ICS 65.080  
 
Reference number
DRS 279: 2020
© RSB 2020
  
 RWANDA 
STANDARD  DRS
279
Second  edition
2020 -mm-dd
Organic fertilizer  — Specification  
   
DRS 279: 2020  
©RSB 2020  - All rights reserved                  ii 
 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 reprodu ce this document should be addressed to : 
Rwanda Standards  Board  
P.O Box 7099 Kigali -Rwanda  
KK 15 Rd, 49  
Tel. +250 788303492  
Toll Free: 3250  
E-mail: info@rsb.gov.rw  
Website: www.rsb.gov.rw  
ePortal: www.portal.rsb.gov.rw   DRS 279: 2020  
iii                ©RSB 2020  - All rights reserved  
 Contents  Page 
1 Scope  ................................ ................................ ................................ ................................ ................ 1 
2 Normative references  ................................ ................................ ................................ ....................... 1 
3 Terms and defi nitions  ................................ ................................ ................................ ...................... 2 
4 Requirements  ................................ ................................ ................................ ................................ ...5 
4.1 General requirements ................................ ................................ ................................ ....................... 5 
4.2 Specific requirements  ................................ ................................ ................................ ...................... 5 
5 Packaging and labelling  ................................ ................................ ................................ ................... 7 
5.1 Packaging  ................................ ................................ ................................ ................................ ......... 7 
5.2 Labelling  ................................ ................................ ................................ ................................ ........... 7 
5.3 Other labelling guidelines  ................................ ................................ ................................ ................ 8 
6 Sampling  ................................ ................................ ................................ ................................ ........... 9 
Annex  A (informative)  Substances that may be used as fertilizers or soil conditioners  ........................  10 
Annex  B (normative)  Test for Faecal Streptococci ................................ ................................ ...................  14 
B.1 Materials and culture media  ................................ ................................ ................................ ...........  14 
B.2 Presumptive test procedure ................................ ................................ ................................ ...........  15 
B.3 Confirmed test procedure  ................................ ................................ ................................ ..............  15 
B.4 Comp uting and recording MPN  ................................ ................................ ................................ ..... 15 
 DRS 279: 2020  
©RSB 2020  - All rights reserved                  iv 
 Foreword   
Rwanda Standard s are prepared  by Technical Committees and approved by Rwanda Standards Board (RSB) 
Board  of Directors in  accordance with the procedures of RSB, in compliance with Annex 3 of the WTO/TBT 
agreement on the preparation, adoption and application of standards.  
The main task of technical committees is to prepare national standards.  Final Draft Rwanda S tandards 
adop ted by Technical committ ees are ratified  by me mbers of RSB Board of Director s for publication and 
gazettment as Rwanda Standards.  
DRS 279 was prepared by Technical Committee RSB/TC 07, Agrochemicals . 
In the preparation of this standard, reference was made to the following standard:  
FDARS 1490 : Organic fertilizer — Specification  
The assistance de rived from the above source is hereby acknowledged with thanks.  
This second edition cancels and replaces the first edition (RS  279: 2019 ), of which has been technica lly 
revised.  
Committee membership  
The following organizations were represented on the Technical Committee on Agrochemicals  (RSB/TC 07) in 
the preparation of this standard.  
One Acre Fund - TUBURA  
Ministry of Environment (MoE)  
University of Rwanda/College of  Agriculture and Veterinary Medicine (UR/CAVM)  
University of Rwanda/College of Sciences and Technology (UR/CST)  
Rwanda Inspectorate, Competition and Consumer Protection Authority (RICA)  
Star Construction and Consultancy (SCC) Ltd  
Rwanda  Standards Board  (RSB) – Secretariat  DRS 279: 2020  
v                ©RSB 2020  - All rights reserved  
 Introduction   
Organic fertilizers are natural products used to provide plant nutrients. There are a number of orga nic fertilizers like 
farmyard manure, green manures, compost prepared from crop residues and other farm wastes, vermicomp ost, oil cakes, 
biological wastes —animal bones, slaughter house refuse and natural mineral deposits that include but not limited to 
phosphate rock, greensand, Epsom salt, calcium, limestone flour.  
This standard has been prepared to promote safe use of organi c fertilizers, promote fair trade practices and ensure safety 
of consumers.  
 
