BS EN 15784:2021

Animal feeding stuffs: Methods of sampling

and analysis - Detection and enumeration
of Bacillus spp. used as feed additive

bsi.
BS EN 15784:2021 BRITISH STANDARD

National foreword
Thi s British Sta nda rd is the UK implementation of EN 15784:2021. It
supersedes BS EN 15784:2009, which is withdrawn.
The UK participation in its preparation was entntsted to Techn ical
Committee AW/10, Animal feeding stuffs.
A list of organizations represented on th is committee ca n be obtained on
request to its comm ittee manager.
Contractual and legal considerations
This publication has been prepared in good fa ith, however no
representation, warranty, assurance or undertaking (express or
implied) is or will be made, and no responsibility or liability is or will be
accepted by BSI in relation to the adequacy, accuracy, completeness or
reasonableness ot this publication. All and any such responsibility and
liability is expressly disclaimed to the full extent permitted by the law.
This publication is provided as is, and is to be used at the
recipient's own risk.
The recipient is advised to consider seeking professional guidance with
respect to its use of this publication.
This publication is not intended to constitute a contract. Users are
responsible for its correct appl ication.
© The British Standards Institution 2021
Published by BS! Standards Limited 2021
ISBN 978 0 580 99829 4
lCS 65.120
Compliance with a British Standard cannot confer immunity from
legal obligations.
This British Sta ndar d was published under the authority of the
Standa rds Policy and Strategy Committee on 30 November 2021.

Amendments/corrigenda issued since publication

Date Text affected
 BS EN 15784:202 1

EUROPEAN STANDARD EN 15784

NORME EUROPEENNE
EUROPAISCHENORM November 2021

ICS 6S.120 Supersedes EN 1S784:2009

Engl ish Version

Animal feeding stuffs: Methods of sampling and analysis -

Detection and enumeration of Bacillus spp. used as feed
additive
Aliments des animaux: M~thodes d'echantillonnage et Futtermittel: Probenahme- und
d'analyse. Detection et dfoombrement des souches de Untersucbungsverfahren • Nachwels und Zahlungvon
naci1lus spp. utilis¢c.s comrne additifs pour Bac:illus spp. als Futtcrmittclz-usat:,;stoff
l'alimentation a nimale

This European Standard was a pproved by CEN on 2 August 2021.

CEN members a re bound to comply with the CEN/CENEl,EC Internal Regulations which stipulate the conditions forg iving this
European Standard the status of a national standard witho·1t any a lteration. Up-to-date lists and bibliogr aphical references
conce rning s uch national standards may be obwined on application to the CEN-CENELEC Management Centre or to any CEN
member.
This European Standard exists in three official versions (English, French, German). A versio11 in any other language made by
rranslatlon under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management
Centre has the same status as the official versions.

CEN members arc the national standards bodies of Austria. llelgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, f'rance, Germany. Greece, Hungary, Iceland, Ireland, Italy, Latvia~ Lithuania, Luxemtourg. Malta, Netherlands, Nonvay,
Poland, Port ugal, Republic of North Macedonia, Romania. Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and
United Kingdom.

a
EUROPEAN COMMIIT66 FORSTAN01\RDIZII.TION
C0~11T£ EUROPEEN DE NORMALISAT I ON
EU ROPli l SC H ES KOMITEE FOR NORMUNC

CEN•CENELEC Management Centre: Rue de l a Science 2 3, B · 1040 Brussels

© 202.1 CEN All rights or exploitation in auy form and by any means reserved Ref. No. EN 15784:2(•2 l E
worldwide for CEN national Members.
BS EN 15784:2021
EN 15784:2021 (E)

Contents Page

European foreword ....................................................................................................................................................... 3

