A Comparison of Six Different Plating Media Used

8/10/2019 A Comparison of Six Different Plating Media Used

1/13

ELSEVIER

Intern ational 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 o o d M i c r o b i o lo g y

c o m p a r i s o n o f s i x d i f f e r e n t p l a t i n g m e d i a u s e d

i n t h e i s o l a t i on o f a lmone l l a

D o n a l d W . W a r b u r to 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 rr 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

Y v o n T r o t t ie r v, B r e n d a D a l y W h e e l e r

q

a Evaluation Division, Bureau o f 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, B2 Y3Z7 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)

b s t r a c t

Seventeen Canadian Federal, Provincial and Public Health Laboratories took part in

different phases of a comparative/collaborative study that evaluated rapid methods to the

* Corresponding author.

0168-1605/94/$07.00 1994 Elsevier Science B.V. All rights reserved

SSDI 0168-1605(94)00017-Z


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278 D.W. Warburton et al. / International Journal of Food Microbiology 22 1994) 277-289

s t anda rd Hea l t h P ro t ec t i on Branch (HPB) me thod fo r t he de t ec t i on o f Salmonella. A

va r i e ty o f com merc i a l m ed i a we re t e s t ed , i nc lud ing Br i ll ian t G reen Su lpha A ga r , B i smu th

Su lph i t e Aga r , Hek toen En t e r i c Aga r , Xy lose Lys ine Deoxycho l a t e Aga r , EF-18 Aga r and

Rambach Aga r . Each l abo ra to ry compared up t o s i x o f t he se d i f f e r en t p l a t i ng med ia .

P la t ing of 123 sa lmonel lae cu l tures and 28 a r t i f ic ia l ly- inocula ted foods showed the recovery

o f Salmonella spp . on the s ix p la t ing media to be wi th in one log . Therefore , quant i ta t ive

t e s ti ng o f t he m ed ia showed t hem to be com parab l e i n t he r ecove ry o f s a lmone l l ae .

Qua l i t a t ive t e s t i ng o f t he s ix med ia d u r ing t he com para t i ve / co l l abo ra t i ve s t udy o f va r i ous

me th ods showed t ha t E F-18 Aga r r ecove red t he g r ea t e s t numb er o f i sol a te s . He k toen

En te r i c A ga r r anked s econd , w ith t he o the r aga r s be ing com parab l e i n the i r r e cove ry o f

Salmonella

spp . P rob l ems wi th the va r i ous med ia a r e summ ar i zed . Based on ou r r e su lt s and

those o f o the r r e sea r ch e r s , i t i s r e com m end ed t ha t B i smu th Su lph i te A ga r be compu l so ry

and t ha t a t l e a s t one o the r aga r be u sed fo r newly deve loped cu l tu r a l p rocedu re s .

Key words: Co m para t i ve / co l l abo ra t i ve st udy; P l a ti ng med ia ; I so l a ti on p rocedu re ; Salmonella

I I n t r o d u c t i o n

T h e u s e o f s el e c ti v e a n d d i f f e r e n ti a l p l a t i n g m e d i a i s a v e r y i m p o r t a n t p a r t o f

s t a n d a r d c u l t u r a l m e t h o d s f o r t h e i s o l a ti o n o f Salmonella f r o m f o o d s a n d e n v i ro n -

m e n t a l s a m p l e s . A w i d e v a r i e ty o f m e d i a h a v e b e e n d e v e l o p e d , d i f f e r in g in th e i r

c o m p o s i t i o n , u s e a n d p e r f o r m a n c e c h a r a c t e r i s t i c s .

B r i ll ia n t G r e e n S u l p h a A g a r ( B G S ; H P B , 1 9 78 ), B i s m u t h S u l p h i t e A g a r (B I S ;

A n d r e w s e t al ., 1 98 4; H P B , 1 97 8), H e k t o e n E n t e r i c A g a r ( H E K ; A n d r e w s e t al .,

1 98 4; A n o n . , 1 99 3) a n d X y l o s e L y s i n e D e s o x y c h o l a t e A g a r ( X L D ; A n d r e w s e t a l.,

1 98 4 ) a r e u s e d i n s t a n d a r d m e t h o d s o f d i ff e r e n t a g e n c i e s in C a n a d a , t h e U n i t e d

