ELSEVIER

International Journal of
Food Microbiology 22 (1994) 277-289
I n t e r n a t i o n a l J o u r n a l
o f F o o d M i c r o b i o l o g y
A comparison of six different plating media used
in the isolation of Salmonella
D o n a l d W . W a r b u r t o n a,* B r u c e B o w e n a A n n e K o n k l e a
C a r o l C r a w f o r d b, S a m i r D u r z i c, R o g e r F o s t e r a, C a t h y F o x e,
L o r r a i n e G o u r f, G a i l K r o h n g, P i e r r e L a C a s s e h, G a e t a n L a m o n t a g n e i,
S h e l a g h M c D o n a g h J, V i c t o r i a A r l i n g J, J a n e t M a c k e n z i e k,
E w e n C . D . T o d d h, J o h n O g g e l ~, R o b e r t P l a n t e m S u s a n S h a w n,
N . P . T i w a r i o, Yvon Trot t ier v, Brenda Daly Wheel er q
a Evaluation Division, Bureau of Microbial Hazards, HPB Health and Welfare Canada, Ottawa,
KIA OL2 Canada;
b FieM Operations Division, HPB Health and Welfare Canada, Burnaby, V5G 4P2 Canada;
Fisheries and Oceans Canada, Toronto, M6N 3E3 Canada;
d Field Operations Directorate, HPB Health and Welfare Canada, Winnipeg, R2J 3YI Canada;
e Field Operations Directorate, HPB Health and Welfare Canada, Dartmouth, B2Y3Z7 Canada;
f Field Operations Directorate, HPB Health and Welfare Canada, Longueuil, J4K 1C7 Canada;
g Regina Veterinary Laboratory, Saskatchewan Agriculture and Food, Regina, $4S OBI Canada;
n Laboratoires d'expertises et d'analyses alimentaires de Sainte-Foy MAPAQ, Sainte-Foy,
G1P 2W8 Canada;
i Laboratoires d'expertises et d'analyses alimentaires de Sainte-Hyacinthe MAPAQ, Sainte-Hyacinthe,
J2S 2M2 Canada;
J Lab Services Division West, Agriculture Canada, Calgary, T2L 2L1 Canada;
k Research Division Bureau of Microbial Hazards, HPB Health and Welfare Canada, Ottawa,
K1A OL2 Canada;
l Laboratory Services Division, Agriculture Canada, Ottawa, K1A 0C6 Canada;
m Service de l'environnement, Communaute urbain de Montreal, Montreal, H4N 2T2 Canada;
n Fisheries and Oceans Canada, Halifax, B3J 2S7 Canada;
o Food Laboratory, Services Branch, Alberta Agriculture, Edmonton, T6H 4P2 Canada;
P Laboratoire d'hygiene veternaire, Agriculture Canada, Sainte-Hyacinthe, J2S 8E3 Canada;
q Fish Inspection Laboratory, Fisheries and Oceans Canada, St. John's A1C 5X1, Canada
(Received 15 December 1993; accepted 21 February 1994)
Abs t ract
Sevent een Canadian Federal, Provincial and Public Healt h Laborat ories took part in
different phases of a comparat ive/collaborat ive study that evaluat ed rapid met hods to the
* Corresponding author.
0168-1605/94/$07.00 1994 Elsevier Science B.V. All rights reserved
SSDI 0168-1605(94)00017-Z
278 D.W. Warburton et al. / International Journal of Food Microbiology 22 (1994) 277-289
standard Health Protection Branch (HPB) method for the detection of Salmonella. A
variety of commercial media were tested, including Brilliant Green Sulpha Agar, Bismuth
Sulphite Agar, Hektoen Enteric Agar, Xylose Lysine Deoxycholate Agar, EF-18 Agar and
Rambach Agar. Each laboratory compared up to six of these different plating media.
Plating of 123 salmonellae cultures and 28 artificially-inoculated foods showed the recovery
of Salmonella spp. on the six plating media to be within one log. Therefore, quantitative
testing of the media showed them to be comparable in the recovery of salmonellae.
