further subcultures for the test (see 4.7), but do not make more than 
six serial s ubcultures from each stock culture. After the sixth serial subculture, resort to a new freeze -dried 
culture.  
4.7 Preparation of cultures for test suspensions  
4.7.1  For each of the test organisms, inoculate a tube that contains 10 ml of Wright and Mundy medium (see 
4.4.6) from a slope subculture (see 4.6.4) and incubate at 30 °C for 24 h.  
4.7.2  For the test, use a 24 -h culture that has been sub -cultured for two successive days. After six 
subcultures, restart the process using a fresh culture (see 4.6.3).  
NOTE  The physiological condition of the test organisms is important and might influence inter -laboratory and intra -
laboratory variations in test results.  
4.8 Minimum inhibitory concentration test  
4.8.1  Preparation of the test suspensions  
Prepare a 1 in 10 dilution in Wrigh t and Mundy medium (see 4.4.6) of a freshly grown subculture of each of the 
test organisms (see 4.7.2).  
Before diluting a Pseudomonas aeruginosa  culture, filter it though a coarse filter paper.  
4.8.2  Preparation of the test sample dilutions  
4.8.2.1  To 5 ml of te st sample in a glass bottle of capacity 30 ml, and which has a standard screwed metal 
cap fitted with a rubber liner (do not use plastics containers or glass containers fitted with plastic tops), add 5 
ml of Wright and Mundy medium (see 4.4.6). Mix well an d transfer 5 ml of this dilution of test sample to a further 
5 ml of Wright and Mundy medium. Repeat the procedure until 10 doubling dilutions of the test sample have 
been prepared. Discard 5 ml of the last dilution (so that each bottle will contain 5 ml o f a dilution of the test 
sample).  
4.8.2.2  Repeat 4.8.2.1  until three sets of 10 doubling dilutions of the test sample have been prepared.  
4.8.3  Test procedure  
4.8.3.1  To each of the 10 dilutions of the test sample (see 4.8.2.1) add 0.02 ml of the Staphylococcus aureus  
test suspension (see 4.8.1). Incubate the inoculated bottles at 30 °C for 72 h and examine the bottles for growth. 
The minimum inhibitory concentration is the highest dilution (minimum concentration) not showing growth.   
9                ©RSB 2021  - All rights reserved  
 4.8.3.2  Repeat the procedure given in 4.8.3.1 but using, successively, the Escherichia coli  and Pseudomonas 
aeruginosa  (see 4.8.1) test suspensions.  
4.8.4   Interpretation of results  
Determine which of the three test organisms is most resistant to the test sample, that is, the organism for w hich 
the minimum inhibitory concentration is the highest. Use this organism for the remainder pf the test (see 4.9)  
4.9 Kelsey Sykes test  
4.9.1  Test organism  
Using the minimum inhibitory concentration test (see 4.8), determine which of the test organisms is the most  
resistant, and use it as the test organism for the determination.  
4.9.2  Preparation of culture for test organism suspensions  
On the day before the test is due to be carried out, inoculate a bottle that contains 10 ml of Wright and Mundy 
medium (see 4.4.6) from a daily subculture of the appropriate test organism which is at least a second and not 
more than a sixth subculture (see 4.6.4) and incubate the inoculated medium at 30 °C for 24 h.  
4.9.3  Preparation of test organism suspension for the test under “clean” conditi ons 
4.9.3.1  After incubation (see 4.9.2), centrifuge the culture of the test organism for 15 min at a resultant 
centrifugal force of 6 kN/kg. Using a sterile Pasteur pipette remove and discard the supernatant liquid and re -
suspend the organism in 10 ml of hard water (see 4.4.2).  
4.9.3.2  Transfer this suspension to a sterile glass bottle (see 4.3.2) of capacity 30 ml.  
4.9.3.3  Vortex for 1 min.  
NOTE  If the test culture is a culture of Pseudomonas aeruginosa , filter it through sterile coarse filter paper befor e 
centrifuging.  
4.9.4  Preparation of test organism suspension for the test under “dirty” conditions  
Obtain a suspension that contains 2% (by mass) of dry yeast by adding 6 ml of the culture of test organism 
suspension (see 4.9.2) to 4 ml of 5% (by mass) of dry y east suspension (see 4.4.7.4) contained in a sterile glass 
bottle (see 4.3.2) of capacity 30 ml. Vortex for 1 min.  
4.9.5  Estimation of the number of viable organisms in the test organism suspension  
4.9.5.1  From each of the test suspensions (see 4.9.3 and 4.9.4) prepare serial dilutions by pipetting 1 ml of 
each suspension into a bottle that contains 9 ml of sterile distilled water and making further dilutions (using a 
fresh pipette each time) by pipetting 1 ml of the first dilution into another bottle that contai ns 9 ml of diluent and 
repeating this procedure until eight successive dilutions have been prepared.   
©RSB 2021  - All rights reserved                  10 
 
