PUBLIC REVIEW DRAFT DRAFT UGANDA STANDARD  
  DUS DEAS 969 
First  Edition  
2019-mm-dd 
 
 Reference number  
DUS DEAS 969: 2019 
© UNBS 2019 
   
Disposable baby  diapers — Specification  
 
 PUBLIC REVIEW DRAFTDUS DEAS 969: 2019 
ii                © UNBS  2019 - All rights reserved 
 Compliance with this standard does not, of itself confer immunity from legal obligations  
A Uganda Standard does not purport to include all necessary provisions of a contract. Users are 
responsible for its correct applicati on 
© UNBS 2019 
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilised in any 
form or by any means, electronic or mechanical, including photocopying and microfilm, without prior written 
permission from UNB S. 
Requests for permission to reproduce this document should be addressed to 
The Executive Director 
Uganda National Bureau of Standards 
P.O. Box 6329 
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Uganda 
Tel: +256 417 333 250/1/2 
Fax: +256 414 286 123 
E-mail: info@unbs.go.ug  
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 PUBLIC REVIEW DRAFTDUS DEAS 969: 2019 
© UNBS  2019 - All rights reserved                iii National foreword 
Uganda National Bureau of Standards (UNBS) is a parastatal under the Ministry of Trade, Industry and 
Cooperatives established under Cap 327, of the Laws of Uganda, as amended.  UNBS is mandated to co-
ordinate the elaboration of standards and is  
(a) a member of International Organisation for Standardisation (ISO) and  
(b) a contact point for the WHO/FAO Codex Alimentarius Commission on Food Standards, and  
(c) the National Enquiry Point on TBT Agreement of the World Trade Organisation (WTO). 
The work of preparing Uganda Standards is carried out through Technical Committees. A Technical 
Committee is established to deliberate on standards in a given field or area and consists of representatives of 
consumers, traders, academicians, manufacturers, government and other stakeholders. 
Draft Uganda Standards adopted by the Technical Committee are widely circulated to stakeholders and the 
general public for comments. The committee reviews the comments before recommending the draft standards 
for approval and declaration as Uganda Standards by the National Standards Council.  
This Draft Uganda Standard, DUS DEAS 969: 2019 , Disposable baby diapers — Specification , is identical 
with and has been reproduced from an International Standard, DEAS 969 : 2019, Disposable baby diapers — 
Specification , and is being proposed for adoption as a Uganda Standard. 
The committee responsible for this document is Technical Committee UNBS/TC 7,  Textiles, leather, paper 
and related products , Subcommittee SC 1 , Textiles and related products.  
Wherever the words, “ East African Standard " appear, they should be replaced by "Uganda Standard."  DEAS 969 : 2019  
ICS nn.nnn.nn  
 