 DRS 279 : 2020  
1                ©RSB 2020  - All rights reserved  
 Organic fertilizer  — Specification  
1 Scope   
This Draft Rwanda Standard specifies the requirements, sampling and test methods of organic fertiliz ers. 
2 Normative references   
The following documents are referred to in the text in such a way that some or all of  their content constitu tes 
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.  
AOAC 2006.03 , Arsenic, cadmium, cobalt, chromium, lead, molybdenum, nickel, and selenium in fertilizers  — 
Microwave digestion and inductively coupled plasma -optical emission spectrometry  
EN 13475, Liming materials — Determination of calcium content — Oxalate method  
EN 16032, Fertilizers — Extraction and determination of elemental sulfur  
EN 16197, Fertilizers — Determination of magnesium by atomic absorption spectrometry  
EN 16198, Fertilizers — Determination of magnesium by complexometry  
EN 16963, Fertilizers — Determination of boron, cobalt, copper, iron,  manganese, molybdenum and zinc 
using ICP -AES 
EN 16965, Fertilizers — Determination of cobalt, copper, iron, manganese and zinc using flame atomic 
absorption spectrometry (FAAS)  
ISO 6579 , Microbiology of food and animal feeding stuffs — Horizontal method f or the detection of Salmonella 
spp. 
ISO 6598 , Fertilizers — Determination of phosphorus content — Quinoline phosphomolybdate gravimetric 
method  
ISO 7251,  Microbiology of food and animal feeding stuffs — Horizontal method for the detection and 
enumeration of presumptive Escherichia coli — Most probable number technique  
ISO 7409, Fertilizers — Marking — Presentation and declaratio ns 
ISO 8157, Fertilizers and soil conditioners — Vocabulary  
ISO 8397, Solid fertilizers and soil conditioners — Test sieving  
ISO 10390 , Soil quality — Determination of pH   
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ISO 10694, Soil quality — Determination of organic and total carbon after dry combustion (elementary 
analysis)  
ISO 11047, Soil quality — Determination of cadmium, chromium, cobalt, copper, lead, manganese, nickel and 
zinc — Flame and electrothermal atomic absorption spectrometric methods  
ISO 11261, Soil quality — Determination of total nitrogen — Modified Kjeldahl method  
ISO 11265, Soil quality — Determination of the specific electrical conductivity  
ISO 11465, Soil quality — Determination of dry matter and water content on a mass basis — Gravimetric 
method  
ISO 14820 -1, Fertilizers and liming materials — Sampling and sample preparation — Part 1:  Sampling  
ISO 14820 -2, Fertilizers and liming materials — Sampling and sample prepar ation — Part 2: Sample 
preparation  
ISO 15178, Soil quality — Determination of total sulfur by dry combustion  
ISO 17318, Fertilizers and soil conditioners — Determination of arsenic, cadmium, chromium, lead and 
mercury contents  
 
ISO 17184, Soil quality — Determination of carbon and nitrogen by near -infrared spectrometry (NIRS)  
3 Terms and definitions  
For the purposes of this standard, the terms and definitions given in ISO 8157 and the following apply.  
3.1 
blood meal  
dried, powdered blood collected from livestock slaughter  houses  used as fertilizer.  
3.2 
bone meal  
fertilizer made from degreased bone which may be degelatinized and has been ground or crushed  
3.3 
compost  
well decomposed organic wastes like plant residues, animal slurry from livestock sheds  DRS 279 : 2020  
3                ©RSB 2020  - All rights reserved  
 3.4 
farmyard manure  
a decomposed mixture of livestock dung and urine with straws and litter used as bedding material and 
residues from the fodder fed to livestock  
3.5 
fertilizer  
substance containing one or more recognized plant nutrient(s), which is used for  its plant nutrient content and 
which is designed for use or claimed to have value in promoting plant growth  
3.6 
fish emulsion fertilizer  
a partially decomposed blend of finely pulverized fish  
3.7 
green manure  
crop that is incorporated into the soil for th e purpose of soil improvement and which may include spontaneous 
crops, plants or weeds  
3.8 
guano  
well decomposed accumulated and mined excrements of birds, bats and seals valued as fertilizers  
3.9 
manure  
mixture of litter and/or dung in process of biological change  
 