S t a t e s a n d i n E u r o p e ( F l o w e r s e t a l. , 1 9 92 ). B G S u s e s s o d i u m s u l f a p y r i d i n e a s t h e

s e l ec t iv e a g e n t a n d b r i l li a n t g r e e n a n d p h e n o l r e d d y e s as th e i n d i c a to r s o f

c a r b o h y d r a t e u t i li z a ti o n ( A n o n . , 1 98 4). B I S u s e s b i s m u t h s u lf it e a n d b r i l li a n t g r e e n

a s i n h i b i t o r y a g e n t s a g a i n s t c o m p e t i n g m i c r o r g a n i s m s a n d f e r r o u s s u l f a t e a s t h e

i n d i c a t o r o f H 2 S p r o d u c t i o n ( D i f c o , 1 9 84 ). H E K r e li e s o n t h e u s e o f b il e s al ts f o r

s e le c ti v it y , b r o m o t h y m o l b l u e a n d a c i d fu s c h i n a s in d i c a t o r s o f c a r b o h y d r a t e

u t il iz a t io n , a n d f e r r ic i r o n a s a n i n d i c a t o r o f f o r m a t i o n o f H 2 S f r o m t h i o s u l p h a t e

( A n o n . , 1 9 9 3 b ) . X L D u s e s s o d i u m d e s o x y c h o l a t e , s o d i u m t h i o s u l f a t e , f e r r i c a m m o -

n i u m c i t ra t e a n d p h e n o l r e d a s s e l e c ti v e a g e n t s , in d i c a t o r s o f H 2 S p r o d u c t i o n a n d

c a r b o h y d r a t e u t i li z a t io n , r e s p e c t i v e l y ( A n o n . , 1 9 8 4 )

E F - 1 8 A g a r ( E F - 1 8 ; D i c k s o n a n d A n d e r s o n , 1 99 1; E n t is , 1 99 0 ; E n t i s a n d

B o l e s z c z u k , 1 99 1; T o d d e t al. , 19 92 ) a n d R a m b a c h A g a r ( R A M ; A n o n . , 1 99 3,

B a n k e s , 1 9 9 2 ; F e n g , 1 9 9 2 ; F r e y d i e r e a n d G i l l e , 1 9 9 1 ; G r u e n e w a l d e t a l . , 1 9 9 1 ;

M a n a f i a n d S o m m e r , 1 9 92 ; R a m b a c h , 1 9 90 ) a r e ne w l y d e v e l o p e d m e d i a a s s o c i a t e d

w i t h t h e r e c e n t l y d e v e l o p e d m e t h o d s ( c i t e d a b o v e ) . E F - 1 8 u s e s s u l fa p y r i d in e , b i le

s a lt s, c r y s ta l v i o le t , n o v o b i o c i n a n d i n c u b a t i o n a t 4 2 C a s d i f f e r e n t i a l / s e l e c t i v e

a g e n t s , w i t h s u c r o s e a n d l y s in e d e c a r b o x y l a s e u t i l i z a t io n a s i ts d i f f e r e n t i a l r e a c t i o n s

( E n t is , 1 99 0) . R A M u s e s t h e s e l e c ti v e a g e n t d e s o x y c h o l a t e , t h e f o r m a t i o n o f a c id

f r o m p r o p y l e n e g ly c o l b y Salmonella a n d a c h r o m o g e n i c i n d i c a to r o f / 3 - g a l a c t o s i -


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D.W. Wa rburton e t a l. / In ternat ion al Journa l o f Food Microbiology 22 1994) 27 7-2 89

279

d a s e t o d i f f e r e n t i a t e Sa lmonel la f r o m Pro teus a n d o t h e r E n t e r o b a c t e r i a c e a e

( A n o n . , 1 9 9 3 a ) .

T h e s e d i f f e r e n t c o m m e r c i a l l y a v a il a bl e p l a ti n g m e d i a w e r e e v a l u a t e d a s p a r t o f

a c o m p a r a t i v e a n d c o l l a b o r a ti v e s tu d y c o m p a r i n g r a p i d m e t h o d s f o r t h e d e t e c t i o n

o f Sa lmonel la t o t h e s t a n d a r d H e a l t h P r o t e c t io n B r a n c h ( H P B ) m e t h o d ( H P B ,

1 9 7 8) i n v o lv i n g C a n a d i a n L a b o r a t o r i e s .