© EAC 2019                                                                                                                               First Edition 2019   
 DRAFT EAST AFRICAN STANDARD  
Disposable baby diapers  — Specification  
EAST AFRICAN COMMUNITY  DEAS 969: 2019  
ii © EAC  2019 – All rights reserved  
 Copyright notice  
This EAC document is copyright -protected by EAC. While the reproduction of this document by 
participants in the EAC standards development process i s 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 permission from EAC.  
Requests for permission to reproduce this doc ument 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  2018  — All rights reserved  
East African Community   
P.O. Box 1096 , 
Arusha  
Tanzania  
Tel: + 255 27 2 162100  
Fax: + 255 27 2162190  
E-mail: eac@eachq.org  
       Web: www.eac -quality.net  
Reproduction for sales purposes may be subject to royalty payments or a licensing agreement.  
Violators may be prosecuted . DEAS 969: 2019  
© EAC 2019 – All rights reserved  iii 
 Contents  Page 
Foreword  ................................ ................................ ................................ ................................ ............................  iv 
1 Scope  ................................ ................................ ................................ ................................ ......................  1 
2 Normative references  ................................ ................................ ................................ ............................  1 
3 Terms and definitions  ................................ ................................ ................................ ...........................  1 
4 Requirements  ................................ ................................ ................................ ................................ .........  2 
4.1 Classification  ................................ ................................ ................................ ................................ .........  2 
4.2 General Requirements  ................................ ................................ ................................ ..........................  2 
4.3 Materials  ................................ ................................ ................................ ................................ .................  2 
4.4 Other physical characteristics  ................................ ................................ ................................ .............  3 
4.5 Microbiological requirements  ................................ ................................ ................................ ..............  4 
5 Packaging and marking  ................................ ................................ ................................ ........................  4 
5.1 Packing and/or packaging  ................................ ................................ ................................ ....................  4 
5.2 Marking  ................................ ................................ ................................ ................................ ...................  4 
Annex  A (normative)  Determination of rate of absorption, absorptive capacity and rewet under 
load ................................ ................................ ................................ ................................ .........................  6 
A.1 Princ iple ................................ ................................ ................................ ................................ .................  6 
A.2 Apparatus and materials  ................................ ................................ ................................ ......................  6 
A.3 Sample preparation and set -up ................................ ................................ ................................ ............  7 
Annex  B (normative)  Microbiological examination  ................................ ................................ ........................  9 
B.1 Apparatus and equipment  ................................ ................................ ................................ ....................  9 
B.2 Media and reagents  ................................ ................................ ................................ ...............................  9 
B.2.1  General  ................................ ................................ ................................ ................................ ...................  9 
B.2.2  Bacteriological peptone  ................................ ................................ ................................ ........................  9 
B.2.3  Plate count agar  ................................ ................................ ................................ ................................ ..... 9 
B.2.4  Neutral red -bile salt peptone glucose medium  ................................ ................................ ..................  9 
B.2.5  Fluid soybean -casein digest medium  ................................ ................................ ...............................  10 
B.2.6  Cetrimide agar medium ................................ ................................ ................................ .......................  10 
B.2.7  Pseudomonas agar medium for detection of fluorescein  ................................ ...............................  10 
B.2.8  Pseudomonas agar medium for detection of pyocyanin  ................................ ................................  11 
B.3 Preparation of test suspension  ................................ ................................ ................................ ..........  11 
B.4 Procedure  ................................ ................................ ................................ ................................ .............  11 
B.4.1  Total viable bacterial count  ................................ ................................ ................................ ................  11 
B.4.2  Examination for the presence of Enterobacteriaceae  ................................ ................................ ..... 11 
B.4.3  Examination for the presence of Staphylococcus aureus  ................................ ..............................  11 
B.4.4  Examination for the presence of Pseudomonas aeruginosa  ................................ ..........................  11 
Bibliography  ................................ ................................ ................................ ................................ ......................  13 
 DEAS 969: 2019  
iv © EAC  2019 – All rights reserved  
 Foreword  
Development of the East African 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 barriers that are encountered when goods and services are exchang ed 
within the Community will be removed.  
The Community has established an East African Standards Committee  (EASC)  mandated to develop and 
issue East African Standards  (EAS) . The Committee is composed of representatives of the National 
Standards Bodies in Partner States, together with the representatives from the public and private sector 
organizations  in the community .  
East African Standards are developed through  Technical Committees that are representative of key 
stakeholders including government, academ ia, consumer groups, private sector and other interested parties. 
Draft East African Standards are circulated to stakeholders through the National Standards Bodies in the 
Partner States. The comments received are discussed and incorporated before finalizat ion of standards, in 
accordance with the Principles and procedures for development of East African Standards. XXXXXX.  
East African Standards are subject to review, to keep pace with technological advances. Users of the East 
African Standards are therefore expected to ensure that they always have the latest versions of the standards 
they are implementing .  
The committee responsible  for this document is Technical Committee EASC /TC 061, Textiles, Textile 
products and Accessories . 
Attention is drawn to the poss ibility that some of the elements of this document may be subject of patent 
rights. EAC shall not be held responsible for identifying any or all such patent rights.  DEAS 969: 2019  
© EAC 2019 – All rights reserved  v 
 
Introduction  
Baby diapers and pants are personal hygiene products that are designed to con tain the baby’s urine and 
faeces in order to avoid soiling the baby’s clothing, beddings and the surrounding environment. They are 
fastened around the baby’s legs and bottom to help prevent leakage. These products aim to provide maximum 
comfort for the bab y and maximum convenience to the care  giver . 
A disposable diaper consists of an absorbent core sandwiched between two or more sheets of nonwoven 
fabric. The core is specifically designed to absorb and retain urine and contain faeces, and the nonwoven 
fabric may be soft and cut in such a way that it is comfortable to wear and prevents leakage. When properly 
fitted, the diaper will retain the body fluids which pass through the top sheet and are absorbed into the core . 
The baby diapers are generally available according to size and weight of the baby. Typical functions of baby 
diapers include : 
 Absorb and retain urine  
 Contain faeces/stool to prevent soiling of the baby’s environment (clothes, bedding)  
 Isolate wetness from the baby’s skin  
 