3.10 
natural mineral fertilizer   
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 materials that are directly mined from mineral deposits and only subjected to physical processes such as 
crushing and drying. Examples of these materials may include: phosphate rock; gypsum; sulphate of 
potassium -magnesia and any other natural mineral deposits.  
3.11 
night soil  
human urine and faeces collected separately from each other or mixed with flush water and amendments  
such as soil, ash other organic matter  
3.12 
organic based product  
product that contains at least 70% organic material  
3.13 
organic fertilizer  
any product in solid or liquid form, of plant (except by -products from petroleum industries) or animal origin that 
has undergone substantial decomposition that can supply available nutrients to  plants. This may be enriched 
by microbial inoculants and naturally occurring minerals but no chemical or inorganic fertilizer mat erial has 
been added to the finished product to affect the nutrient content.  
3.14 
organic matter  
biomass of animals and plants. For this reason, only products that are solely derived from organic m atter may 
be identified or described as "organic".  
3.15 
soil conditioner  
material (could be inorganic or organic) added to soils to improve the physical an d/ or chemical properties, 
and/ or the biological activity of soils without a declarable content of nutrients  
3.16 
sewage sludge  
recycled product of sewage treatment plants  
3.17 DRS 279 : 2020  
5                ©RSB 2020  - All rights reserved  
 synthetic  
substance that is formulated or manufactured by a chemical process or by a process that chemically changes 
a substance extracted from naturally occurring plant, animal, or mineral sources, except for those s ubstances 
created by naturally occurring biological processes.  
3.18 
vermicompost  
product or process of composting us ing various worms such as earthworms to create a heterogenous mixture 
of decomposing vegetable or food waste, bedding materials . 
4 Requirements  
4.1 General requirements  
4.1.1  The organic fertilizer shall be in solid or liquid form.  
4.1.2 The organic fertilizer shall be free from foul odour.  
4.1.3 The organic fertilizer shall be homogenous in texture.  
4.1.4 The organic fertilizer shall be free from contaminants which include but not limited to residual 
hormones, antibiotics, pesticides and disease organism.  
4.1.5 Only allowed raw materials shall be used (Annex A). Those restricted shall be used after having 
recommended treatment or qu ality control (Annex A).  
4.1.6 The inoculants if used shall comply with the relevant standards.  
4.2 Specific requirements  
4.2.1  Organic fertilizers shall conform to the composition requirements set out in Table 1.  
Table 1 — Specific requirements for organic fertilizers  
S/N Parameter  requirements  Test Method  
i) pH 6 – 9 ISO 10390  
ii) Carbon: Nitrogen ratio,  ≤ 20:1  ISO 17184  
iii) Moisture content,(solid) %, m/m  10-35 ISO 11465  
iv) Total Nitrogen, %, m/m, min.  >1 ISO 11261  
v) Dry matter content (solid), %, m/m, min.  ≥70 ISO 11465  
vi) Organic carbon, %, m/m, min.  12 ISO 10694  
vii) Total primary nutrients — N-P2O5-K2O (solid and 
liquid organic fertilizer),%, m/m, min  5  ISO 11261, ISO 
6598   
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 viii) Stones >5 mm, %, m/m, max.  5 ISO 8397  
ix) Foreign matter > 2 mm, % m/m, max  0.5 ISO 8397  
x) Seed, number/kg, max  5  
xi) Soluble salts (conductivity),µSm -1 max.  5 ISO 11265  
* For pelletized fertilizer, moisture content should be ≤10%.  
4.2.2  For organic plant food supplement products such as seaweed extract, fermented products, blood 
meal, and bone meal, any claim should be verifiable, while products with at least 3% total (soluble)  N will be 
subjected to other c onfirmatory test.  
4.2.3  Secondary plant nutrients must not be identified and guaranteed if they are not present in at least the 
following minimum concentrations as given in table 3:  
Table 2 — Minimum nutrient percentage guarantee for organic fertilizers  
S/N Nutrients  Limit  Test Method  
i) Calcium, as Ca, %, m/m,  ≥1.0000  EN 13475  
ii) Magnesium (%)  ≥0.5000  EN 16197 / EN 16198  
iii) Sulphur (%)   ≥1.0000  ISO 15178 / EN 16032  
iv) Boron mg/kg  20-140 EN 16963 / EN 16965  
v) Cobalt ( mg/kg ) 0.5-1.0 ISO 11047  
vi) Copper (mg/kg ) 8-300 ISO 11047  
vii) Iron ( mg/kg ) 1000 -2500  EN 16963 / EN 16965  
viii) Manganese ( mg/kg )  200-800 ISO 11047  
ix) Molybdenum ( mg/kg ) 0.5-1.0 AOAC 2006.03  
x) Zinc ( mg/kg ) 40-1000  ISO 11047  
4.2.4  Metal contaminants , if present , shall conform to the following limits  as given in Table 3. 
Table 3 — Minimum nutrient percentage guarantee for organic fertilizers  
S/N Heavy metals   Acceptable maximum 
limits  (mg/kg, dry weight)  Test method  