Introduction .................................................................................................................................................................... 4
1 Scope .................................................................................................................................................................... S
2 Normative references ................................................................................................................................... 5
3 Terms and definitions .................................................................................................................................. 5
4 Principle ............................................................................................................................................................. S
5 Diluent and culture me dium ...................................................................................................................... 6
5.1 Diluent. ................................................................................................................................................................ 6
5.2 Culture medium t r yptone soy agar (TSA) ............................................................................................. 6
6 Apparatus ........................................................................................................................................................... 7
7 Sampling............................................................................................................................................................ 8
8 Preparation oftestsample ......................................................................................................................... 8
9 Procedure........................................................................................................................................................... 8
9.1 Preparation of poured agar plates for spr ead plate method .......................................................... 8
9.2 Preparation of the initial suspension and decimal dilutions .......................................................... 9
9.3 Inoculation and incubation of plates ................................................................................................... 10
9.4 Enumeration of colonies ........................................................................................................................... 10
9.5 Connrmatton ................................................................................................................................................. 11
10 Expression of results .................................................................................................................................. 11
11 Precision ......................................................................................................................................................... 12
11.1 General...................................................................................................................................... .................. 12
11.2 Interlaboratory studies............................................................................................................................. 12
11.3 Repeatability.................................................................................................................................................. 12
11.4 Reproducibility ............................................................................................................................................. 12
12 Test report ..................................................................................................................................................... 12
Aunex A (i11fon11~live) Notes 0 11 the p ..ocedu,·e .............................................................................................. 13
Annex B (informative) Results of the interlaboratory studies ................................................................... 14
B.1 General ............................................................................................................................................................. 14
B.2 Data obtained from VDLUFA collaborative studies ......................................................................... 14
Bibliography................................................................................................................................................................. 16
 BS EN 15784:2021
EN 15784: 20 21 (E)

European foreword

This document (EN 15784:2021) has been prepared by Technical Committee CEN/TC 327 "Animal
feeding stuffs • Methods of sampling and analysis", the secretariat of which Is held by NEN.

This European Standard shall be given the status of a national standard, either by publication of an
identical text or by e~dorsement, at the latest by May 2022, and conflicting national standards shall be
w ithdrawn at the latest by May 2022.

Attention is drawn t o the possibility that some of the elements of this document may be the subject of
patent rights. CEN sha ll not be held responsible for identifying any or all such patent rights.

This docume nt supersedes EN 15784:2009.

The main changes compared to the previous edition are as follows:

Amendment of t he title;

Extension of the scope of application to all Bacilli used as feed additive and to mineral feeds;

Updating of normative cross references;

Add ition of 0,2 % NaOH as diluent for initial suspension and serial dilutions;

Removal of the necessity of a heating step;

Unific:ition of thHre:itment of :ill m:itrice~;

Replacement of the required laboratory mixer with a rotation speed of 18 000 min•1 :o
22 000 min• 1 by homogenization devices, for example according to EN ISO 7218, w ith a maximal
requested rot'dtion speed of 10 000 min• 1;

Addition of the option to use a spiral plater for plating;

Addition of validation data derived from VDLUFA ring trials of different feeding stuff matrices
including mineral feed;

Adjustment of t he range of accepted colony numbers for counting from ' 2: 30 to s 300' to '2: 10
to :s 100' colonies per plate.

Any feedback and questions o n th is document should be di rected to the users' national standards body.
A complete listing of :hese bodies can be found on the CEN website.

Acco rding to the CEN -CENELEC Internal Regulations, the national standards organisations of the
following countr ies are bound to implement th is European Standard: Austria, Belgium, Bu lgaria, Croatia,
Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Icela nd, Ireland,
Italy, Latvia, Lithuania, Luxembourg, Malta, Netherla11ds, Norway, Poland, PortLisgal, Republic of North
Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United
Kingdom.

3
BS EN 15784:2021
EN 15784:2021 (E)

Introduction

This methodology has been developed to enumerate ·Jacilli spores used as feed additives capable of
germinati ng, to enable the Eu ropean Comm ission to control proper labelling of animal feeding prod ucts.
It was compiled first during the EU project SMT4-CJ"98-2235 "Methods for the official control of
probiotics used as feed additives" (1). During the revision of t he method it was adjus ted to VDLUFA
method 28.2.2 "Enumeration of Bacillus licheniformis and Bacillus subtilis" and completed with validation
data from interlaboratory studies with commercial feed products (2). The method is validated in this
project for two strains of Bacillus subtilis (DSM 5750 and DSM 15544) and one strain of Bacillus
licheniformis (DSM S749). It can be assumed that the method is suitable also for other Bacillus strains
used as feed additives. However, the applicability of the method to the determination of Bacillus spp. in
specific feed add itive preparations may need to be demonstrated based on a case by case decis ion.
Vegetative cells are not taken into account in this method, as all approved Bacillus species products at
present are spores.
S,iores of Bacillus species survive a treatment with 0,2 % sodium hydroxide solution and the Bacillus
species characteristic colony morphology of the individually authorized strains is examined using the
proposed method [3].
Th is method is not selective for bacilli used as feed additives but can be applied to enumerate Bacillus
spp. in feeding stuffs assuming that the added bacilli are present in far higher numbers than any other
bacilli.
Th is method is not applicable for the detection of any ubiquitous or pathogenic Bacillus spp. in food and
animal feeding stuffs.
 BS EN 15784:2021
EN 1571:14::l O:ll (t:)