 
 
 
 
 
 
 
 
 
 
 
Figure 1 — Baby diaper  
 DRAFT EAST AFRICAN STANDARD  DEAS 969:2019 
 
© EAC 2019 – All rights reserv ed 1 
 Disposable baby diapers  — Specification  
1 Scope   
This draft East African Standard specifies the requirements and test methods for disposable baby diapers.  
2 Normative references   
The following documents are referred to in the text in such a w ay 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 217 -1, Metho ds for the microbiological examination of foods — Part 1: General procedures and 
technique  
EAS 217 -5:2001, Methods for the microbiological examination of foods — Part 5: Enumeration of coagulase -
positive staphylococci in foods   
ISO 1 39, Textiles — Standar d atmospheres for conditioning and testing  
ISO 3071, Textiles — Determination of pH of aqueous extract  
ISO 4833 -2, Microbiology of the food chain — Horizontal method for the enumeration of microorganisms — 
Part 2: Colony count at 30 C by the surface platin g technique  
ISO 21149, Cosmetics — Microbiology — Enumeration and detection of aerobic mesophilic bacteria  
ISO 22717, Cosmetics — Microbiology — Detection of Pseudomonas aeruginosa  
ISO 22718 , Cosmetics — Microbiology — Detection of Staphylococcus aureus  
3 Terms and definitions   
For the purposes of this document, the following terms and definitions apply.  
ISO and IEC maintain terminological databases for use in standardization at the following addresses:  
— ISO Online browsing platform: available at http://www.iso.org/obp  
3.1  
disposable baby diapers  
disposable hygienic pad for babies having capability to absorb urine and prevent stool and fluid from leaking  
3.2  
Super Absorbent Polymer (SAP)  
granular cross -linked sodium polyacrylates  material  used as absorbent core with high retention capacity in 
disposable diapers   
2 © EAC  2019 – All rights reserved  
 3.3  
Acquisition/Distribution  Layer (ADL)  
component of an absorbent hygiene product through which the fluid is transferred and distributed within the 
absorbent  core 
3.4  
top sheet (coverstock)  
the outer layer of an absorbent hygiene product that is in direct intimate contact with th e user’s skin. It allows 
gradual  transfer of the fluid from the point of contact to the inside of the product  
3.5  
back sheet  
imperm eable  layer of an absorbent hygiene product made of either polymer film or nonwoven film designed to 
prevent wetness transfer from the wearer to their bed or clothes  
4 Requirements  
4.1 Classification  
Diapers shall be classified according to the weight of t he baby. The intend ed baby weight shall be declared on 
the label . 
4.2 General Requirements  
Baby diapers shall be manufactured, stored and packed under hygienic conditions to minimise contamination 
of the product  and shall be disposable. The diapers shall present a neat, well finished appearance and shall 
be free from all defects which might affect the functionality of the diaper .  
4.3 Materials  
The materials used for making the diapers shall be  free from harmful foreign materials  and shall not harm the 
baby’s skin . 
4.3.1 The absorbent core shall:  
a) consist of cellulose fibres  fluff pulp and superabsorbent polymer for absorption of fluids   
b) be clean  and  free from lumps, splits, holes, and protruding points when visually examined; and  
c) be arranged in a manner t hat will speed up the absorption of urine and keep it away from the baby’s 
skin. 
4.3.2 The top sheet (the layer which contacts the baby’s skin)  shall: 
a) be of material capable of allowing fluid to pass readily through to the next layer and shall  resist 
moist ure return to the skin. It shall  have no harmful effects ; 
b) cover the absorbent core completely and prevent the core from reaching the baby’s skin or clothes 
under normal handling ; and  
c) be soft and shall cause no irritation to the skin .  
4.3.3 The back sheet (outer cover) shall 
a) prevent direct contact of the absorbent core with the baby’s clothing and there shall be no liquid 
leakage out of the diapers ; and   
© EAC 2019 – All rights reserved  3 
 b) allow for air circulation  and be comfortable for the baby . 
4.3.4 There shall be a device e.g. elastic ban d or sticky fasteners for ensuring a good fit of the diapers on 
the baby’s femurs and to prevent leakage at the femurs without causing rubefacient effects.  
4.3.5 There shall be a suitable device for fastening the diaper at the waist for secure use without causing 
rubefacient effects.  
4.3.6 Adhesive used shall prevent shifting of the absorbent core . 
4.3.7 Each material component of the diaper should be bound to the adjacent component to enhance 
strength and prevent shifting of the absorbent core  
4.4 Other ph ysical characteristics  
Other physical characteristics of the diapers shall comply with t he requirements given in Table 1 .  
4 © EAC  2019 – All rights reserved  
 Table  1 — Characteristics of disposable diapers  
Characteristics  Weighta 
kg Test methods  
Up to 4  4.1 - 6 6.1 - 9 9.1 – 15 Above 15  
Rate of absorption per gush, 
min, (max)  2.5 2.5 2.5 2.5 2.5 Annex A  
Absorptive capacity, mL 
(min)  3 x 20  3 x 35  3 x 50  3 x 60  3 x 70 Annex A  
Rewet under load,g (max)  5 5 5 5 5 Annex A  
pHb  6 – 8.5 6 – 8.5 6 – 8.5 6 – 8.5 6 – 8.5 ISO 3071  
NOTE  Re-wet t est: the purpose of this test is to examine the ability of diapers’ top  sheet to resist transportation back  on 
to the skin of a liquid which has already penetrated the top  shee t. The rewet under load  simulates the effect of a baby 
sitting on a wet diaper. The lesser the rewet value, the better the performance of the diaper.  
a The weight is the upper limit of the declared weight by the manufacturer.  
b For products containing SAP or jelly forms, dilute with more distilled water before determining the pH.  
 
