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ñlozor.A
E'r Ar..:
JOURNAL
oF
AOAC lN'r¡nNxltot'JRt-
VoL.
97,
No.
3,
20
l4
Table
1.
lnclusive
and
exclusive isolates
used in the ANSR Salmonella
collaborative study
Organism
lD
No.
Source
Origin
(if known)
Salm
on el
la
enlerica subsp
Sa
lm
onella enferica subsp
Sa
lmon ell
a
enfeíca
subsp
Salmonella bongori
Sal
monella enlerlca subsp
Sa
lmon
ella enterlca subsp
Enterobaçter cloacae
Escherichia coli
Proteus vulgaris
Prov¡denc¡a alcalifaciens
Citrobacter freundi¡
Klebsiella pneumoniae
anzonae
enter¡ca
Set
enterica
Ser
enlerica
Ser
enler¡ca
Set
Typh¡murium
Cubana
Cerro
Enteritid¡s
700
1
50
2356ô
12007
4397
5
10723
4931
13047
25922
29905
27970
8090
1
3883
ATCC"
ATCC
ATCC
ATCC
ATCC
ATCC
ATCC
ATCC
ATCC
ATCC
ATCC
ATCC
Poult
Unknown
Unknown
Unknown
Unknown
Human Gl tracl
Human CSF
Human
Unknown
Feces
Unknown
Unknown
'
American Type Culture Collection,
[/anassas,
VA.
deox,vcholate
agar
(XLD),
bisniuth sullìte
agar
(IlS).
brilliant
gleen sulfä
agar
(BGS). xvlose lysine
telgitol
agar
(XLT-4).
and
double-nrodilìed
lysine
iron
agar
(DMLIA)]
and tested
in
the
ANSR
assay.
'fhe
fomrer
thlee uredia
are
specifìed
f-or
use
in the
BAM
refèrence nrethod,
u'hile
the latter three
are
specified
in
the
MLG
rnethod. One
hundred
and
twelve Salntonella
spp. strains
produccd
positive
resulls
from all
seven
medi4
for
inclusivity
of
99,l
%.
One strain
of
.S.
\&'eslaco,
previoì.rsly
identified
as a
non-
inclusivc
strain lacking the genetic targel
for
the
ANSR
assay.
produced negâtive
lesults fronr
all
seven media.
In
testing
of
exclr.rsive
strains. 248
of25
I
assays
produced negative results,
f'or
accuracy o1'98.8%.
'l'he precollaborative sludy report
is included
a-s
Appendix
I
on
J.
ÁOAC
/¡¡,
rvebsite.
http://aoac.publisher.
i
ngentaconnect.
com/contenlaoaci
j
aoac.
Ilere
rve
rcpofl
results
ofan
intcrlaboratorl collaboralive
stud¡,
conducted
in
I
8
laboratories
for
fifiher
evaluation
ofthe
assay
as
a
colon¡
confìnnation tool.
Collaborative Study
Study Design
'I-his collaborative study
rvas
conducted
in
accordance rvith
fhe
AOÁ(-
INTER^IATIONAL lçlethods Contmiuee
Guidelines
Jòr
l:alidation
oJ
lt4icrobiological
il,Íefhods
Jor
l:ood
and
llnvironntental
Su(Ltces.
Appenclix.t (8).
Eighteen
laboratories
piìrticipated
in
the
collaborative study.
representing
induslr-v,
academic,
go\/elnment.
and privale testing
laboratories.
All
collabolators
rvere
either
established users
of
the
ANSI{
test
syslem
or
rvele
expressly
trained
fbl
the
collaborative
study
priol
to
ils
cornmencelnent.
A
detailed set
of
instructions
and
datir
recolding fbrrns
rvere sent
to
each
collaborator
in
advance
ofthe
snrclv.
Collaborators rvere
provided rvith all
necessâry
agar'
plating
media
test kits.
ANSIì
s¡'51e¡1
instrumentation, and
a
blind-coded
set
o1'l2
baclerial cullures
f'or
analvsis.
P
rep
a
ration of /so/afes
Âll
isolates
rvelc
fi'on'l
the
Neogen
Corp. culture
collection
and consisted
ofsix
diverse slrains
olS
etlterica
and S.
bongori,
¿rnd
six
strains
ol
Dnterobacteriaceae
belonging
to
other
genera
('làble
l).
All
strains rvere
obtained directly
from
the
Arnerican
'lype
Culnrre Collection
(AfCCl
Manassas.
VA).
Identity
ol
isolates u'as
confirnred by
API
20li
testing.
Sa/rrorel/a
isolates
rvere
also
verified
by O group serology. Isolates q'ere culturecl
on
l'SA
slants
for
I
8-24
h
at 36
+
I
oC. Slant
cultures
rvere labeled
rl,ith
a
trvo-digit
alphabetical
code,
Distribution of
I
solates
Cultures
rvere shipped to collaborators
via overnight delively,
at
ambient
temperature,
using
Categor-"r
B
Dangerous
Goods
packaging
as set
fbrth
b),
Intemational
Air
Transport Association
regulations. Collaborators were instructed
to
storc the
cultures
at
2-8'C until
initiation
of
the
anaìytical
work
(4-5
da1,s).
Collaborators
rvere
provided u,ith
a
"Sample Receipt
ltronr."
to
be
completed and returned to
the
Study
Director
by
email or fzx,
acknorvledging that the
samples rvere
leceived
in good
condition,
Analysis of /so/afes
To
initiate
the
analysis. collaborators streaked each
of
the
l2
bacterial
isolates
to
each
ofthe
severr
agar
media, stleaking
fot
isolated colonies.
Collaborators
rvere
plovided rvith
a sarnple
randomization
schenrc
by
the Stud)'
Dilector
¿ìncl
rvere instructerl
to
blind-code each strain-ägal nrcdiunr contbination
ri'ith
a
unique nurnber
l-84. 'lhis
rvas perl'ormed
by
"Operator
1."
rvho rvould
have
no
involvernent
in
the
actual
ANSR
analyscs.
Plates rvere
incubated
for 24+2
h
at
35.l
loC
and
examined
l'or
the
presence
ol
isolated colonies.
Plates
rvithout
isolated colonies
rvere reincubated
for
an additional
l8-24
h.
Plates
containing
distinct
isolated
colonies
after
24 h
rvcre stored at
2-8oC.
Atier
a
maximunr of
48
h
incubation"
plates
without
grorvth
or
isolated
colonies were noted
as
such
on
the Data
Recorcling
Fon¡
ancl
analysis
continued.
Operalor
I
then
picked
a
single
colony from
each
plate,
íncluding the refiigelatcd
plates, using an
inoculating
loop or needlc, and resuspcndcd lhe
colony
in 0.5
ntl,
phosphatc-
bulÌered saline
(PBS).'l'he
coded tubes
rvere
h'ansl-erred to
"Operator
2."
rvho then
perf'orrlcd
the
ANSIì
analyses.
ANSIì
testing was perl'olmecl
in
blocks
of'up
to l6
strrtrples.
stitrting
rvith
sample nurrrbel
I
and continuing through sample
number'