Qualitative testing of the six media during the comparative/collaborative study of various
methods showed that EF-18 Agar recovered the greatest number of isolates. Hektoen
Enteric Agar ranked second, with the other agars being comparable in their recovery of
Salmonella spp. Problems with the various media are summarized. Based on our results and
those of other researchers, it is recommended that Bismuth Sulphite Agar be compulsory
and that at least one other agar be used for newly developed cultural procedures.
Key words: Comparative/collaborative study; Plating media; Isolation procedure; Salmonella
I. Int roduct i on
The use of select ive and different ial plat ing medi a is a very i mport ant part of
st andard cult ural met hods for t he isolat ion of Salmonella from foods and environ-
ment al samples. A wide variet y of medi a have be e n devel oped, differing in t heir
composit ion, use and pe r f or ma nce charact erist ics.
Brilliant Gr e e n Sul pha Agar (BGS; HPB, 1978), Bismut h Sulphit e Agar (BIS;
Andrews et al., 1984; HPB, 1978), He kt oe n Ent eri c Agar (HEK; Andrews et al.,
1984; Anon. , 1993) and Xylose Lysine Desoxycholat e Agar (XLD; Andrews et al.,
1984) are used in st andard met hods of different agencies in Canada, t he Uni t ed
St at es and in Eur ope (Flowers et al., 1992). BGS uses sodium sulfapyridine as t he
select ive agent and brilliant green and phenol red dyes as t he indicat ors of
carbohydrat e ut ilizat ion (Anon. , 1984). BIS uses bi smut h sulfite and brilliant green
as inhibit ory agent s against compet i ng mi crorgani sms and ferrous sulfat e as t he
indicat or of H2S product i on (Difco, 1984). HEK relies on t he use of bile salts for
selectivity, bromot hymol blue and acid fuschin as indicat ors of carbohydrat e
ut ilizat ion, and ferric iron as an indicat or of format i on of H2S from t hi osul phat e
(Anon., 1993b). XLD uses sodium desoxycholat e, sodium t hiosulfat e, ferric a mmo-
nium cit rat e and phenol red as select ive agent s, indicat ors of H2S product i on and
carbohydrat e ut ilizat ion, respect ively (Anon., 1984)
EF-18 Agar (EF-18; Dickson and Anderson, 1991; Entis, 1990; Ent is and
Boleszczuk, 1991; Todd et al., 1992) and Ra mba ch Agar (RAM; Anon. , 1993,
Bankes, 1992; Feng, 1992; Freydi ere and Gille, 1991; Gruenewal d et al., 1991;
Manafi and Sommer, 1992; Ra mba ch, 1990) are newly devel oped medi a associat ed
wit h t he recent ly devel oped met hods (cit ed above). EF-18 uses sulfapyridine, bile
salts, cryst al violet, novobiocin and incubat ion at 42C as di fferent i al / sel ect i ve
agent s, wit h sucrose and lysine decarboxylase ut ilizat ion as its different ial react ions
(Ent is, 1990). RAM uses t he select ive agent desoxycholat e, t he format i on of acid
from propyl ene glycol by Salmonella, and a chromogeni c indicat or of/ 3-gal act osi -
D.W. Warburton et al. / International Journal of Food Microbiology 22 (1994) 277-289 279
dase t o different iat e Salmonella from Proteus and ot her Ent erobact eri aceae
(Anon., 1993a).
These different commercially available plat ing media were evaluat ed as part of
a comparat ive and collaborat ive st udy comparing rapid met hods for t he det ect ion
of Salmonella to t he st andard Heal t h Prot ect i on Branch (HPB) met hod (HPB,
1978) involving Canadi an Laborat ories.
2. Materi al s and met hods
Sevent een Canadi an Federal, Provincial and Public Heal t h Laborat ori es t ook
part in different phases of a comparat i ve/ col l aborat i ve st udy t hat evaluat ed rapid
met hods t o t he st andard HPB met hod for t he det ect ion of Salmonella. During t he
different part s of this st udy a variet y of commercially available media were t est ed,
including BGS (Difco Laborat ories, Det roit , MI), BIS (Merck, Darmst edt , Ger-
many; Difco Laborat ories), HEK (Merck; Difco Laborat ories; Uni pat h Inc., Ne-
pean, Ont.), XLD (Merck; Difco Laborat ories), EF-18 (QA Life Sciences Inc., San
Diego, CA) and RAM (Merck). The first four media are associat ed with st andard
met hods (Flowers et al., 1992) while t he last two are associat ed with met hods being
t est ed in this study. Each laborat ory compared up to six of t hese different plat ing
media.