4.5 Microbiological requirements   
The microbiological limits shall be as defined below;  
a) the total viable bacterial count, when  determined in accordance with B .4.1 shall not exceed 100 CFU / 
g; and 
b) when tested in accordance with ISO 21149 , disposable diapers shall be free from E. coli , 
Staphylococcus aureus, and Pseudomonas aeruginosa  and C. albicans in 1 g of product   
5 Packaging and marking  
5.1 Packing and/or packaging  
Diapers shall be packed in a suitable package that shall protect them from any form of con tamination and 
damage.  Packaging for shipment shall be in accordance with the manufacturer's standard practice and in a 
manner readily accepted by  the market.  Within the shipping carton, units shall be packed in manner designed 
to minimize damage during sh ipment due to rough or improper handling.  
5.2 Marking  
The diaper packs shall be marked with legible and indelible pre -printed marking or a securely affixed and 
durable label bearing the following information:  
a) name of contents;  
b) name and address of the manu facturer;  
c) number of diapers;  
d) intended baby weight  in kilograms ; 
e) instruction for storage and disposal;   
© EAC 2019 – All rights reserved  5 
 f) instruction for use; 
g) date of manufacture and expiry;  
h) batch/ lot number;  
i) country of origin; and  
j) any perfume, lotion , powder and any other substance ad ded on to the diaper shall be declared.  
 
  
6 © EAC  2019 – All rights reserved  
 Annex  A 
(normative)  
 
Determination of rate of absorption, absorptive capacity and rewet under 
load 
A.1 Principle  
The intention of this method is to test the minimum fluid handling performance requirement of diapers ; the 
outcome of the test will be a “Pass” or “Fail” result.  
This test simulates the introduction of urine into a diaper under the following conditions;  
a) The diaper is tested under pressure to simulate real wearing conditions  i.e. to mimic the pressure that  
the baby applies onto the diaper when wearing it.  
b) Specific loading volumes are used for each diaper size  
A.2 Apparatus and materials  
A.2.1  A rigid cover plat e with a weight as shown in Figure A.1  
Dimensions of the plate (200 mm ± 2 mm) x ( 70 mm  ± 2 mm) . Inner diameter of cylinder 40 mm  ± 2 mm , total 
Weight: 6300 g  ± 10 g  representing a pressure of 4.41 kPa  ± 0.05 kPa  (0.64 psi  ± 0.0 07 psi ) for all sizes  
A.2.2  Tray  
A.2.3  Filter paper , having a diameter of 110 mm and conditioned togethe r with the test sampl es. 
A.2.4 Graduated cylinder (1 ml graduation)  
A.2.5 Stopwatch  
A.2.6 Ruler (at least 2 cm longer as absorbent core of the sample, 1mm graduation)  
A.2.7 Pen 
A.2.8 Weighing scale  
A.2.9 0.9% Saline solution (22 – 24 ºC) : 9 g sodium chloride, and 1 g of blue d ye added into 200 mL 
distilled water at ambient temperature after which the solution is made up to 1 L. The pH shall be between 6.2 
– 6.7. When not within this range adjust by using 0.1 mol/L sodium hydroxide solut ion or acetic acid as 
required.  
Depending on the size of the diaper, the following amount of s aline solution  shall be used  
Table A.1: Volume  of saline solution per size of Diaper  
Category (informative)  Baby weight,  
Kg Volume of saline 
solution (mL)   
© EAC 2019 – All rights reserved  7 
 Newborn / Extra small  Up to 4  3 x 20  
Small  3 – 6 3 x 35  
Medium  4 – 9 3 x 50  
Large  7 and above  3 x 60  
   