 4.9.5.2  From each dilution of the suspension, starting at the sixth dilution, take two 1 -ml volumes and transfer 
each volume to a Petri dish. For each plat e one 15 -ml volume of nutrient agar (see 4.4.3), melt, temper it to 
45 °C, add it to the Petri dish, and mix. Avoid spilling any of the contents of the Petri dish during this procedure. 
Allow the agar to set and incubate the Petri dishes at 32 °C ± 1 °C for 24 h ± 1 h. Ensure that the total period 
between the preparation of the dilutions of the suspension and the final plating does not exceed 30 min.  
4.9.5.3  After incubation count the colonies on each plate and record the results given by plates that contain  
between 30 colonies and 300 colonies.  
4.9.5.4  Multiply each of the recorded counts by the dilution factor involved (for example, for a dilution of 1 in 
107, multiply the average plate count by 107), and take the average of the number of organisms in each millilitre 
of test suspension.  
NOTE 1  If the number of viable organisms in a test suspension is less than 108 per ml or more than 1010 per ml, the 
results of a test (see 4.9.7 and 4.9.8) in which the suspension was used shall be considered invalid.  
NOTE 2  Nephelometric or turbidimetric methods may be used instead of the procedure given in 4.9.5.1 to 4.9.5.4 to 
determine the number of viable organisms in the culture from which  the test suspension is prepared if these can be shown 
to give satisfactory results . 
4.9.6  Preparation of test solutions  
Using hard water (see 4.4.2) and glass bottles of capacity 30 ml, and which have standard screwed metal caps 
fitted with rubber liners, prepare three different concentrations A, B and C of the test sample 
(disinfectant/sanit izer) that are such that  
a) concentration B is that which is expected or claimed to pass the test,  
b) concentration A is concentration B less 50%, and  
c) concentration C is concentration B plus 50%.  
For example, if a test sample (disinfectant/sanitizer) 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.  
4.9.7  Test procedure under “clean” conditions  
4.9.7.1  Dispense 3 ml of each concentration of the test sample (see 4.9.6) into glass bottles or test tubes of 
capacity 30 ml, and which have standard screwed metal caps fitted with rubber liners, and label these bottles 
A, B and C, as relevant.  
4.9.7.2  Place these bottles and the bottle that contains the test organism suspension  (see 4.9.3) for at least 
30 min in a water bath maintained at 22 °C ± 2 °C. To maintain reproducibility of the test, adhere strictly to the 
temperature state.  
4.9.7.3  Then, without removing the bottle that contains test concentration A  from the water bath , add 1 ml of 
the test organism suspension (see 4.9.3) and start a stop -watch (zero time) simultaneously. Remove the bottle 
from the water bath, vortex well and immediately return it to the water bath.   
11                ©RSB 2021  - All rights reserved  
 4.9.7.4  One minute after zero time add, in the same wa y, 1 ml of the test suspension to the bottle that contains 
test concentration B.  
4.9.7.5  Five minutes after zero time add, in the same way, 1ml of the test suspension to the bottle that contains 
test concentration C.  
4.9.7.6  Eight minutes after zero time, transfer 0.02 ml from the bottle that contains test concentration A to each 
of five tubes of re -culture medium (see 4.4.5) each of which has been labelled A1.  
4.9.7.7  Ten minutes after zero time add, in the same way as described in 4.9.7.3, a further 1 ml of test organism 
suspension (see 4.9.3) to the bottle that contains test concentration A.  
4.9.7.8  Eighteen minutes after the zero time transfer 0.02 ml from the bottle that contains test concentration A 
to each of five tubes of re -culture medium each  of wh ich has been labelled A2.  
4.9.7.9  Concurrently with 4.9.7.6 to 4.9.7.8, treat the test concentration B and C in the same way, but basing 
the time intervals on the times at which the first additions of test suspensions were made, and labelling the sets 
of tubes of re -culture medium B1 and B2, and C1 and C2 respectively. Thus, in the case of test concentration 
B addition and transference will be made 1 min later than the times given for test concentration A (that is, at 9 
min, 11 min and 19 min after zero tim e) and in the case of test concentration C additions and transference will 
be made at 13 min, 15 min and 23 min after zero time.  
NOTE  In order to obviate errors in transference, a copy of the test timetable (see table 1) should be used during each 
test. Ea ch step of the test should be kicked off on the timetable as it is carried out.  
Table 1 – Kelsey Sykes test timetable  
Time 
(min)  Test sample suspension 
A Time 
(min)  Test sample suspension 
B Time 
(min)  Test sample suspension 
C 
0 1 ml test suspension to A  1 1 ml test suspension to B  5 1 ml test suspension to C  
8 0.02 ml transferred from A 
to re-culture medium A1  9 0.02 ml transferred from B 
to re-culture medium B1  -  
10 1 ml test suspension to A  11 1 ml test suspension to B  13 0.02 ml transferred from C 
to re-culture medium C1  
-  -  15 1 ml test suspension to C  
18 0.02 ml transferred from A 
to re-culture medium A2  19 0.02 ml transferred from B 
to re-culture medium B2  23 0.02 ml transferred from C 
to re-culture medium C2  
4.9.7.10  Incubate all the inoculated tubes of re -culture medium at 30 °C for 48 h.  
4.9.7.11  After incubation examine the tubes of re -culture medium for growth and record the results as shown in 
the example given in table 2.  
4.9.7.12  If test concentration B passes t he test (see 4.9.9), repeat steps 4.9.7.1 to 4.9.7.11 on two subsequent 
days. If test concentration B fails the test and the minimum concentration that will pass the test is required, 
repeat the entire test on concentrations of greater strength until a val ue for concentration B is found that passes 
three successive tests.  
4.9.8  Test procedure under “dirty” conditions  
Carry out the procedure described in 4.9.7 but use the test organism suspension described in 4.9.4.   
©RSB 2021  - All rights reserved                  12 
 