1 Scope
This document specifies general rules fo r the enumeration of bacill i in feeding stuffs (additives,
premixtures and compound feeds includ ing m ineral feeds) [4) that conta in bacill i as a single
microorgan ism component or in a mixtu re with other microorganisms. There are different categories of
feed samples:
a) Additives contairting about 10 10 colony forming units (CFU)/g;

b) Prem ixtures co ntaining about 1011 CFU/kg;

c) Compound feeds. meal or pellets containing about 109 CFU /kg.

2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constit:utP.s requirements of this document. For d~ted references, only the P.dition cited ~pplies. For
undated references, the latest edition of the referenced document (including any amendments) applies.
EN ISO 6498, Animal,feeding stuffs- Guidelines for sample preparation {ISO c498)

3 Terms and definitions

For the purposes of t his 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: ava ilable at https://www.iso.org/obp

IEC Electropedia: available at https://www.electropedia.org/

3.1
Bacillus strains
genus of Gram -positi•te, rod-shaped bacteria

Note 1 to entry: This description is based on their characteristics as used forthis document.

Note 2 to entry: Bacillus species can be either obligate aerobes or facultative anaerobes. Cultured Bacillus species
~rP. r.a t:1:hu~"'•pnsitivP. if .-·ulriv:itPrl in thP. prP.~"'nrP. of nxyeP.n.

Note 3 to entry: Bacilli can form oval endospores.

Note 4 to entry: Bacilli form colonies on the surface of tryptone soy agar (TSA) after incubation at a temperature of
37 •c under aerobic conditions for 16 h to 24 h fitting the description given in 9.5.

4 Principle
a) Preparation of sterile and dry poured plates;

b) Drawing a representative test sample under aseptic conditions;

c) Preparation of the initial suspension with a tempered 0,2 % sodium hydroxide diluent to obtain a
homogeneous distribution of bacterial cells from the test portion and to reduce the vegetative
bacterial flora in the suspension;

5
BS EN 15784:2021
EN 15784:ZO:ll (t:)

d} Preparation of further decimal dilutions of the initia l suspens ion in or der to reduce the number of
microorganisms per unit volume t o allow, after incubation, the counting of colonies;

e) Inoculation of the prepared poured plates with an al iquot of the optimum dilutions and dispersion
of the inocu lum by using a sterile spreader;

I) Incubation of inverted plates for 16 h to 24 hat 37 °C ± 1 °C under aerobic cond itions;

g) Counting of typical colonies, considering the specific properties of bacilli;

h} Confirmation of exemplary isolates by microscopy or biochemica l properties if necessary;

i) Calculation of the colony forming units of Bacillus S?P- per gram or kilogram or feed sample.

5 Diluent and culture medium

5.1 Diluent

This diluent, a sodium hydroxide solution, is used for the preparation of the initial suspension and for the
preparation of further decimal dilutions. The composition of the diluent is given in Table 1.
Table 1- Compone nts of a 0,2 % s odium hydroxide solution
Sodium hydroxide NaOH 2,0 g
Polyoxyethylene (20) sorbitan monooleate (Tween® 80) 1 C64H124026 1 ml
Water, distilled or deionized H20 1000 ml

Dissolve the components (see Table 1) in water. Fil l the solution into appropriate containers (e.g. bottles,
llasks, or test tubes) and sterilize at 121 °C ± 3 °C for 15 min. To avoid loss during autoclaving, screw cap
bottles are recommended.
Fo r immed iate use, hold at 40 •c ± 1 °Ci na water bath or incubator.
5.2. Culture medium tryptone soy agar (TSA) 2
5.2,-1 Composition

The composition of the cu lture medium tryptone soy agar is give n in Table 2. The resu lti ng pH value at
2s •c is 7,3 ± 0,2.

Tween® 80 is an example of a suitable product available commercially. This information is given for the
convenience of users of this document and does not constitute an endorsement by CEN of this product.
2 The TSA medium is commercially ready made available from various suppliers.
 BS EN 15784:2021
EN 15784:2021 (E)

Table 2 - Composition of the tryptone soy agar (TSA)

Tryptone 15,0 g
Sodium chloride 5,0 g
Soja peptone 5,0 g
Agar agar 12 g to 15 g•
Water, distilled or deionized l 000 ml
• Depending on the gel strength of the agar.