 
A.3 Sample preparation and s et-up 
A.3.1  Condition the test samples in accordance with ISO 139 before taking the test specimens required for 
the tests  
A.3.2  Take 5 test specimens randomly from th e test samples  
Determination of absorption capacity  
A.3.3  Mark the loading point in the middle of the absorbent core. Note that the absorbent core does not 
cover the full length of the diaper but it is more concentrated in the front of the diaper. Therefor e, the middle of 
the absorbent core will not be the same as the middle of the diaper: Measure the length and width of the 
absorbent core. Mark the midpoint, which will be the loading point.  
A.3.4  Place core with top  sheet facing upward on the tray  
A.3.5  Place the rigid cover plate  onto the diaper ensuring that  the plate is centered towards the width of the 
diaper core and the cylinder opening is pl aced over marked loading point as per Figure A.1. The diaper should 
be stretched showing minimum number of wrin kles. 
 
Fig A.1: apparatus and set up  
A.3.6  Gently place weights on the plate (ideally use weight in a ring form to allow for equally applied 
pressure)  
A.3.7  Fill the measuring cylinder with respective amount of saline solution  
A.3.8  Set the stopwatch to  5 minutes  
A.3.9  Gently pour the saline solution onto the diaper via cylinder and immediately start the stopwatch.   
8 © EAC  2019 – All rights reserved  
 A.3.10  If the saline solution is absorbed after 5 minutes (i.e. no more liquid observed in the cylinder and no 
liquid on the tray outside the  diaper), then proceed to the next step.  
A.3.11  Leave the diaper undisturbed for 5 min  
A.3.12  Repeat step A.3.7  to A.3.11  twice  more on the same diaper  (i.e. a total of 3 loads of saline solution 
with 5 -minute intervals between successive loads) and observ e for any liquid leaking out of the absorbent 
core. 
Note your observations after a total of 3 loads of the specified amount of fluid and score the diaper based on 
the minimum volumes specified in Table A.1  
A.3.13  Acceptance criteria  
Pass : No fluid observed  leaking out of the diaper  
Report the time it takes for the fluid to be completely absorbed . 
Determination of rewet under load  
A.3.14  Weigh a stack of 5 dry filter paper s and record as weight “W1”.  
A.3.15  Lift the weight and cover plate and p lace the filt er paper stack on the absorption point of the diaper.  
A.3.16  Place the cover plate and weight  on top of the stack of filter paper and leave undisturbed for 2 min . 
A.3.17  After 2 min, remove the weight and cover plate and immediately determine and record th e weight of 
the wet filter paper stack as “W2”. 
A.3.18  Repeat A.3.13 to A.3.16  on the remaining 4 test specimens  and record  each result separately.  
A.3.19  Calculation of results  
A.3.18.1  Rewet  (g) = W2 ( Weight of stack of wet filter paper ) - W1 ( Weight of stack of dry filter paper).  
Record the average rewet of the 5 samples tested and score the samples as follows:  
Pass:  Average rewet weight is less than or equal to the maximum limit specified in Table 2.  
Fail: Average rewet weight exceeds the maximum limit specified in Table 2 . 
  