2.1. Microorganisms
One hundred and sevent een Salmonella and 49 non-Salmonella cult ures were
used (Table 1). These cult ures were used to artificially cont ami nat e samples, and
as positive or negat ive cont rols for t est ing bot h media and met hods.
2.2. Samples tested
Seven hundred and thirty-five nat urally-cont aminat ed and 551 artificially-con-
t ami nat ed food and environment al samples were t est ed in this st udy (Table 2). In
all cases t he foods were analysed by t he HPB met hod and one ot her met hod.
Oft en, several met hods were used simult aneously. In ot her cases, samples found
positive by one met hod, were oft en re-t est ed by ot her met hods so t hat 1622
nat urally-cont aminat ed and 1271 art ificially-cont aminat ed samples were t est ed by
t he six met hods.
2.3. Artificial contamination of the foods
The met hod used was t he same as t hat followed for an earlier st udy with
Listeria (Warburt on et al., 1992) and is summarized below: The Salmonella
cult ures were grown in Trypt ose Phosphat e Brot h (TPB) for 24 h at 35C. Aft er a
second subcult ure in TPB (24 h at 35C), t he cult ures were serially dilut ed in 0.1%
pept one wat er and 0.5 ml of t he 10 -s dilut ion was inoculat ed int o 25 g of food (so
~.80 D. W. Wa r b u r t o n et al. / I n t e r n a t i o n a l J o u r n a l o f F o o d Mi c r obi ol ogy 22 (1994) 2 7 7 - 2 8 9
Fable 1
List of cultures used (numbers of isolates)
~almonellae used
~almonellae(13) Agriculture Canada and HPB
ret yped isolates,
~almonellae Grp B(4) chicken fluff, chicken,
:urkey and pork isolates,
~almonellae Grp C(2) chicken isolates,
~almonellae rough(2),
~. a l a c h u a
~. a l b a n y
~. agona,
~. a n a t u m,
~. ar i z onae ( 4) ,
~. ar i z onae 1,
~. a r i z o n a e II(2),
g. a r k a n s a s ,
g. barei l l y( 2) ,
5. bredeny,
5. br aender up,
5. cerro( 3) ,
5. chest er,
5. chol era- sui s,
5. dubl i n( 2) ,
S. eal i ng( 2) ,
S. e as t bour ne ( 2) ,
S. e nt e r i t i di s ( 2) 1 H2S- ,
S. f l i n t ( 2 ) ,
S. gal l i nar um,
S. git,e,
S. godesburg,
S. haar dt ,
S. h a d a r ( 6 ) ,
S. hal~ana(2),
S. hei del berg,
S. i ndi ana H2S-Suc+
S. i nf ant i s ( 5) ,
S. j o h a n n e s b e r g ( 5 ) ,
S. k e n t u c k y ( 2 ) 1 Lac +,
S. l andow,
~. mb a n d a k a ( 5 ) ,
S. mont et ; i deo,
S. mi n n e s o t a ( 2 ) ,
S. muens t er ,
S. newi ngt on,
S. newport ,
S. ohi o,
S. orani enburg,
S. pi nz a,
S. p u l l o r u m,
S. r e adi ng,
S. s c h wa r z e n g r u n d ( 4 )
S. s e n f t e n b e r g ( 6 ) 1 H2S-Lys-, 1 775W, 1 Suc+
S. s t . - paul ( 2) ,
S. t a k s o n y ( 2 ) ,
S. thomasL, ille(2),
S. t h o mp s o n ( 5 ) ,
S. t yphi ,
S. t y p h i mu r i u m( 4 ) , 1 ATCC 14028,
S. urbana,
S. was s e naar Grp II,
S. wel t eureden,
S. wor t hi ngt on( 2) ,
Other species
A c h r o mo b a c t e r xyl os oxi dans ,
Ci t r obac t e r dit,ersus,
C. f r e u n d i i ( 3 ) 1HzS +,
En t e r o b a c t e r aer ogenes ( 3) ,
E. aggl ome r ans (5),
E. cl oacae( 2) ,
E. georgenes,
E. haf ni a,
E. t ayl orae,
En t e r o c o c c u s f aecal &,
Er wi ni a carotoL,ora(2),
Es c he r i c hi a col i (5),
Ha f n i a ah, ei ( 3) ,
Kl ebsi el l a oxyt oca,
K. p n e u mo n i a e ( 2 ) ,
Oerskot~ia sp.,