5.2.2 Preparation

Dissolve a ll components (Table 2) in water unde~ heating and fill into appropriate contain ers ( e.g. bottles
or flasks with non-toxic meta l screw-caps). If necessa,y, adjust to a final pH of 7,3 ± 0,2 at 25 °C after
sterilization. Sterilize at 121 •c ± 3 •c for 15 min.

6 Apparatus
Usual microbio logical laboratory equ ipment and, in particular, the following:
6.1 Equipment for dry sterilization (oven) and wet sterilization (autoclave), for exam ple
according to EN ISO 7218 (S].

6.2 Incubator, capable of maintaining a temperature of 37 •c ± 1 °C. Optionally also capable of

maintain ing a t emperature of 40 °c ± 1 °C and/or between 44 °C and 47 °c.

6.3 Water bath, capable of maintai ning a temperature of 40 •c ± 1 °C and between 44 °C and 4 7 °C.

6.4 Blending equi()ment.

The following apparatus may be used accord ing to EN ISO 7218 (SJ:
a rota1y homogenizer (b lender) with a notiona l variable speed of 3 000 mi n• 1 to 10 000 mi11• 1, as
well as aseptic glass or metals bowls equipped with covers; or

a peristalti c homogenizer with steri le bags (paddle homogenizer), possibly with the optio n to adjust
blending speed and time; or

a vibrational mixer with sterile bags; or

any other homogenizing system w ith equivalent efficiency ( e.g. a hand ble nder w ith aseptic beaker).

6.5 Mechanical stirrer.

A mechanical stirrer (e.g. Vo rtex Mixer) facilitates the homogenous mixing of decima l dilutions, as
described in e.g. EN ISO 7218 [5].
6.6 Balances, of the requ ired range and accuracy, for example according to EN ISO 7218 [5], for the
different products to be weighed.

6.7 Flasks or screw-cap bottles, of app ropriate capacities.

6.8 Test tubes, of appropriate capacities.

7
BS EN 15784:2021
EN 15784:2021 (E)

6.9 Pipettes or pipettor and sterile tips, to dis pense 0,1 ml to 1 ml.

6.10 Sterile pipettes, to dispense 5 ml, for full outlet with wide (approx. 3 mm) tips (e.g. serological
pipette).

NOTE As alternative, 5 ml graduated pipettes without tips can be used.

6.11 Spreading spatula, sterile L- or triangular-shaped spreaders from glass or metal or sterile
disposable plastic spreaders.

NOTE As alternatives. a spiral plater with a sanitized dis pensing system or d isposable one-way micro syringes
can be used.

6.12 Steri.le Petri dishes, with triple vents (plates), 90 mm in diameter.

6.13 Laminar flow cabineL

6.14 Microscope, capable of phase-contrast microscopy at a magnification of 600x to 1 000x.

6.15 pH meter, having an accuracy of ca libration of± 0,1 pH unit at 20 °C to 25 °C.

7 Sampling
Carry out the sampling procedure in accordance with the specific standa rd appropriate to the product
concerned. If such a specific standard is not available, it is recommended that agreement be reached on
this subject among the parties concerned. Apply community rules [1) for official control sampling of
animal feeds.
NOTE Sampling can be done according to EN ISO 6497 (6). Although EN ISO 6497 is not specifically applicable
to microorganisms, due to the lack of other references it seems the most suitable protocol to be taken into account
for microorganisms as feed additives.

Take precautions to avoid potentia l cross-contamination of samples with microorganisms, particularly

after sampling additives and premixtures supplemented with microorganisms. When required, clean and
disinfect the sampling equipment between each sample, particularly after the sampling of additives and
premixtures containing microorganisms.
Put the sample in a steri le container.

8 Preparation of test sample

The test sample preparation shall be done in accordance with EN ISO 6498 and the congruent product
standard.
NOTE EN ISO 6498 gives general guidelines on test sample preparation.