2 M a t e r i a l s a n d m e t h o d s

S e v e n t e e n C a n a d i a n F e d e r a l , P r o v i n ci a l a n d P u b l i c H e a l t h L a b o r a t o r i e s t o o k

p a r t i n d if f e r e n t p h a s e s o f a c o m p a r a t i v e / c o l l a b o r a t i v e s t u d y th a t e v a l u a t e d r a p i d

m e t h o d s t o th e s t a n d a r d H P B m e t h o d f o r t h e d e t e c t i o n o f Sa lmonel la . D u r i n g t h e

d i f f e r e n t p a r t s o f th i s s t u d y a v a r i e t y o f c o m m e r c i a l l y a v a il a b le m e d i a w e r e t e s t e d ,

i n c lu d i n g B G S ( D i f c o L a b o r a t o r i e s , D e t r o i t , M I ) , B I S ( M e r c k , D a r m s t e d t , G e r -

m a n y ; D i f c o L a b o r a t o r i e s ) , H E K ( M e r c k ; D i f c o L a b o r a t o r i e s ; U n i p a t h I n c. , N e -

p e a n , O n t . ), X L D ( M e r c k ; D i f c o L a b o r a t o r i e s ), E F - 1 8 ( Q A L i f e S c i e n c e s I n c. , S a n

D i e g o , C A ) a n d R A M ( M e r c k ) . T h e f ir st f o u r m e d i a a r e a s s o c i a te d w i th s t a n d a r d

m e t h o d s ( F l o w e r s e t al. , 1 99 2 ) w h i l e t h e l a st tw o a r e a s s o c i a t e d w i t h m e t h o d s b e i n g

t e s t e d i n t h is s t u d y . E a c h l a b o r a t o r y c o m p a r e d u p t o s ix o f t h e s e d i f f e r e n t p l a t in g

m e d i a .

2.1 . Microorganisms

O n e h u n d r e d a n d s e v e n t e e n

Sa lmonel la

a n d 4 9

n o n S a l m o n e l la

c u l t u r e s w e r e

u s e d ( T a b l e 1 ). T h e s e c u l t u r e s w e r e u s e d t o a r ti f ic i a ll y c o n t a m i n a t e s a m p l e s , a n d

a s p o s i t i v e o r n e g a t i v e c o n t r o l s f o r t e s t i n g b o t h m e d i a a n d m e t h o d s .

2.2 . Samples tes ted

S e v e n h u n d r e d a n d t h i r t y - fi v e n a t u r a l l y - c o n t a m i n a t e d a n d 5 5 1 a rt if i c ia l l y -c o n -

t a m i n a t e d f o o d a n d e n v i r o n m e n t a l s a m p l e s w e r e t e s t e d i n th is s t u d y ( T a b l e 2 ). In

a ll c a s es th e f o o d s w e r e a n a l y s e d b y t h e H P B m e t h o d a n d o n e o t h e r m e t h o d .

O f t e n , s e v e r a l m e t h o d s w e r e u s e d s i m u l ta n e o u s ly . I n o t h e r c a s es , s a m p l e s f o u n d

p o s it iv e b y o n e m e t h o d , w e r e o f t e n r e - t e s t e d b y o t h e r m e t h o d s s o t h a t 1 62 2

n a t u r a l l y - c o n t a m i n a t e d a n d 1 27 1 a r t i f i c ia l l y - c o n t a m i n a t e d s a m p l e s w e r e t e s t e d b y

t h e s i x m e t h o d s .

2 .3 . Ar t i f ic ia l con tamina t ion o f t he f oo ds

T h e m e t h o d u s e d w a s t h e s a m e a s t h a t f o l lo w e d f o r an e a r l i e r s tu d y w it h

Listeria ( W a r b u r t o n e t a l . , 1 9 9 2 ) a n d i s s u m m a r i z e d b e l o w : T h e Sa lmonel la

c u l t u r e s w e r e g r o w n i n T r y p t o s e P h o s p h a t e B r o t h ( T P B ) f o r 2 4 h a t 3 5 C . A f t e r a

s e c o n d s u b c u l t u r e i n T P B ( 2 4 h a t 3 5 C ), th e c u l t u r e s w e r e s e r i a ll y d i l u t e d i n 0 . 1 %

p e p t o n e w a t e r a n d 0 .5 m l o f t h e 1 0 - s d i l u t i o n w a s i n o c u l a t e d i n t o 2 5 g o f f o o d (s o


