DEAS 794: 2021 
ICS 71.100.40  
© EAC 2021                                                                                                                         Second  Edition 2021  
 
 DRAFT EAST AFRICAN STANDARD  
Determination of the microbial inhibition of cosmetic soap bars and 
liquid hand and body washes — Test method  
EAST AFRICAN COMMUNITY  
 
 
 
 
 
 
 
   
  DEAS 794: 2021 
ii © EAC 2021 – All rights reser ved 
 Copyright notice  
This EAC document is copyright -protected by EAC. While the  reprodu ction of this do cument by 
participants in the EAC standards development process is permitted without prior permission from EAC, 
neither this document nor any extract from it may be reproduced, stored or transmitted in any form for 
any other purpose  without  prior written p ermission from EAC.  
Requests for permission to reproduce this document for the purpose of selling it should be addressed 
as shown below or to EAC’s member body in the country of the requester:  
© East African Community  2021 — All rig hts rese rved 
East Africa n Community   
P.O.Box 1096  
Arusha  
Tanzania  
Tel: 255 27 2504253/8  
Fax: 255 27 2504481/2504255  
E-mail: eac@eachq.org  
       Web: www.eac -quality.net  
Reproduction for sales purposes may be subject to royalty payments or a licensing agre ement.  
Violators may be persecuted  
 DEAS 794: 2021 
© EAC 2021 – All right s reserved  iii 
 Foreword  
Development of the East A frican Standards has been necessitated by the need for harmonizing requirements 
governing quality of products and services in the East African Community. It is envisaged that through 
harmonized standardization, trade barrie rs that are encountered when good s and services are exchanged within 
the Community will be removed.  
The Community has established an East African Standards Committee (EASC) mandated to develop and 
issue East African St andards (EAS) and other deliverables. The Com mittee is composed of rep resentatives of 
the National Standards Bodies in Partner States, tog ether with the representatives from the public and private 
sector organizations in the community.  
East African Standa rds are developed through Technical Committee s that are representative  of key 
stakeholders including government, academia, consumer groups , private sector and other interested parties. 
Draft East African Standards are circulated to stakeholders through the  Nationa l Standards Bodies in the 
Partner Sta tes. The comments receive d are discussed and incorporated before finalization of standards, i n 
accordance with the Principles and procedures for development of East African Standards.  
East African Standards and  other d eliverables are subject to review, to  keep pace with technolog ical 
advances. Users of the East African Standards are therefore exp ected to ensure that they always have the 
latest versions of the standards they are implementing.  
The committee respo nsible f or this document is Technical Committ ee EASC/TC 074, Surface a ctive agents  
Attention is drawn to the possibility that some of the elements of this document may be subject of patent 
rights. EAC shall not be held responsible for identifying any or al l such p atent rights.  
This second  edition can cels and replaces the first edition (EAS 794:2013), which has been technically revised.  
 DEAS 794: 2021 
iv © EAC 2021 – All rights reser ved 
 Introduction  
Cosmetic soap bars and liquid hand and body washes normally have antibacterial agents incorporated into 
them . They n ot only clean the skin, but also reduce drastically the bacterial count on the skin. This prevents 
skin infections and p erspiration odour caused by the decomposition of sweat by bacteria.  They are especially 
effective against Staphylococcus  and si milar ba cteria which have the habit of residing in the under layers of 
the skin.  
This standard specifies a method of test for de termination  of the microbial inhibition of cosmetic soap bars and 
liquid hand and body washes.  
  DRAFT EAST AFRICAN STANDARD  DEAS 794: 2021 
 
© EAC 2013  – All rights reserved  1 
 Determination of the microbial inhibiti on of cosmetic soap bars and 
liquid hand and body washes — Test method  
1 Scope  
This Draft  East African Standard prescribes  a method for testing and comparing the microbial inhibition 
properties of cosmetic soap bars and liquid hand and body washes.  
2 Princ iple 
When soap bars and liquid hand and body washes that have antimicrobial properties are inoculated into 
medium No.1 (see 3.2.1), specific active ingredients diffuse into the surrounding agar thereby creating a zone 
of inhibition around the produ ct, whic h is measured as an indication of the microbial inhibition properties of the 
product.  
NOTE  The organisms referenced in this s tandard are the indicator organisms used when testing for hygiene purposes.  
3 Test method  
3.1 General  
Sampling and testing shall be  carried out by personnel familiar with microbiological procedures.  
3.2 Culture medium, reagents, reference cultures and controls  
3.2.1 Culture medium  
The culture medium used shall be Medium No . 1 or any other suitable agar medium can be used for the  test. 
 