© EAC 2019 – All rights reserved  9 
 Annex  B 
(normative ) 
 
Microbiological examination  
B.1 Apparatus and equipment  
Use apparatus and equipment complying with the releva nt requirements of EAS 217 -1. 
B.2 Media and reagents   
B.2.1 General  
Ensure compliance with the general requirements for the ingredients and for the preparation of media and 
reagents given in EAS 217 -1.  
B.2.2 Bacteriological peptone  
Peptone               10 g  
Disodium phosphate dodecahydrate         1 g  
Sodium chloride             5 g  
Mono -potassium phosphate          1.5 g  
Dissolve the ingredients in distilled water and make up to 1 L. Adjust the pH value to be 7.0 ± 0.1 after 
sterilization. Dispense 300 mL volumes into flasks of capacity 500 mL and steri lize by autoclaving at 121 °C ± 
2 °C for 20 min.  
B.2.3 Plate count agar  
Agar               15 g  
Glucose               1 g  
Tryptone               5 g  
Yeast extract              2.5 g  
Dissolve the ingredients in distilled water, made up to 1litre, and adju st the pH value to 7.2 ± 0.2. Dispense 15 
mL volumes into bottles and sterilize by autoclaving at 121 °C ± 2 °C for 20 min.  
B.2.4  Neutral red -bile salt peptone glucose medium  
Peptone               20 g  
Glucose               10 g  
Bile salts No. 3             1.5 g   
10 © EAC  2019 – All rights reserved  
 Sodium chloride           5 g  
Neutral red            0.03 g  
Crystal violet            0.002 g  
Dissolve the ingredients in 400 mL of distilled water and make up to 500 mL boiling to aid solution. Adjust the 
pH value to 7.4 and filter to a clear so lution. Dispense 10 mL volumes into bottles each containing a Du rham 
tube and sterilize by auto claving at 121 °C ± 2 °C for 20 min.  
B.2.5 Fluid soybean -casein digest medium  
Pancreatic digest of casein         17 g 
Papaic digest of soybean meal        3 g  
Sodium chloride           5 g  
Dibasic potassium phosphate        2.5 g  
Dextrose             2.5 g  
Dissolve the ingredients in distilled water and make up to 1 L, warming slightly to aid solution. Cool the 
solution to room temperature and adjust the pH valu e to be 7.3 ± 0.2 after sterilization. Filter to clarify (if 
necessary), dispense into suitable containers, and sterilize by autoclaving at 121 °C ± 2 °C for 20 min.  
B.2.6 Cetrimide agar medium  
Pancreatic digest of gelatine        20 g  
Magnesium chloride          1.4 g  
Potassium sulphate          10 g  
Agar             13.6 g  
Cetyl trimethylammonium bromide  (Cetrimide)    0.3 g  
Glycerine            10 mL  
Dissolve all the solid ingredients in distilled water, make up to 1 L, and then add the glycerine. Hea t, agitating 
frequently, and boil for 1 min. Adjust the pH value to be 7.2 ± 0.2 after sterilization. Dispense into suitable 
containers and sterilize by autoclaving at 121 °C ± 2 °C for 20 min .  
B.2.7 Pseudomonas agar medium for detection of fluorescein  
Pancreatic digest of casein         10 g  
Peptic digest of animal tissue        10 g  
Anhydr ous dibasic potassium phosphate      1.5 g  
Magnesium sulphate (MgSO
4.7H
2O)      1.5 g  
Glycerine            10 mL  
Agar             15 g   
© EAC 2019 – All rights reserved  11 
 Dissolve all the solid ingredie nts in distilled water, make up to 1  L, and then add the glycerine. Heat, agitating 
frequently, and boil for 1 min. Adjust the pH value to be 7.2 ± 0.2 after sterilization. Dispense into suitable 
containers and sterilize by autoclaving at 121 °C ± 2 °C for  20 min.  
B.2.8 Pseudomonas agar medium for detection of pyocyanin  
Pancreatic digest of casein           20 g  
Anhydrous magnesium chloride          1.4 g  
Anhydrous potassium sulphate          10 g  
Agar               15 g  
Glycerine              10 Ml  
Dissol ve all the solid ingredients in distilled water, make up to 1 L, and then add the glycerine. Heat, agitating 
frequently, and boil for 1 min. Adjust the pH value to be 7.2 ± 0.2 after sterilization. Dispense into suitable 
containers and sterilize by autocla ving at 121 °C ± 2 °C for 20 min.  
B.3 Preparation of test suspension  
Transfer 300 mL of the sterile soluti on of bacteriological peptone (B .2.2) to a sterile wide -mouthed jar of 