4/13

~.80 D . W . W a r b u r t o n e t al. / I n t e r n a t io n a l J o u r n a l o f F o o d M i c ro b i o lo g y 2 2 ( 1 99 4 ) 2 7 7 - 2 8 9

Fab le 1

Li s t o f cu l tu res used (numbers o f i so la t es )

~ a l m o n e l la e u s e d

~ a l mo n e l la e ( 1 3 ) A g r i c u l t u r e Ca n a d a a n d H PB

re typed i so la t es ,

~a lmo nel l ae Grp B(4) ch icken f lu ff , ch icken ,

:u rkey and pork i so la tes ,

~a lmonel l ae Grp C(2) ch icken i so la t es ,

~a lmonel l ae rough(2) ,

~ . a l a c h u a

~ . a l b a n y

~ . a g o n a ,

~ . a n a t u m ,

~ . a r i z o n a e ( 4 ) ,

~ . a r i z o n a e 1 ,

~ . a r i z o n a e II(2) ,

g . a r k a n s a s ,

g . b a r e i l l y (2 ) ,

5 . b r e d e n y ,

5 . b r a e n d e r u p ,

5 . c e r r o (3 ) ,

5 . ches ter ,

5 . c h o le r a - s u i s ,

5 . d u b l i n ( 2 ) ,

S . e a l in g (2 ) ,

S . e a s t b o u r n e ( 2 ) ,

S . e n t e r i t i d i s ( 2 ) 1 H 2 S - ,

S . f l i n t ( 2 ) ,

S . g a l l i n a r u m ,

S. git,e,

S . g o d e s b u r g ,

S . h a a r d t ,

S . h a d a r ( 6 ) ,

S . h a l~ a n a (2 ) ,

S . h e id e lb e r g ,

S . i n d i a n a H 2 S - S u c +

S . i n f a n t 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 L a c + ,

S . l a n d o w ,

~ . m b a n d a k a ( 5 ) ,

S . m o n t e t ; i d e o ,

S . m i n n e s o t a ( 2 ) ,

S . m u e n s t e r ,

S . n e w i n g t o n ,

S . n e w p o r t ,

S . oh io ,

S . o r a n i e n b u r g ,

S . p in z a ,

S . p u l l o r u m ,

S . r e a d i n g ,

S . s c h w a r z e n g r u n d ( 4 )

S . s e n f t e n b e r g ( 6 ) 1 H 2 S - L y s - , 1 7 75 W , 1 S u c +

S . s t . - p a u l ( 2 ) ,

S . t a k s o n y ( 2 ) ,

S . t h o m a s L , i l l e (2 ) ,

S . t h o m p s o n ( 5 ) ,

S . typh i ,

S . t y p h i m u r i u m ( 4 ) , 1 AT CC 14028 ,

S . u r b a n a ,

S . w a s s e n a a r G r p I I ,

S . w e l t e u r e d e n ,

S . w o r t h i n g t o n ( 2 ) ,

ther spec i e s

A c h r o m o b a c t e r x y l o s o x i d a n s ,

C i t r o b a c t e r d i t ,e r s u s,

C . f re u n d i i ( 3 ) 1 H z S + ,

E n t e r o b a c t e r a e r o g e n e s ( 3 ) ,

E . a g g l o m e r a n s (5),

E . c l o a c a e ( 2 ) ,

E . g e o r g e n e s ,

E . h a f n i a ,

E . t a y lo r a e ,

E n t e r o c o c c u s f a e c al ,

E r w i n i a c a r o to L , o r a (2 ) ,

E s c h e r i c h i a c o l i ( 5 ) ,

H a f n i a a h , e i( 3 ) ,

K l e b s i e l la o x y t o c a ,

K . p n e u m o n i a e ( 2 ) ,

O e r s k o t~ ia sp.,

P r o t e u s sp.,

P . m i r a b i l i s ( 3 ) ,

P . m o r g a n i i ( 2 ) ,

P . r e t tg e r i (2 ) ,

P . t , u lg a r i s (2 ) ,

P r o L , id e n c ia spp. ,

P r o v i d e n c i a a l c a l i f a c i e n s ,

P s e u d o m o n a s a e r u g i n o s a ,

P . f l u o r e s c e n s ,

S e r r a t i a f o n t i c o l a ,

S . m a r c e s c e n s ( 2 ) ,

S h i g e l l a b o y d i i ,

S . d y s e n te r ia e ,

S . f l e x n e r i ,

S . s o n n e i ,

S t a p h y l o c o c c u s a u r e u s ,


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D.W . W arburton et al. / International Journal of Food M icrobiology 22 1994) 277-289 281