3.2.1.1 Ingredients  
The ingredient s for Medium No. 1  shall inclu de the following:  
a) agar, 15.0 g ; 
b) beef extract , 15.0 g ; 
c) glucose , 1.0 g ; 
d) pancreatic digest of casein , 4.0 g ; 
e) peptone , 6.0 g ; 
f) yeast extract , 3.0 g ; and   
2 © EAC 2021 – All rights reser ved 
 g) water, 1 000 mL . 
NOTE 1  The culture med ium is commercially avai lable in dehydrated form and is made up in ac cordance with the 
manufacturer’s instructions.  
NOTE 2  Glass di stilled water or deionised water should be used  
3.2.1.2 Preparation  
3.2.1.2.1  Dissolve the ingredients in approximately 900 m L of water and mix.  
3.2.1.2.2  Make up to one litre . 
3.2.1.2.3  Adjust the pH so that after sterilization the pH value is 6.6  ± 0.05.  
3.2.1.2.4  Dispense 20 mL ± 0.2 mL volumes into suitable containers and sterilize in an autoclave for 15 min 
± 0.5  min at 12 1 °C. 
3.2.2 Horse serum  
Sterile inactivated horse serum that is free f rom preservatives  shall be used . 
3.2.3 Reference cultu res 
3.2.3.1 Test organisms  
Use the following test organisms  or their equivalen ts 
• Staphylococcus aureus : ATCC 6538;  
• Escherichia coli :   ATCC 8739; and  
• Pseudomonas aeruginosa : ATCC 1 5442.  
NOTE Other reference organi sms may also be used in addition to the on es speci fied in 3.2.3.1. 
3.2.3.2 Preparation of test organism suspensions  
3.2.3.2.1  Nutrient medium  
3.2.3.2.1.1  Ingredients  
The ingr edien ts for the nutrient medium  shall include th e following:  
• peptone:    5.0 g ; 
• sodium chloride:  5.0 g ; 
• yeast extract:   2.0 g ; 
• beef ex tract:   1.0 g ; and  
• water:    1 000 mL . 
NOTE  The test organisms should be sub -cultured from a newly opened freeze -dried cultur e or recently received agar 
culture into bottles  of 10 mL nutrient medium.   
© EAC 2021 – All right s reserved  3 
 3.2.3.2.1.2  Procedure  
Dissolve the ingredients in  the wat er and adjust the pH value to 7.1. Dispense in 10 mL volumes into suitable 
bottles an d sterilize by autoclaving at  121 ± 3 °C for 15 min.  Incubate the bottles at 37  °C ± 2 °C for 24 h. 
Subculture onto nutrient agar slopes. Incubate the slo pes at 37 °C  ± 2 °C for 24 h.  
3.2.3.2.2  Nutrient agar  
3.2.3.2.2.1  Ingredients  
The ingredient s for nutrient agar  shall include the following : 
• 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 m L 
3.2.3.3.2.2 Preparatio n 
Dissolve the ingredients in the water and adjust the pH value to 7.1.  Dispense 10 m L and 15 mL volumes into  
suitable bottles and sterilize by autoclaving at  121 ± 2 °C for 15 minutes. Allow only the 10 mL volumes t o 
solidify in a sloped p osition. From each of these slope cultures, prepare four subcultures (stock cultures)  of 
each test organism o nto 10 mL nutrient ag ar slopes. Incubate the stock cul tures at  37 °C ± 2 °C for 24 h and 
then store in a refrigerator maintained a t 4 °C  ± 2 °C, except for Pseud omonas  aeruginosa  which is stored at 
ambient temperature.  Use the stock cultures to p repare further subcultur es for the test, but do not make more 
than six serial subcultures from each stock culture. After the sixth serial subcultur e, resort to n new freez e-
dried culture.  
3.2.3.3 Preparation of cultures for test suspensions  
3.2.3.3.1 For e ach of the test organism s, inoc ulate a nutrient agar slope from a stock culture and incubate 