 4.9.9  Interpretation of results  
Deem the initial conce ntration of disinfectant/sanitizer (concentration B – see 4.9.6,a) to pass the test if there is 
no growth of the test organism in at least two of the five tubes of re -culture medium in sets inoculated at  
a) the eighth, 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.  
Table 2 – Test sample tested under “clean” conditions  
Test No  Test sample 
concentration % 
(by volume)  Test 
suspension 
organisms 
(ml) Re-culture medium  
Result  1 2 
1 0.8 
1.6 
2.4 6.9 x 108 
6.9 x 108 
6.9 x 108 +++++  
- -+++ 
- - - - - +++++  
- ++++  
- - - - - Fail 
Fail 
Pass  
Test solution at 1.6% (by volume) concentration has failed  
Test repeated at higher concentrations  
1 0.9 
1.8 
2.7 8.4 x  108 
8.4 x 108 
8.4 x 108 - ++++  
- - - - + 
- - - - - ++ - + - 
- - +++ 
- - - - -  Fail  
Pass  
Pass  
2 0.9 
1.8 
2.7 2.1 x 109 
2.1 x 109 
2.1 x 109 - ++++  
- - - - + 
- - - - - +++++  
- - +++ 
- - - - - Fail 
Pass  
Pass  
3 0.9 
1.8 
2.7 5.7 x 108 
5.7 x 108 
5.7 x 108 - - +++ 
- - - - - 
- - - - - +++++  
- - - ++ 
- - - - - Fail 
Pass  
Pass  
Test organism: Pseudomonas aeruginosa  
NOTE:  
+ refers to growth of the test organism; and  
- refers to no growth of the test organism  
 
 
   
13                ©RSB 2021  - All rights reserved  
  
 
 
  
©RSB 2021  - All rights reserved                  14 
 
 Bibliography  
[1] ISO/IEC Directives, Part  2, Rules for the structure and drafting of International Standards , 2016  
[2] ISO/IEC  TR 10000 -1, Information technology  — Framework and taxonomy of International Standardized 
Profiles  — Part 1: General principles and documentation framework  
[3]