Pr ot e us sp.,
P. mi r abi l i s ( 3) ,
P. mor gani i ( 2) ,
P. ret t geri (2),
P. t , ul gari s(2),
ProL,idencia spp.,
Pr ov i de nc i a al cal i f aci ens,
P s e u d o mo n a s aerugi nosa,
P. f l uor es cens ,
Serrat i a f ont i c ol a,
S. mar c e s c e ns ( 2) ,
Shi gel l a boydi i ,
S. dysent eri ae,
S. f l exneri ,
S. sonnei ,
S t a p h y l o c o c c u s aureus,
D.W. Warburton et al. / International Journal of Food Microbiology 22 (1994) 277-289 281
t hat the inoculat ion was < 1 cf u/ g) and blended (or mixed using a st omacher) with
the preenri chment brot h. To ensure a high compet it or level, eit her 1 ml of a
non- Sal mone l l a cult ure or 2 g of soil (previously t est ed and found negative for
Sal mone l l a) was added to t he food mixture. Incubat ion and analysis cont inued as
appropriat e for t he met hod st udied (described below).
To det ermine t he inoculum level, 0.1 ml of the 10 -6 to 10 -8 dilutions were
plat ed ont o BIS and t he ot her agars. The agars were incubat ed at 35C for 18-48
h, except for EF-18 which was incubat ed at 42C. At 24 h, plates were examined
and confirmat ion st art ed on typical colonies. As needed, BIS and ot her agars were
re-incubat ed overnight , and visible colonies count ed.
2.4. Me t h o d s t est ed ( qual i t at i ve t est i ng)
During the comparat i ve/ col l aborat i ve testing six met hods were used. Five
met hods (t he modified 1-2 Test (Feng, 1992; Nat h et al., 1989; Oggel et al., 1990;
BioCont rol Systems Inc., Bot hell WA), EF-18 and HGMF met hod (Entis, 1990;
Entis and Boleszczuk, 1991; Feng, 1992; Gel man Sciences Inc.), the ELA met hod
(Todd et al., 1993), Gene-Trak (Feng, 1992; BDH Inc.), the Merck Salmosyst brot h
and tablet met hod (Ossmer, 1992; BDH Inc.)) were compared to the HPB
st andard met hod MFHPB-20 (HPB, 1978). The results of t he compa r i son/
collaborat ive testing of t he met hods will be published elsewhere (Warburt on et al.,
1994b,c,d,e). Each laborat ory could st reak out the selective brot hs, from t he HPB
and ot her met hods tested, ont o at least BIS and BGS, plus t he media st ipulat ed
for t hat met hod and any ot her agars t hey wished to test. A represent at ive number
of typical colonies (up to five per plate) and atypical colonies, if desired, were
Table 2
Foods tested and number of samples found to contain Salmonella sp. by one or more methods
Sample Naturally- Artificially-
contaminated contaminated
Controls ND 124
Dairy: cheese ND 52
milk/cream ND 87
Egg/egg powder 3 17
Environmental 15 3
Feeds 366 10
Seafood 17 2
Meat 15 11
Other 35 21
Poultry 62 ND
Spices 221 7
Vegetables/fruit ND 10
Water ND 10
Grain/flour 1 197
Total 735 551
ND, not done.
282 D.W.. Warburton et al. / International Journal of Food Microbiology 22 (1994) 277-289
purified for confirmat ion. Biochemical t est ing and serology were done according to
t he HPB met hod (HPB, 1978). Ot he r confirmat ion tests included t he use of
API-20E (API Laborat ory Product s Lt d, St -Laurent , Que.), Micro-ID (Organon
Tekni ka Inc., Scarborough, Ont.), Ent erot ube (Roche Diagnost ic Systems, Nut ley,
N J), as well as t he mini-Vidas and t he Vit ek (bioMerieux USA, Hazelwood MI).