that the inoculation was < 1 cfu/g) and blended (or mixed using a stomacher) with

the preenrichment broth. To ensure a high competitor level, either 1 ml of a

n o n - S a l m o n e l l a culture or 2 g of soil (previously tested and found negative for

S a l m o n e l l a ) was added to the food mixture. Incubation and analysis continued as

appropriate for the method studied (described below).

To det ermi ne the inocu lum level, 0.1 ml of the 10 -6 to 10 -8 dilutions were

plated onto BIS and the other agars. The agars were incubated at 35C for 18-48

h, except for EF-18 which was incubated at 42C. At 24 h, plates were examined

and confirmation started on typical colonies. As needed, BIS and other agars were

re-incubated overnight, and visible colonies counted.

2 .4 . M e t h o d s t e s t e d q u a l i t a t i v e t e s ti n g )

During the comparative/collaborative testing six methods were used. Five

methods (the modified 1-2 Test (Feng, 1992; Nath et al., 1989; Oggel et al., 1990;

BioControl Systems Inc., Bothell WA), EF-18 and HGMF method (Entis, 1990;

Entis and Boleszczuk, 1991; Feng, 1992; Gelman Sciences Inc.), the ELA method

(Todd et al., 1993), Gene-Trak (Feng, 1992; BDH Inc.), the Merck Salmosyst broth

and tablet me thod (Ossmer, 1992; BDH Inc.)) were comp ared to the H PB

standard m etho d MFHP B-20 (HPB, 1978). The results of the com pa ris on /

collaborative testing of the methods will be published elsewhere (Warburton et al.,

1994b,c,d,e). Each laboratory could streak out the selective broths, from the HPB

and other methods tested, onto at least BIS and BGS, plus the media stipulated

for that method and any other agars they wished to test. A representative 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.


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D.W.. Warburton et al. / International Journal of Food Microbiology 22 1994) 277-289

p u r i f i e d f o r c o n f i rm a t i o n . B i o c h e m i c a l t e st in g a n d s e r o lo g y w e r e d o n e a c c o r d i n g to

t h e H P B m e t h o d ( H P B , 1 97 8). O t h e r c o n f i r m a t i o n te s ts i n c lu d e d t h e u s e o f

A P I - 2 0 E ( A P I L a b o r a t o r y P r o d u c ts L t d , S t -L a u r e n t , Q u e . ), M i c ro - I D ( O r g a n o n

T e k n i k a I n c . , S c a r b o r o u g h , O n t . ) , E n t e r o t u b e ( R o c h e D i a g n o s t i c S y s t e m s , N u t l e y ,

N J ), a s w e ll a s t h e m i n i- V i d a s a n d t h e V i te k ( b i o M e r i e u x U S A , H a z e l w o o d M I ) .

T h e a b il it y o f s t a n d a r d

non Salmonel la

t o g r o w o n t h e p l a t i n g m e d i a w a s t e s t e d

a s f o ll o w s . T h e c u l t u r e s , l i st e d in T a b l e 1 a s o t h e r s p e c i e s , w e r e s u b c u l t u r e d a n d

s t r e a k e d o n t o t h e p l a t i n g m e d i a w h i c h w e r e i n c u b a t e d a s d e s c r i b e d a b o v e .