at 37 °C for 24 h.  
3.2.3.3.2 For the test, use a 24  h culture that has been su b-cultured for two successive days. After six 
subcultures, restart the process using a fresh stock culture.  
NOTE The physiological condition of the t est organisms is important and might influence inter -laboratory and intra -
laborator y variations in test res ults. 
3.2.3.3.3 After incubation, wash the bacterial growth from the slope using 10 m L sterile water and, if 
necessa ry, scrape the agar surface. Care fully decant the suspension into a sterile conical  flask and shake 
vigorously to  suspend all growth in t he water. Standardize the suspension, by using a spectrophotometer in 
conjunction wit h a standard curve, a ha emocytomet er, Petroff -Hausser co unting chamber or any other 
suitable means, so that it contains 10 5 cfu/mL ± 10 4 cfu/mL . Use the suspension within 3 h of preparation.  
3.2.4 Control  
3.2.4.1 Positive control  
This shall be n atural honey with no additives, for example  colourants .   
4 © EAC 2021 – All rights reser ved 
 3.2.4.2 Negative control  
This shall be s terile deionised or sterile distilled water .3.4 Procedure  
3.3.1 Melt t he content s of a bottle of the med ium No. 1 (see 3.2.1.1) and cool to 45 °C ± 2 °C. Add 1 mL of 
the sterile inactivated horse serum ( see 3.2.2) and 1 mL o f S. aureus  test organism suspension prepared in 
accordance with 3.2.3.2.  Mix well and avoid the form ation of a ir bubbles, then pour about 25 ml to 30 ml of 
the mixture into a Petri dish of 90 mm diameter or about 60 ml to 70 ml into a 150 - mm Petri di sh and allow to 
solidify. 3.3.2 Using as sterile cutter that produ ces cylindrical  wells (holes) of 8 mm diameter, make five evenly 
spaced straight cylindrical wells in the solidified medium .  Remove and discard th e plugs of medium.  Seal the 
bottom of each w ell using one or two drops of the molten medium in 3.2.1.1 and a llow to solidif y. 
3.3.3 Introduce the test sample into the wells in such a way that each well is completely filled with the  
sample, taking care not  to form bubbles.  When t he test sample is a soap bar, grind a sufficient amount to a 
powder, using a sterile  pestle and mor tar, and introduce the powder into the wells.  Ensure that the surface of 
the agar remains free from the sam ple. 
3.3.4 Incubate the petri dish at 37  °C ± 1 °C for 18 h to 24 h.  
3.3.5 At the end of this period (see 3.4.4), remove the Petri di sh from the incubator.  Measure the diameter 
of the zone of inhibition diagonally across the well to the neares t millimetre  
3.3.6 Take the average of the five diameters and  record this to the  nearest millimetres . 
3.3.7 Repeat the procedure described in 3.3.1 to 3.3.6 (inclusive) using the  E. coli  and P. aeruginosa test 
suspensions successively.  
3.3.8 Repeat the pro cedure descr ibed in 3.3.1 to 3.3.6 (inclusive) for the positi ve control and the negative 
control . 
4 Interpretation of results  
4.1 For the product  to pass the test, the average diameter of the zone of inhibition for each of the test 
organisms shall be at le ast 10 mm.  
4.2 If the zones are not clearly defined for one o f the reference organisms (for example,  hazy, incomplete 
zone, or distorted shape), t he test shall be repeated for the specified reference organism.  
 DEAS 794: 2021   
 
© EAC 2021 – All rights reserved