9 Procedure
9-1 Preparation of poured agar plates for spread plate method
The culture medium is prepared as described in 5.2.2 or according to the manufacturer's directions. Cool
after autoclaving in a water bath to a temperature between 44 °C and 4 7 •c. Pour portions of
approximately 15 ml into each plate (6.1 2) under steri:e conditions and spread to give a homogeneous
layer.
 BS EN 15784:2021
EN 15784:2021 (E)

When the medium has solidified, pile quantities of four inverted plates on each other and dry at room
temperature or in an incubator at 37 •c ± 1 •c for approximately 12 h or overnight. Alternatively, spread
the plates out in a laminar flow cabinet and dry the agar surface with the lids partially removed for about
30min.
Check the dried plates for s terility. If correctly protected aga inst dehyd rati on, dried plates may be stored
in a refrigerator at 5 •c ± 3 •c for up to two weeks. Bring t he plates to room temperatu re before use.
On agar plates with a wet surface or containing media portions of more than 15 ml per plate, Bacillus
species can very often form swarm ing colon ies possibly covering the entire plate and precl uding the
enumeration of colony formi ng units on these plates.
9.2 Preparation of the initial suspension and decimal dilutions

Table 3 gives recommended sample quantities and corresponding volumes of tempered d iluent for the
preparation of initial suspensions for va rious feeds and t reatment of the initial suspension.
Table 3 - Recommended sample quantities and corresponding volumes of tempered diluen t for
the preparation of initial suspensions for various feeds and treatment of initial suspe nsion

Nature of s ample Sample quantity Diluent for Dilution factor Treatment of

gorml suspension of initial initial
(5.1) suspension suspension

Additives
5,0 ± 0,25 (495 ± 9,9) ml 1: 100
Premixtu res
Mixer or paddle
Mineral feeds 20,0 ± 1,0 {380 ± 7,6) ml 1:20
blender 5 min
Compound feeds 50,0 ± 2,5 (450 ± 9) ml 1: 10
Milk replacers 50,0 ± 2,5 (450 ± 9) ml 1: 10
{90 ± 1,8) ml +
Paste-like and Paddle blender
5,0 ± 0,25 5 g of Tween® 80 1:20
oleaginous feed 5min
(6]
Weigh the recommended quantity of sample according to Table 3 into an aseptic blender bowl, beaker or
plastic bag. Add the corresponding amount of tempered diluent (5.1). If encapsulated bacilli are analysed,
an adequate amount of sample material depending on the size of the capsules has to be used for the
prepa ration of the initial suspension. The number of capsules should be greater than 25, to obtain a
standard deviation in repeated analysis ofless than 20 % for a good repeat of analysis.
If a significantly lower value than expected is obtained in a first ana lysis, it is recommended to repeat the
analysis with a wider ratio of sample mass to diluent volume (5.1) for initial suspension or to blend the
sample with low-germ grain bran or any other neutral carrier to reduce the influence of interfering
s ubstances.
For compound feed, it is strongly recommended to perform the analysis in duplicate to minimize
variation. The result will be the average of both samples. For pelleted compound feeds, a low speed dry
grinding for 30 s is recommended to obtain an unrefined powder. Avoid excessive heating that can
damage the microorganisms.
According to specific procedures for dehydrated products and products with low water activity m
EN ISO 6887-4 (7] it is recommended to weigh out the sample accurately imo a pre-dispensed volume of
tempered diluent (5.1) to minimize osmotic shock to the microflora.

9
BS EN 15784:2021
EN 15784:2021 (E)

Blend the samples for 5 min in a mixer at a speed of 3 000 min•l to 5 000 min•l for 5 min (see 6.4) or
homogenize on high speed in a paddle blender.
For recovery, foam break-up and rehydration it is recommended to leave the initial suspension for 15 min
ac laboratory ambient temperature (18 °C to 27 °C) before preparing any further dilutions.
NOTE Intense foaming in initial suspensions during blending can be avoided by ming a few drops of an
appropriate antifoaming agent (e.g. :;iii con antifoaming agcttt or equivalent).