2.5 . Quanti ta t ive tes t ing of the media

O n e h u n d r e d a n d t w e n t y - t h r e e s a l m o n e l l a e c u l t u r e s a n d 2 8 i n o c u l a t e d f o o d

s a m p l e s w e r e p l a t e d o n B I S , B G S , E F - 1 8 , H E K a n d R A M . A n a d d i ti o n a l 4 6

s a l m o n e l l a e w e r e p l a t e d o n t h e s e a g a r s a n d X L D . T h e f o o d s c o n s i st e d o f n i n e

i n o c u l a t e d h o m e m a d e c h o c o l a t e a n d e g g li q u e u r s a m p l e s, a s w e l l a s 1 9 i n o c u l a t e d

d i s t i l l e d w a t e r s a m p l e s ( W a r b u r t o n e t a l . , 1 9 9 3 , 1 9 9 4 a , b ) . T h e s a l m o n e l l a e i n t h e s e

Table 3

Recovery of Salmonellae by different selective plating media

Plating medium tested

Num ber of positive plates (% recovery) a

BGS BIS HEK EF-18 b RAM XLD

BGS vs BIS vs HE K vs 139 136 142 149 130 132

EF-18 vs RA M vs XL D (93.3 a) (91.3) (95.3) (100) (87.3) (88.6)

BGS vs BIS vs HE K vs 596 605 613 624 602 ND

EF-18 vs RA M (95.5) (97.0) (98.2) (100) (96.5)

BGS vs BIS vs HE K vs 102 119 116 ND 95 ND

RA M (85.7) (100) (97.5) (79.8)

BGS vs BIS vs RA M 238 251 ND ND 249 ND

(94.8) (100) (99.2)

BGS vs BIS vs EF-18 vs 91 92 ND 96 ND 96

XL D (94.8) (95.8) (100) (100)

BGS vs BIS 1125 1119 ND ND ND ND

(100) (99.5)

BGS vs BIS vs HE K 301 267 318 ND ND ND

(94.7) (84.0) (100)

BGS vs BIS vs HE K vs 114 53 c 114 ND 100 117

RA M vs XL D (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 1 1 99.2 1

a AS compared to the media with the highest number of positive plates. Plating media were streaked

from either the side-arm of the 1-2 Test, the Salmosyst broth, Tetrathionate brilliant green broth,

Mueller-Kaufman Tetrathionate broth, Selenite cystine broth or GN broth.

b The results for the EF-18 a gar are fro m streaked plates, the results using the HGM Fs will be

presented elsewhere.

c One lab was unfamiliar with BIS, and had proble ms with false-positives and false-negatives (see text).

ND, not done.


7/13

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 stored in a low nutrient

environment for 60 days, respectively. Counts were obtained by plating serial

dilutions of the foods.

Quantitative recovery from broth cultures was studied using the method used

for

Lis ter ia

(Warburton et al., 1992) and is summarized as follows. After being

subcultured twice in TPB, 24 h at 35C, the

Salmone l la

cultures were then serially

diluted in 0.1% pept one water and 0.1 ml o f the 10 -6 to 10 -8 dilutions were

plated onto BIS and the other agars, which were incubated as above.

3 R e s u l t s

Table 3 shows the results of the qualitative testing of the six media on the

different foods tested (Table 2). The % recovery was determine d by comparin g the

number of positive plates recovered by each medium to the highest number of

positive plates possible in each comparison. The range of the % recovery of

Salmone l la

varied between plating media (Table 3). Streaked plates of EF-18 agar

recovered the greatest number of isolates, showing 100% recovery. HEK ranked

second with 95.3-100% recovery, and the others 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 laboratory was unfamiliar with BIS, despite the fact that they tested for

Salmone l la .

[Other laboratories noted some problems (Table 5) with the different

media, but not to this extent]. Subsequently, this laboratory 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.


8/13

284 D . W . W arbur ton e t a l. / I n t e rna t ion a l Journa l o f Food M ic rob io logy 22 1994 ) 27 7 - 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) Unipath( 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 (H 2S - , Lac+), S. ty-

p h i m u r i u m 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 Salmone l la 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 para ty ph i , 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 dif ferentiate between non- and salmonellae, false-positives a problem.

f Colour may change at 4C storage, repicking due to false-positives, extremely good different iation

between non- and salmonellae, P s e u d o m o n a s 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/S C

Raw meats TBG

SC

Miscellaneous TBG/SC

TBG/SC

TBG/SC

Meats and animal feeds TB G/ SC /R V

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


9/13

D.W. Warburton et al. /In ternational Journal of Food Microbiology 22 1994) 277-289 285

t ia t in g b e t w e e n

Salmonella

a n d o t h e r b a c t e r i a , s o t h a t a n u m b e r o f f a l se - p o s i ti v e s

a n d f a l s e - n e g a t i v e s w e r e n o t e d w i t h th i s m e d i a , b u t n o t w i t h o t h e r s . I f t h e l o w e s t

r e c o v e r y r a t e f o r B I S (4 5 . 3 % ) is r e g a r d e d a s a n a n o m a l y (d u e t o t h e p r o b l e m s t h e

o n e l a b h a d w i t h t hi s m e d i u m ) t h e n t h e % r e c o v e r y r a n g e f o r B IS is 8 4 . 0 - 1 0 0 % .