The ability of st andard non-Salmonella t o grow on t he plat ing media was t est ed
as follows. The cult ures, listed in Tabl e 1 as " ot he r species", were subcult ured and
st reaked ont o t he plat ing media which were incubat ed as described above.
2.5. Quantitative testing of the media
One hundred and t went y-t hree salmonellae cult ures and 28 inoculat ed food
samples were plat ed on BIS, BGS, EF-18, HEK and RAM. An addit ional 46
salmonellae were plat ed on t hese agars and XLD. The foods consist ed of nine
inoculat ed homemade chocolat e and egg liqueur samples, as well as 19 inoculat ed
distilled wat er samples (Warburt on et al., 1993, 1994a,b). The salmonellae in t hese
Table 3
Recovery of Salmonellae by different selective plating media
Plating medium tested Number of positive plates (% recovery) a
BGS BIS HEK EF-18 b RAM XLD
BGS vs BIS vs HEK vs 139 136 142 149 130 132
EF-18 vs RAM vs XLD (93.3 a) (91.3) (95.3) (100) (87.3) (88.6)
BGS vs BIS vs HEK vs 596 605 613 624 602 ND
EF-18 vs RAM (95.5) (97.0) (98.2) (100) (96.5)
BGS vs BIS vs HEK vs 102 119 116 ND 95 ND
RAM (85.7) (100) (97.5) (79.8)
BGS vs BIS vs RAM 238 251 ND ND 249 ND
(94.8) (100) (99.2)
BGS vs BIS vs EF-18 vs 91 92 ND 96 ND 96
XLD (94.8) (95.8) (100) (100)
BGS vs BIS 1125 1119 ND ND ND ND
(100) (99.5)
BGS vs BIS vs HEK 301 267 318 ND ND ND
(94.7) (84.0) (100)
BGS vs BIS vs HEK vs 114 53 c 114 ND 100 117
RAM vs XLD (97.4) (45.3) (97.4) (85.5) (100)
BGS vs BIS vs EF-18 26 34 ND 36 ND ND
(72.2) (94.4) (100)
Range of % recovery 72.2- 45.3- 95.3- 100 79.8- 88.6-
100 100 100 99.2 100
a AS compared to t he media with t he highest number of positive plates. Plating media were streaked
from eit her the side-arm of the 1-2 Test, t he Salmosyst broth, Tet rat hionat e brilliant green broth,
Mueller-Kaufman Tet rat hionat e brot h, Selenite cystine brot h or GN brot h.
b The results for the EF-18 agar are from streaked plates, the results using the HGMFs will be
present ed elsewhere.
c One lab was unfamiliar with BIS, and had problems with false-positives and false-negatives (see text).
ND, not done.
D.W. Warburton et al. / International Journal of Food Microbiology 22 (1994) 277-289 283
foods were stressed due to exposure to alcohol, and being st ored in a low nut rient
environment for 60 days, respectively. Count s were obt ained by plat ing serial
dilutions of t he foods.
Quant it at ive recovery from brot h cult ures was st udied using the met hod used
for Li st eri a (Warburt on et al., 1992) and is summarized as follows. Aft er being
subcult ured twice in TPB, 24 h at 35C, t he Sal monel l a cult ures were t hen serially
dilut ed in 0.1% pept one wat er and 0.1 ml of t he 10 -6 to 10 -8 dilutions were
plat ed ont o BIS and t he ot her agars, which were incubat ed as above.
3 . R e s u l t s
Table 3 shows t he results of t he qualitative t est ing of the six media on the
different foods t est ed (Table 2). The % recovery was det ermi ned by comparing the
number of positive plat es recovered by each medi um to the highest number of
positive plat es possible in each comparison. The range of t he % recovery of
Sal monel l a varied bet ween plat ing media (Table 3). St reaked plat es of EF-18 agar
recovered t he great est number of isolates, showing 100% recovery. HEK ranked
second with 95. 3-100% recovery, and t he ot hers as follows: XLD with 88. 6-100%,
RAM with 79.8-99.2%, BGS with 72. 2-100% and BIS with 45. 3-100% recovery.