Prepare a first dilution from the treated initial suspension. Use a 1 ml pipette (6.9) for additives or a 5 ml
pipette (6.10) for compound feeds and pre mixtures. Transfer 1 ml or 5 ml of the initial suspens ion into a
vessel containing 9 ml or 45 ml of sterile 0,2 % sodium hydroxide sol ution brought to room temperature
and mix with a mechanica l stirrer. Weigh the dispensed pipette vo lume in order to apply a mass
correction factor for the calcu lation of the counts of Bacillus species.
Suspensions containing microbial additives tend to settle qu ickly, see Annex A. Be sure that the initial
suspension is homogenous before pipetting subsamples for further dilutions. T his is done by letting the
mixer run slowly.
Prepare further decimal dilutions (serial dilutions) using a sterile 1 ml pipette such as a micro dispenser
set at 1 ml. Transfer 1 ml of the first dilution into a t ube contain ing 9 ml of sterile 0,2 % sodium hydroxide
solution (5.1) and mix with a mechanical stirrer. Repeat this procedure until the appropriate estimate fo r
the number of cells is obtained.
9.3 Inoculation and incubation of plates

The time elapsing between the start of the preparation of the initial suspension and the moment when
the plates are inoculated shall not exceed 45 min.
NOTF, 1 The hydration pericxl of 15 min is included In the total period of 45 min.

To check sterility, prepare a control spread plate.

Selected dilutions shall be homogeneous prior to transferring a subsample onto plates.
Prepare at least two plates per su itable dilution. Three or four plates per dilution minimize the variation
and increase the co nfidence level.
The lower limit of 10 expected colonies per plate corresponds to the recommendations of EN ISO 7218
[5]. For a higher precis ion and increasing confidence level, a lower limit of 20 expected colonies per plate
is recommended.
Transfer 0,1 ml to 0,25 ml of each suitable dilution step onto at least 2 plates, each prepared with culture
medium (5.2.1) and distribute the inoculum using a sterile spreader or a spiral plater, so that not less
than 10 colonies and not more than 100 colonies per plate are to be expected.
Incubate the inverted plates aerobica lly at 37 °C ± 1 •c for 16 h to 24 h. Up to four plates can be stacked
for incubation.
NOTE 2 Depending on the growth rate of specific Bacillus spp., the incubation time might be adapted to yield
colonies of appropriate size for counting.

9.4 Enumeration of colonies

After incubation under the conditions specified above, all plates showing more than 10 and not more than
100 presumptive bacilli colonies according to the phenotypic characterization (9.5) are used for the
enumeration of CFU.
For the calculation of the number of bacilli per un it of sample (CFU/g or CFU/ml), a lower lim it of
10 colonies per plate corresponds to the recommendations of EN ISO 7218 (5]. For a higher precision and
increased confidence level, a lower limit of 20 colonies per plate is recommended.
 BS EN 15784:2021
EN 15784:2021 (E)

If possi ble, count at least fou r plates (of two successive dilutions) or three plates (of one dilution) . If no
suitable incubated plates with colony counts between;, 10 and,; 100 a re available, t he analysis has to be
repeated with appropr iate di lutions.
9.5 Confirmation
The colony morphology of different Bacillus strains that belong to the same species can be highly diverse.
The method was validated with strains of Bacil!us liche11iformis DSM 5749 {CH 200) as well as Bacillus
subti/is DSM 5750 (CH 201) and Bacillus subtilis C-3102 (DSM 15544) .
These Bacillus strains showed the following colony morphology on TSA:
a) Bacillus subtilis: 3 mm to 8 mm in diameter, round, surface dull, opaque, wrinkled a nd cream or
brown coloured; colonies of one strain often s how tv,o different morphologies (DSMZ 5750).

b) Bacillus licheniformis: 4 mm to 8 mm diameter, convex, from matt, chondroid, to very mucous,

lobular, shape surrounded by smaller sub-colon ies. The centra l mucous part of a colony can dry up
and become flat, white and opaque while the colonies are still surrounded by small, mucous sub-
colonies. Some parts of a colony will adhere more strongly to the substrate than others.

Depending on the number of different colony morphologies appearing after incubation, two to five
colonies of each morphology type are selected for Gram staining.
Microscopic observation of the Gram stained microorganism confirms the bacteria. Only Gram -positive,
rod shaped bacteria, which can {or cannot) form endospores, are considered as bacilli.
In case of doubt, the prese nce of bacilli is confirmed by biochemical cha racte rization (for exa mple
commercial available t est kits), polymerase chain reaction (PCR) analysis or via matrix assisted laser
desorpt ion ioniwtion time offlight (MALOJ-TOF) mass spectrometry of exempl~ry colon ies.