i Arsenic (As)  10 ISO 17318  
ii Lead (Pb)  30 ISO 17318  
iii Chromium (Cr)  50 ISO 17318  
iv Nickel (Ni)  50 ISO 11047  
v Mercury (Hg)  0.5 ISO 17318  
vi Cadmium (Cd)  5 ISO 17318  DRS 279 : 2020  
7                ©RSB 2020  - All rights reserved  
 4.2.5  The organic fertilizer shall be free from pathogenic organisms.  Where applicable,  the product shall 
also comply with microbiological limits in the following table  4: 
Table 3 — Minimum nutrient percentage guarantee for organic fertilizers  
Microorganisms  Acceptable limits  Test Method s  
E. coli 1000 cfu/g  ISO 7251  
Salmonella spp  Absent in 25 g fresh mass  ISO 6579  
Faecal streptococci  <500 cfu/g  Annex B  
Total coliforms  Nil ISO 4831  
5 Packaging and labelling  
5.1 Packaging  
The organic fertiliser shall be pack aged in clean, non -defective and strong  packages . The material for which 
the package  is made shall be such as to protect the contents from moisture  and also not lead to easy rupture 
during handling, transportation and storage.  
5.2 Labelling  
5.2.1  Each package of the fertiliser shall be legibly and indelibly marked in accordance with ISO 7409 and 
the following particulars:  
a) Name of product as Organic fertilizer  
b) nutrient content  (N-K-P) 
c) carbon  : nitrogen ratio ; 
d) dry matter content ; 
e) batch number /lot number;  
f) percentage foreign;  
g) name and physical address  of the manufacturer/packer/importer ; 
h) date of manufacture ; 
i) best before date or expiry date;  
j) instructions for use , storage and handling; and   
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 k) precautions /warnings  
5.2.2  Where the organic fertilizer  is distributed in bulk, the marking  information shall acco mpany the delivery 
note to  the purchaser.  
5.3 Other labelling guidelines  
5.3.1  Testimonials/endorsements : The public has no way of evaluating the status of the endorser in relation 
to a product. For this reason, testimonials and endorsements will be viewed as claims and evaluated 
accordingly.  
5.3.2  Other claims : Any reference to the activity of a product containin g plant nutrients that is not generally 
associated with its nutritional value must be substantiated with statistically significant efficacy data 
derived from field trials.  
5.3.3  Nutrient guarantees : Any product represented as a source of plant nutrients mu st carry a guaranteed 
analysis.  
5.3.4  Directions for use : All specialty fertilizers must carry instructions for use. These instructions must 
specify both the rate and frequency of application. Suggested rates of application must provide an 
adequate quantit y of nutrients to the plants concerned.  
5.3.5  Where the product does not contain all 3 major plant nutrients, the label should carry a statement 
indicating that some plants may require an additional source of the nutrient(s) that are lacking.  
5.3.6  Any pro duct containing composted materials or plant nutrients may represent a potential hazard when 
misused. In order to avoid giving the impression that reasonable precautions are unnecessary, 
blanket statements suggesting that the product is completely safe and  non-toxic to humans, animals 
or the environment must not appear on the label.  
5.3.7  Miscellaneous terms : Words, such as balanced and healthy, should be avoided as they are often 
misunderstood and consequently misleading. Also objectionable are comparative s such as best, 
superior, and greener, as they imply a comparison without indicating the basis of this comparison  
5.3.8  Environmentally beneficial : Any product represented as "environmentally sound", etc. must identify 
the rationale for the claim and list all ingredients in order to allow the consumer to determine the 
validity of the statement.  
5.2.9  Slowly available plant nutrients : Only products providing at least 70%of a plant nutrient in a slowly 
available form may indicate this on the label. Such a cla im must be accompanied by the associated 
guarantee.  
5.2.10  Low leaching potential : Only products containing at least70% of a nutrient in a slowly available form 
may indicate that nutrient will be slowly available and thus unlikely to leach when used in accordance with 
label instructions.  
5.3.11  Improving soil structure : In order to suggest that the use of an organic or organic based product will 
improve the structure of a soil, it must be recommended for use at rates that could be expected to s ignificant ly 
increase the organic matter level of the soil. Such as 1kg per square metre or 10 metric tonnes per hectare . DRS 279 : 2020  
9                ©RSB 2020  - All rights reserved  
 6 Sampling  
Sampling and sample preparation for inspection and testing shall be carried out in accordance with ISO 14820 
parts 1 and 2 .  
©RSB 2020  - All rights reserved                  10 
 Annex  A 
(informat ive) 
 