One laborat ory was unfamiliar with BIS, despit e t he fact t hat t hey t est ed for
Sal monel l a. [Ot her laborat ories not ed some problems (Table 5) with the different
media, but not to this extent]. Subsequent ly, this laborat ory had problems differen-
Table 4
Microorganisms giving false-positive reactions on the plating media a
Plating media
BGS BIS HEK EF-18 RAM XLD d
M
i
C
r
0
0
r
g
a
n
i
S
m
S
C. malonaticus Aeromonas spp. C. diversus C. freundii
C. freundii C. freundii C. freundii E. aerogenes
E. agglomerans E. agglomerans P. mirabilis E. agglomerans
E. taylorae E. eoli E. coli
K. oxytoca H. alvei E. fergusonii
Kluyvera spp. K. pneumoniae H. alveii
P. mirabilis P. mirabilis Providencia
P. morganii P. fluorescens
Pseudomonas spp.
E. taylorae
H. alvei
Proteus spp. b
Providencia c
Pseudomonas
spp.
C. freundii
a Microorganisms giving false-positives were isolated from this study, and were part of the qualitative
testing (Table 1).
b,c Clear colonies (typical of some Salmonella): b p. morganii and P. rettgeri; c p. alcalifaciens.
d Some false-positives that were red (no H2S) that might be picked included /~ fluorescens, S.
dysenteriae, A. xylosoxidans, E. hafnia and P. morganii.
284 D.W. Warburton et al. / I nt ernat i onal Journal of Food Microbiology 22 (1994) 277-289
Table 5
Summary of the questionaire rating the plating media
Media BIS BGS RAM XLD HEK EF- 18
Company Difco(8), Difco(10) Merck(7) Difco(4), Difco(2), QA Life
(# labs using Merck(2), Merck(2) Unipath(2), Sciences(6)
product) Unipat h(l) Merck(3)
Selectivity High(4), High(l), High(2), Medium(5), Medium(4), High(l),
Rating Medium(6) Medium(6), Med-high(2), Low(l) Low(3) Medium(3),
Low(4) Medium(3) Low(2)
High(2), High(4), High(2), High(3), High(2),
Medium(7), Medium(3) Medium(3), Medium(3), Medium(3),
Low(1 ) Low(1) LOw(1 ) Low( 1 )
Yes(9) High, Yes(3) Low to Yes(4) Low to Yes(5) Low to
No(I) high, high, high, No(5)
No(4) No(2) No(2)
High(2), High(4), High(l), High(3), High(l),
Medium(5), Med-high(1), Medium(3), Medium(2), Medium(4),
Low(3) Medium(2) Low(l) Low(2) Low(l)
Yes(4), Yes(7) Yes(3), Yes(4), Yes(2),
No(4), Maybe(l), No(3) Maybe(2),
Only with No(2) No(2)
others(I)
Differentia- High(3),
tion rating Medium(6),
Low(I)
Used before'? Yes(9) High,
No(l)
Overall High(3),
rating Medium(7)
Would you Yes(7),
use it Maybe(l),
routinely? No(l),
Only with
others(I)
Comments? a b c d e f
a Fussy preparation and storage, short expiry, detects atypical salmonella ( H2S- , Lac+), S. ty-
phimurium at times difficult to detect, isolations easy to make, deteriorates with age quickly, need to
reincubate plates, too many false-positives.
b Lactose fermenters a problem, had few Salmonella colonies compared to the others, many mimics, not
suitable for S. typhi, Pseudomonas a problem, not vary selective.
c Looks promising, still being evaluated, expensive, clear differentiation between non- and salmonellae,
atypical reactions for S. typhi and paratyphi, a good medium for a novice.
d Discontinued its use, salmonellae very characteristic.
Discontinued its use, isolations were made easier with this agar, least number of false-negatives,
extremely easy to differentiate between non- and salmonellae, false-positives a problem.
f Colour may change at 4C storage, repicking due to false-positives, extremely good differentiation
between non- and salmonellae, Pseudomonas a problem.
NB: not all participants completed the evaluation forms. Ratings above were assigned by the participat-
ing labs.