10 Expression of results
The number of colony forming units {CFU) per gram or per millilitre of feed {N) is calculated according
to the recommendation in EN ISO 7218 (5) using Formula (1):

(l)

where

N is the number of colony forming units (CFU) per g or ml of feed;

1:C is the sum of colonies counted on all the plates;
V is the volume of inoculum applied to each plate, in ml;
n1 is the number of plates retained at t he fi rst countable dilution;
n2 is the number of plates retai ned at the second countable dilution;
r/ is the d ilu tion far.t or of the firs t cons idered dil ution from which r.ounts we re obtai ned.
If the number of one of the counted plates is incoherent within the set of considered plates, the incoherent
result has to be discarded. Nevertheless at least three plates (n 1 + n2 ;, 3) have to be taken into account
for the calcu lation. In these cases the formu la has to be adapted accordingly, e.g. with 111 =1 and 11 2 = 2 or
n 1 = 2 and 112 = 1.

11
BS EN 15784:2021
EN 15784 :2021 (E)

Alternatively, the number ofCFU per g or ml sample (NJ can be calculated from only one dilution. In this
case 11 1 should be higher or equal to 3 and 11 2 is set to zero in Formula (1 ).
The calculated result is rounded as described in, for example, EN ISO 7218 [SJ.
TI1e result is the number of microorganisms per g ram or millilitre of the sample, exp ressed as a number
between 1.0 and 9,9 multiplied by the appropriate power of 10. For premixtures and compound feed, it
is recommended to trans late the result to CFU/kg according to the declaration.

11 Precision
11.1 General

The precision of the method in terms of repeatability and reproducibility was determined in
interlaboratory studies [2].
11.2 lnterlaboratory studies

Details of the interlaboratory studies on the precision of the method are published in [2] and are
summarized in Annex B. Precision data were determined using nine samples (two additives, two
premixture, two mineral feeds and three compound feeds). The values derived from the interlaboratory
studies are not applicable to concentration ranges and matrices other than those given.
11.3 Repeatability

The absolute difference between two independent single test res ults (number of bacilli per gram or per
millilitre), obtained using the same method on identical test material in the same laboratory by the same
operator using the same apparatus within the shortest feasible time interva l, will in no more than S % of
the cases exceed the repeatability limit r.
11.4 Reproducibility

The absolute difference between two single test results (number of bacilli per gram or per millilitre),
obtained using the same method on identical test material in different laboratories with different
operators using different equipment, will in no more than 5 % of cases exceed the reproducibility limit R.

12 Test report

The test report shall at least specify the following aspects:

a) information necessary for the complete identification of the sample;

b) the test method used, with reference to this document (including it~ year of publication);

c] the sampling method used, if known;

d) operating details not specified in this document, or regarded as optional;

e) details of any incidents which can have influenced the test result(s);

t) the test result(s) obtained, or, if the repeatability has been checked, the fi nal quoted result obtained
(i ncluding a reference to the clause which explains how the results were calcu lated);

g) any unusual features observed;

h) the date of the test.

 BS EN 15784:2021
EN 15784:2021 (E)

Annex A
(informative)

Notes on the procedure

Most vegetative cells from microorganisms will be inactivated by the alkaline treatment used in this
method.
Colloidal precipitations, formed by flocculation of hyd roxides and phosphates of multivalent cations, can
occur in initial suspensions with alkaline pH values. In these floes a part or the spores may be enclosed or
bound by adsorption, resulting in a sediment which is - compared to the supernatant - enriched in
bacteria l s pores. As the floes can settle down quickly, the initial suspensions should be kept homogenous
during pipetting using appropriate measures.
High contents of copper and other toxic substances shou ld not affect the survival of bacterial spores in
initial suspensions. However, if in a first analysis a significantly lower value than expected is obtained, it
is recommended to re peat the analysis with a wider ratio or sample mass to diluent vol ume (5.1 ) for
initial suspens ion or to blend the sample with low-germ grain bran or any other neutral carrier to reduce
the influence of interfering substances.

13
BS EN 15784:2021
EN 15784:2021 (E)

Annexe
(informative)

Results of the interlaboratory studies

B.1 General
The following data were obtained in in terlaboratory studies organized by the VDLUFA between 2004 and
2010, in which one Austrian, one Swiss and eight to eleven German laboratories participated. They were
evaluated according to ISO 5725-2 [8].

B.2 Data obtained from VDLUFA collaborative s tudies

The interlaboratory tests were conducted with commercial feed products, which were tested for stability
and· homogeneity. Each sample was analysed in triplicate by the respective participating laboratories. As
the .obtained data were distributed normally, the specifications of the method were calculated in non-
logarithmic form. The precision data derived from the study are presented in Table B.1.
 F.N 1 S7R4:2021 (F.)