Substances that may be used as fertilizers or soil conditioners  
Substance  Description; Compositional requirements; Conditions of 
use 
Plant and animal origin   
Farmyard and poultry manure  Products comprising a mixture of animal excrements and 
vegetable matter (animal bedding).  
Indication of animal species.  
Coming from extensive farming, but if sourced from intensive 
farming or not sourced from organic production systems, 
need recognition by the approved certifying organisation and 
shall be comp osted.  
Slurry or urine (not from human 
origin)  If not from organic farming sources, need recognition by the 
approved certifying organisation.  
Use after controlled fermentation and/or appro priate dilution.  
Factory farming sources not permitted.  
Indication of animal species.  
Composted animal excrements, 
including poultry manure  Need recognition by the approved certifying organisation.  
Indication of animal species.  
Dried farmyard manure and 
dehydrated poultry manure  Need recognition by the approv ed certifying organisation.  
Indication of animal species.  
Coming from extensive farming, but if from intensive farming 
sources it must be composted.  
Guano  Need recognition by the approved certifying organisation.  
Straw  Need recognition by the approved c ertifying organisation.  
Composts from spent 
mushroom and dejecta of 
worms and insects (vermiculture 
substrates)  The initial composition limited to products on this list.  
Composted or fermented 
organic household refuse  Organic vegetable and animal waste separated from 
household waste, which has been subjected to composting or 
anaerobic fermentation for biogas production.  
Need recognition by the approved certifying organisation.  
Maximum concentrations in mg/kg of dry matter: Cadmium: 
0.7; Copper: 70; Nicke l: 25; Lead: 45; Zinc: 200; Mercury: 
0.4; Chromium (total): 70; Chromium (VI):  0(*).  
(*) = limit of determination.  
Composted or fermented plant 
residues  Need recognition by the approved certifying organisation.  
Mixtures of plant matter which has been sub jected to 
composting or anaerobic fermentation for biogas production.  
Products and by -products of Need recognition by the approved certifying organisation.  DRS 279 : 2020  
11                ©RSB 2020  - All rights reserved  
 Substance  Description; Compositional requirements; Conditions of 
use 
animal origin from 
slaughterhouses & fish 
industries:  
 blood meal  — 
 hoof meal  — 
 horn meal  — 
 bone  meal or degelatinized 
bone meal  Heavy metal contamination monitoring necessary  
 fish meal  — 
 meat meal  — 
 feather, hair and "chiquette" 
meal  maximum concentration in mg/kg of dry matter of Chromium 
(VI):0 (*)  
(*) = limit of determination   wool 
 fur 
 hair — 
 dairy products  — 
By-products of food & textile 
industries  Not treated with synthetic additives.  
Need recognition by the approved certifying organisation.  
Seaweeds and seaweeds 
products  Need recognition by the approved certifying organisation.  
Directly obtained by — physical processes; extraction with 
water or acid and/or alkaline solution; and fermentation.  
Sawdust, bark and wood waste  From wood not chemically treated after felling.  
Wood ash  From wood not chemically treated after felling.  
Crop residues / by -products 
(from oil, palm, coconut and 
cocoa (including empty fruit 
bunch, coir, husks), palm oil mill 
effluent (pome), cocoa peat, 
empty cocoa pods, straw, 
peanut hulls, sugar cane trash, 
straw, mud press, etc.)   
Green manure / legumino us 
crops   
Azolla  (mosquito fern, 
duckweed fern, fairy moss, 
water fern)   
Plant preparations and extracts   
Segregated biodegradable 
market waste   
Calcium lignosulfate  Recognized by the competent authority  
Naturally occurring biological   
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 Substance  Description; Compositional requirements; Conditions of 
use 
organisms e.g. worms  
Peat Should not be extracted or treated using inorganic chemicals; 
permitted for seed, potting module composts.  
By-products of industries 
processing ingredients from organic 
agriculture  Need recognition by the approved certifying organisation.  
Night soil -faeces and material 
containing faecal matter  Subjected to either of the following treatments: composting, 
incineration/drying, anaerobic digestion and ammonia treatment  
Sewage Sludge  Subjected to anaerobic digestion/fermentation, composting or long 
time treatments  
Human urine  Proper storage (based on the action of ammonia in combination with 
temperature.  
Stillage and stillage extract  Ammonium stillage excluded.  
ii. Mineral origin   
Natural phosphate rock  Need recognition by the approved certifying organisation.  
Cadmium should not exceed 90mg/kg P2O5 
 