capacity not less than 1 L and not more than 2 L. The jar shall have a mouth of diameter not less than 150 
mm and not more than 250 mm, and is fitted with a hermetically closing glass or metal -and-glass lid. 
Aseptically place the diaper under test in the solution in the jar, fit the lid, agitate the contents of the jar for 2 
min an d then allow the jar to stand for 10 min. Repeat this agitating and standing procedure twice more. 
Aseptically remove about 100 mL of the test suspensi on for testing as described in B .4 below.  
B.4 Procedure  
B.4.1 Total viable bacterial count  
Into each o f three sterile petri dishes aseptically pipette a 1 mL portion of the test suspension. To each dish, 
add 15 mL of fr eshly melted plate count agar (B .2.3) that has been cooled to 45 °C, and mix well. Incubate, 
count and calculate the total count as describ ed in ISO 4833  Part 2  
B.4.2 Examination for the presence of Enterobacteriaceae  
Aseptically add 10 mL of the test suspension to a bottle that contains neutral red -bile salt peptone glucose 
medium (B .2.4). Incubate the bottle for 24 h to 36 h at 37 °C ± 0.5 °C and examine for the presence of 
Enterobacteriaceae as evidenced by  the formation of acid and gas.  
B.4.3 Examination for the presence of Staphylococcus aureus  
Use the media, reagents and procedure descri bed in ISO 22718  to ex amine the test suspension (s ee B.3). As 
a control, pipette 0.1 mL of a 1:1000 dilution of an 18 h to 24 h culture of Staphylococcus aureus SATCC Sta 
10 into Staphylococcus medium and proce ed as with the test suspension.  
B.4.4 Examination for the presence of Pseudomonas aeruginosa  
B.4.4.1 Use the media, reagents and procedure descri bed in ISO 22717. Aseptically pipette 10 mL of the 
test suspension into 90 mL of fluid  soybean -casein digest medium (B .2.5) and mix well. Incubate for 24 h at 
30 °C to 35 °C. By means of an inoculating loop  transfer a portion from the 24 h incubated sample tube of fluid 
soybean -casein digest medium to the dry surface of Petri dishes each containing approximately 2 0 mL of  
12 © EAC  2019 – All rights reserved  
 Cetrimide agar medium (B .2.6). Incubate at 30 °C to 35 °C and examine after 24h, and aga in after 48 h 
incubation, for suspect colonies, bearing in mind that in general greenish fluorescent colonies are typical of 
Pseudomonas aureginosa and that in its presence a gram stain examined microscopically will reveal gram -
negative slender rod -shaped cells.  
B.4.4.2  As a control, add 0.1 mL of a 1:1 000 dilution of an 18 h to 24 h culture of Pseudomonas 
aeruginosa SATCC Pse 11, mL to 100 mL of fluid  soybean -casein digest medium (B .2.5), and proceed as with 
the test suspension.  
B.4.4.3  If none of the c olonies obtained from the test suspension conform s to the description given in 
(B.4.4.1) above and the control culture has been satisfactorily recovered, deem the test sample to be free 
from Pseudomonas aeruginosa.  
B.4.4.4  If colonies conforming to the de scrip tion given in (B .4.4.1) above are found, streak representative 
suspect colonies from the Cetrimide agar onto the surfaces of Pseudomonas agar medium for  the detection of 
flourescein (B .2.7) and Pseudomonas agar medium f or the detection of pyocyanin (B .2.8) to obtain isolated 
colonies. Cover and invert the Petri dishes and incubate at 30 °C – 35 °C for at least three days. Examine the 
streaked surfaces under ultraviolet light for suspect c olonies, as described in Table C .1. 
Table B .1 — Description of co lonies  
Medium  Description of colonies  
Pseudomonas agar for the detection of fluorescein  Generally colourless to yellowish  
Yellowish fluorescence in ultra violet light  
Pseudomonas agar for the detection of pyocyanin  Generally greenish. Blue fluorescence  in ultraviolet light  
 
If any further doubt exists as to the identity of the colonies, obtain final confirmation by inoculating the suspect 
colonies to the wells on commercially available diagnostic kits in accordance with the manufacturer’s 
instructions.   
© EAC 2019 – All rights reserved  13 
 Bibliography  
[1] US 950:2011, Disposable baby diapers (First Edition)  
 DEAS 969: 2019   
 
© EAC 2019 – All rights reserved