Table B.1 - Precision data obta ined from VDLUFA collaborative studies (2)
TSAmedium
Test no 335 M·d 381Q 335 M-a 353Q-a 328 Q-a 360 Q-c 353 Q-b 353 Q-c 384Q
Year 2004 2010 2004 2006 2004 2007 2006 2006 2010

Matri_x Turkey feed Turkey feed Piglet feed

Ad.ditive Premixture Mineral fe.ed
meal pelleted pelleted
Number of laboratories 9 13 12 13 13 13 13 12 14
Number of single values 25 39 36 39 39 39 39 36 42
Unit CFU/g CFU/kg
Concentration E<-ll E<-10 E<-11 E<-l2 E<-10 E<-10 E<-09 E<-09 E<-09
Mean value 4,45 0,92 5,54 2,03 3,23 3,62 1,46 1,27 0,90
Standard deviation of
repeatability Sr 0.50 0.14 0.67 0.40 0.30 0.39 0.21 0,19 0,08

Repeatability limit r
1,40 0,39 1,88 1,12 0,84 1,09 0,59 0,5 3 0,2 2
(: 2,8 X s,)

Standard deviation of
1,24 0,21 1,88 0,64 0,87 0,71 0,34 0,22 0,17
reproducibility SR

Reproducibil ity limit R

3,47 0,59 5,26 1,79 2,44 1.99 0,95 0,62 0,48
(=2,8 X SR)

Repeatability relative
sta ndard deviation RSD, l 1,1 14,9 12,1 19,6 9,4 10,6 14,1 15,2 9,1
(%)
Reproducibility relative 0:,
standard deviation, RSDR 27,9 22,S 33,9 31,4 26,9 19,6 23,0 17,1 18,5 V,
t'!1
(%) z
,...
Vl
'-I
.,.;._:,
0:,

0
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BS EN 15784:2021
EN 15784:2021 {E)

Bibliography

[1] European Community Project SMT4-CT98-2235, Methods for the official control ofprobiotics used
as feed additives (vo l.1 -3). 2004. Report EUR 20873/1 -3. Office for Official Publications of the
European Communities. ISBN 92-894-6249-3 (set)

[2] Verband Deutscher Landwirtschaftl icher Unter;uch ungs- und Forschungsanstalte n (VDLUFA),
Method 28.2.2: £numeration of Bacillus subti/is and Bacillus /icheniformis, in. Methods book Vol. Ill
- 28.2.2

[3) Leuschner R.G. K., Bew J., Cruc A., Enumeration ofprobiotic bacilli spores in animal feed:
A collaborative study. In:]. AOAC. 2003, 86 pp. S68- 575

[4) Regulat ion (EC) No 767 /2009 of the European Parlia ment and of the Council of 13 Ju ly 2009 on
t he placing on the market and use of feed, amending European Parliament and Council
Regu lation (EC) No 1831/2003 and repealing Counci l Directive 79/373/EEC, Commission
Directive 80/511/EEC, Council Directives 82/471/EEC, 83/228/EEC, 93/74/EEC, 93/113/EC
and 96/25/EC and Commission Decision 2004/217 /EC, Official Journal of the European Union,
L 229/1, 1.9.2009. Available at https://data.europa.eu/eli/ reg/2009/767 /oj

[SJ EN ISO 7218, Microbiology offood and animal feeding stuffs· General requirements and guidance
for microbiological examinations (ISO 7218}

[6] EN ISO 6497, Animal feeding stuffs · Sampling {ISO 6497)

[7] EN ISO 6887-4, Microbiology ofthe food chain · Preparation of test samples, initial suspension and
decimal dilutions for microbiological examination • Part 4: Specific rules for the preparation of
miscellaneous products {ISO 6887-4)

[8) ISO 5725-2, Accuracy (trueness and precision) ofmeasurement methods and results - Part 2:
Basic method for the determination of repeatability and reproducibility ofa standard measurement
method

(9) EN ISO 6887-1, Microbiology ofthe food chain • Preparation of test samples, initial suspension and
ue<:imul uilutium; fur micrubiuluyicul exumif1utiu11 - Puri 1: Ge11erul ruft:y fur i/1,: prepuruiiu11 uf ilte
initial suspension and decimal dilutions {ISO 6887-1)

[10) EN ISO 11133, Microbiology offood, animal feed and water - Preparation, production, storage and
performance testing ofculture media {ISO 11133)
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