Table 6
Other comparisons of various plating media (from D' Aoust 1989)
Food Enrichment broths Ranking
Frogs legs TBG/ SC
Raw meats TBG
SC
Miscellaneous TBG/ SC
TBG/ SC
TBG/ SC
Meats and animal feeds TBG/ SC/ RV
Meats and dry products TBG/ SC
BIS > HEK = XLD
XLD > HEK
HEK > BGS > XLD
BIS > BGS
BIS > BGS
BIS > XLD > HEK
BIS = BGS
BIS = HEK = XLD
D.W. Warburton et al. /International Journal of Food Microbiology 22 (1994) 277-289 285
t iat ing bet ween Salmonella and ot her bact eria, so t hat a number of false-positives
and false-negat ives were not ed with this media, but not with ot hers. If t he lowest
recovery rat e for BIS (45.3%) is regarded as an anomaly (due t o t he problems t he
one lab had with this medi um) t hen t he % recovery range for BIS is 84. 0-100%.
During t he quant it at ive testing, t here was a < 1 log difference bet ween t he
count s on five media (BIS, BGS, EF-18, HEK and RAM) with 120 (out of 123)
salmonellae cult ures (dat a not shown). Test ing of an addit ional 46 Salmonella
cult ures on six media (t he above including XLD) result ed in 43 with a < 1 log
difference in count s. Re-t est ing of t he six cult ures (with differences in count s > 1
log) on t he six media result ed in a < 1 log difference (dat a not shown). Similarly,
plat ing t he 19 wat er and t he nine liquor samples cont aining st ressed bact eria
result ed in 25 having < 1 log difference on t hese five media (dat a not shown).
Therefore, t he quant it at ive recovery of Salmonella on t he six media appears
comparable.
The st ock cult ures (Table 1) and isolates found to give false-positive react ions
on t he various plat ing media are listed in Tabl e 4. All media had from t hree to
nine different non-Salmonella t hat gave false-positive react ions. Alt hough t he list
of t he false-positives is not exhaust ive, it does show t hat each medi um may have
problems with compet it ive microorganisms.
4. Di scussi on
Tabl e 5 gives a summary of t he rat ings given to t he various media by t he
part icipat ing laborat ories. This Tabl e includes ratings for selectivity, different iat ion
and overall performance with comment s showing t he pros and cons of each media.
BIS and RAM had t he best overall rat ings (medi um to high) while t he rat ings of
ot her media varied from low t o high. D' Aoust (1989) summarized t he performance
of various plat ing media, with t he pert i nent informat ion being shown in Table 6.
The i mport ance of BIS in t he isolat ion of Salmonella from various t est mat erials
under different enri chment condit ions is clearly evident from Tabl e 6 (D' Aoust ,
1989). However, based on t he overall informat ion provided by this Tabl e BIS,
BGS, HEK and XLD are fairly comparabl e in t heir ranking.
BGS, HEK and XLD are similar in t heir response t o carbohydrat e (lact ose and
sucrose) ut ilizat ion, have a medi um t o low selectivity, and can cont ain numerous
false-positives (D' Aoust , 1989; Tabl e 5). These agars, as well as RAM and EF-18,
do not readily ident ify atypical lact ose-posit ive (lac + ) and sucrose-posit ive (suc + )
salmonellae (Table 7). This fact support s t he use of BIS in st andard met hods due
to its nonsaccharide different ial system (D' Aoust , 1989). However, BIS is not
wit hout its problems (Tables 4 and 5) and is unable t o det ect some strains of
H2S- Salmonella ( HEK and XLD may have problems det ect ing H2S-
Salmonella as well). Problems with preparat i on, st orage and aging of BIS before
use have been not ed (D' Aoust , 1989). Aft er complet ion of this study, t he pot ent ial
use of Novobiocin-Brilliant Green-Gl ucose Agar (Devenish et al., 1986) to det ect
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D.W. Warburton et al. / International Journal of Food Microbiology 22 (1994) 277-289 287
lact ose-posit ive Salmonella was not ed. Furt her comparisons of this agar t o BIS
would be beneficial.
Despit e t hese problems, various combinat ions of BIS, BGS, HEK and XLD are
r e comme nde d for use by various st andard met hods organizat ions 1 in Canada and
t he Uni t ed St at es (Flowers et al., 1992): AOAC, APHA and FDA recommend BIS,
HEK and XLD; HPB recommends BIS and BGS; ICMSF and ISO recommend
BIS and brilliant green agar (BGA) with a t hird opt ional agar; NAS recommends
BIS and BGA; while t he USDA recommends BIS, BGS and XLD.