May contain elevated levels of trace elements. Detailed 
chemical analysis is necessary.  
 
Their widespread extraction can also deplete the natural 
deposits and may cause negative environmental  impact. 
Rate of extraction is subject to regulations.  
Basic slag  Need recognition by the approved certifying organisation.  
Rock potash, mined potassium 
salts (e.g. kainite, sylvinite)  Need recognition by the approved certifying organisation.  
Calcareous  and magnesium 
amendments  Recognized by the competent authority  
Limestone (dolomite, 
calcite), marl, chalk, 
lime Recognized by the competent authority  
Calcium chloride  
 
Chloride of lime  Only from natural sources/origin  
Gypsum (calcium sulphate)  Only from natural sources/origin  
Magnesium rock, kieserite 
and Epsom salt 
(magnesium sulfate)  Only from natural sources/origin  
Rock potash, mined 
potassium salts (e.g. kainite, 
sylvinite)  Less than 60% chlorine  
Sulphate of potash  Obtained by physical procedures but not enriched by DRS 279 : 2020  
13                ©RSB 2020  - All rights reserved  
 Substance  Description; Compositional requirements; Conditions of 
use 
chemical processes to increase its solution  
Sulfur  Allowed if from natural source  
Pulverized rock, stone meal  
May contain elevated levels of trace elements. Detailed 
chemical analysis is necessary.  
 
Their widespread extraction can also deplete the natural 
deposits and may cause negative environmental impact.  
 
Rate of extraction is subject to regulations.  
Clay (e.g. bentonite, perlite, 
vermiculite, zeolite)   
Sodium chloride  Only mined salt  
Trace elements (e.g. boron, 
copper, iron, manganese, 
molybdenum, zinc)   
Stillage and stillage extract  Ammonium stillage excluded  
Aluminum calcium phosphate  Cadmium should not exceed 90mg/kg P2O5 
  
iii. Microbiological   
Biodegradable processing by -
products of microbial origin, e.g. 
by-products of brewery or 
distillery processing   
Microbial preparations (i.e. 
Trichoderma, Rhizobia, 
Mychorrizae , others) of non -
GMO origin   
  
iv. others   
Biodynamic and Agnihotra 
preparations   
 
 
   
©RSB 2020  - All rights reserved                  14 
 Annex  B 
(normative)  
 