No st udies have been published comparing eit her EF-18 or RAM to ot her
st andard media in t heir ability to isolat e salmonellae from foods, however, t here
are a few papers t hat discuss t he isolat ion of salmonellae from stools and marine
wat ers on RAM. Rambach and co-workers (Laudat and Rambach, 1991; Prere et
al., 1992) have compared RAM t o HEK in t he isolation of Salmonella from stool
samples in several studies. They found t hat RAM had a sensitivity of 98% and a
specificity close to 100%, was able t o increase t he number of Salmonella isolates,
and had less false-positives. Dusch and Alt wegg (1993) compared RAM t o HEK
(and anot her media not used in this study) in t heir t est ing of 504 stool samples via
direct plat ing for Salmonella. The sensitivities and specificities were 69 and 98%
for RAM and 100 and 79% for HEK. They concluded t hat RAM should not be
used for primary plat ing of stool samples because of its lack of sensitivity.
However, t hey also concluded t hat RAM might be used for subcult uring of
enri chment brot h cult ures because of its high specificity (compared with HEK) and
t hat this could subst ant ially reduce t he work load in a laborat ory. During this
study, it was found t hat RAM was one of t he easiest media t o use in purifying
isolates and stock cult ures.
Alonso et al. (1992) found t hat , in general, RAM performed as well as HEK in
t he isolat ion of Salmonella while t est ing marine wat ers and t hat t here was no
significant difference in Salmonella recovery bet ween t he two media. RAM was
more efficient t han HEK when t here was low fecal cont aminat ion but, in t he
presence of high levels of compet ing bact eria, HEK showed great er selectivity.
Dusch and Alt wegg (1993) found RAM to be very specific, having obt ai ned only
10 false-positives from 504 stool samples. These included Escherichia coli, four
Citrobacter freundii isolates, t hree Citrobacter amalonaticus isolates, Klebsiella
oxytoca and E. agglomerans. Freydi ere and Gille (1991) found t hat Pseudomonas
and Acinetobacter gave false-positives react ions on RAM. Many of t hese non-
Salmonella give false-positive react ions on t he ot her media as well (Table 4).
Flowers et al. (1992) st at e t hat none of t he generally used agars (BIS, BGS,
HEK, XLD and ot hers) are ideal for all situations, justifying t he recommendat i on
in many reference met hods for t he use of two or more agar media. Based upon our
1 AOAC, Association of Official Analytical Chemists; APHA, American Public Healt h Association;
FDA, US Food and Drug Administ rat ion; HPB, Healt h Protection Branch; ICMSF, Int ernat ional
Commission on Microbiological Specifications for Food; ISO, Int ernat ional Organization for St andard-
ization; NAS, Nat ional Academy of Sciences (US); USDA, US Depart ment of Agriculture).
288 D.W. Warburton et al. /International Journal of Food Microbiology 22 (1994) 277-289
r e s ul t s a n d t h os e f r om ot h e r s t udi e s , a n d t h e f ol l owi n g f a ct s: (1) BI S is c ur r e n t l y a
me di um of c h oi c e i n Ca n a di a n s t a n da r d me t h ods due t o i t s n on - s a c c h a r i de
di f f e r e n t i a l s ys t e m; a n d (2) n e wl y de v e l ope d or t e s t e d me di a mus t be e x t e n s i v e l y
f i e l d t e s t e d be f or e i n c or por a t i on i n t o Ca n a di a n s t a n da r d me t h ods us e d i n c ompl i -
a n c e a ct i vi t y, our r e c omme n da t i on f or n e wl y de v e l ope d l a bor a t or y pr oc e dur e s is
t h a t us e of BI S be c ompul s or y a n d a t l e a s t on e ot h e r a ga r ( BGS, HEK, XLD,
RAM a n d EF- 18) be us e d as we l l .
Acknowl edgement s
A t h a n k you is e x t e n de d t o al l s t a f f wh o a i de d i n t h e pr e pa r a t i on of me di a a n d
t h e s ubc ul t ur i n g of s a mpl e s t o a ddi t i on a l t e s t me di a .
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EF-18 agar/Hydrophobic Grid Membrane Filter (HGMF) method and the enzyme linked antibody
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Warburton, D.W., Bowen, B., Durzi, S., Lamontagne, G., et al. (1994e) A comparison study of the
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