Test for Faecal Streptococci  
B.1 Materials and culture media  
Preferably use commercially available medium. Follow manufacturer’s instructions for storing and dis carding 
after preparation. If the medium must be prepared from basic ingredients, follow directions below.  
B.1.1  Azide dextrose broth:  
Beef extract .....................................................................................4. 5 g 
Tryptone or polypeptone ..............................................................15.0 g  
Glucose................................ ............................................................7.5 g  
Sodium chloride, NaCl ...................................................................7.5 g  
Sodium azide, NaN3 .......................................................................0.2 g  
Reagent -grade water .......................................................................1 L  
CAUTION!  Sodium azide is a dangerous chemical requiring special attention and care. It is toxic and  
mutagenic. Take precautions to avoid contact with this compo und. Azide also can form explosive  compounds 
if it contacts metal pipes.  
Adjust pH so it is 7.2 ± 0.2 at 25°C after sterilization. If pH is out of range, adjust and retest pH; discard if pH 
remains out of range. The media described in this section are avai lable commercially; follow manufacturer’s 
instructions for storage and disposal after preparation . 
B.1.2  Bile esculin azide agar  
Yeast extract................................................................................ 5.0 g  
Proteose peptone No. 3..... .......................................................... 3.0 g  
Tryptone ......................................................................................17.0 g 
Oxgall .................................................................................. ........10.0 g  
Esculin......................................................................................... 1.0  g DRS 279 : 2020  
15                ©RSB 2020  - All rights reserved  
 Ferric ammonium citrate ............................................................ 0.5 g  
Sodium chloride ............................... ........................................... 5.0 g  
Sodium azide............................................................................... 0.15 g  
Agar.............................................................................................15. 0 g 
Reagent-grade water .....................................................................1 L  
CAUTION!  Sodium azide is a dangerous chemical requiring special attention and care. It is toxic and  
mutagenic. Take precautions to avoid contact with this compound. Azide also can form explosive compounds 
if it contacts metal pipes.  
B.2 Presumptive test procedure  
Inoculate a series of tubes of azide dextrose broth with appropriate graduated quantities of sample.  Use 
sample volumes of 10 mL or less. Use double -strength bro th for 10 -mL inocula. The sample portions used will 
vary in size and number with the sample character. Use only decimal multiples of  
1 mL. Incubate inoculated tubes at 35  ± 0.5°C. Examine each tube for turbidity at the end of 24 ±  2 h. If no 
definite turb idity is present, reincubate, and read again at the end of 48 ±  4 h. 
B.3 Confirmed test procedure  
After 24 or 48 h incubation, subject all azide dextrose broth tubes showing turbidity to the confirm ed test for 
streptococci. Streak a portion of growth from each  positive azide dextrose broth tube on bile esculin azide 
agar (BEA). Invert and incubate the dish at 35 ± 0.5  °C for 24  ± 2 h. Brownish -black  colonies with brown halos 
confirm the presence of fecal streptococci. Then, transfer brownish -black colonies with  brown halos to two 
tubes of brain –heart infusion (BHI) broth: one with 6.5% NaCl and one without NaCl. If growth is observed 
when tube is incubated at 35 ± 0.5  °C after 48  ± 4 h (BHI broth with 6.5% NaCl) or 24 ± 2 h (BHI broth without 
NaCl), the colony i s confirmed as a member of the Enterococcus genus. The aforementioned procedure is 
expected to offer an acceptably accurate confirmation of the presence of the faecal Streptococci . However, 
more accuracy ( ≥90%) can be achieved by doing all of the following:  
observing gram -positive cocci, a catalase -negative reaction, growth on BHI agar at  
10 ± 0.5  °C, positive pyrrolidonylarylamidase (PYR) activity, and positive leucine aminopeptidase (LAP) 
reaction 3,4 using a commercially available test kit.  
B.4 Com puting and recording MPN  
Calculate the total fecal streptococci density from the number of confirmed positive cultures on bil e esculin 
azide agar and corresponding positive tubes of BHI broth with 6.5% NaCl at 35  ± 0.5 °C after 48 ±  4 h. 
Compute the combin ation of positive and negative tubes and record as the most probable number (MPN).   
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 Bibliography  
[1] ISO/IEC Directives, Part  2, Rules for the structure and drafting of International Standards , 2016  
 DRS 279: 2020  
 
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