D u r i n g t h e q u a n t i t a t i v e t e s ti n g , t h e r e w a s a < 1 l o g d i f f e r e n c e b e t w e e n t h e

c o u n t s o n f iv e m e d i a ( B I S , B G S , E F - 1 8 , H E K a n d R A M ) w i th 1 20 ( o u t o f 12 3)

s a l m o n e l l a e c u l t u r e s ( d a t a n o t s h o w n ) . T e s t i n g o f a n a d d i t i o n a l 4 6 Salmonella

c u l t u r e s o n s ix m e d i a ( t h e a b o v e i n c l u d i n g X L D ) r e s u l t e d in 4 3 w i t h a < 1 l o g

d i f f e r e n c e i n c o u n t s . R e - t e s t i n g o f t h e s ix c u l t u r e s ( w i t h d i f f e r e n c e s i n c o u n t s > 1

l o g ) o n t h e s ix m e d i a r e s u l t e d i n a < 1 l o g d i f f e r e n c e ( d a t a n o t s h o w n ) . S i m i l a rl y ,

p l a t i n g t h e 1 9 w a t e r a n d t h e n i n e l i q u o r s a m p l e s c o n t a i n i n g s t r e s s e d b a c t e r i a

r e s u l t e d i n 25 h a v i n g < 1 l o g d i f f e r e n c e o n t h e s e f iv e m e d i a ( d a t a n o t s h o w n ) .

T h e r e f o r e , t h e q u a n t i t a ti v e r e c o v e r y o f Salmonella o n t h e s ix m e d i a a p p e a r s

c o m p a r a b l e .

T h e s t o c k c u l t u r e s ( T a b l e 1 ) a n d i s o la t e s f o u n d t o g iv e f a l s e- p o s i ti v e r e a c t i o n s

o n t h e v a r i o u s p l a t i n g m e d i a a r e l i s t e d i n T a b l e 4 . A l l m e d i a h a d f r o m t h r e e t o

n i n e d i f f e r e n t non Salmonella t h a t g a v e f a ls e - p o s i ti v e r e a c t io n s . A l t h o u g h t h e l is t

o f th e f a l s e - p o s i ti v e s is n o t e x h a u s t i v e , it d o e s s h o w t h a t e a c h m e d i u m m a y h a v e

p r o b l e m s w i th c o m p e t i t i v e m i c r o o rg a n i s m s .

4 D i s c u s s i o n

T a b l e 5 g i ve s a s u m m a r y o f t h e r a t i n g s g i v e n t o t h e v a r i o u s m e d i a b y th e

p a r t i c i p a t i n g l a b o r a t o r i e s . T h i s T a b l e i n c l u d e s r a t in g s f o r s e le c t iv i ty , d i f f e r e n t i a t i o n

a n d o v e r a l l p e r f o r m a n c e w i t h c o m m e n t s s h o w i n g t h e p r o s a n d c o n s o f e a c h m e d i a .

B I S a n d R A M h a d t h e b e s t o v e ra l l r a ti n g s ( m e d i u m t o h ig h ) w h i l e t h e r a ti n g s o f

o t h e r m e d i a v a r ie d f r o m l ow to h ig h . D ' A o u s t ( 19 8 9 ) s u m m a r i z e d t h e p e r f o r m a n c e

o f v a r io u s p l a t in g m e d i a , w i t h t h e p e r t i n e n t i n f o r m a t i o n b e i n g s h o w n in T a b l e 6 .

T h e i m p o r t a n c e o f B I S in t h e i s o la t io n o f Salmonella f r o m v a r i o u s t e s t m a t e r i a l s

u n d e r d i f f e r e n t e n r i c h m e n t c o n d i t io n s is c l e a rl y e v i d e n t f ro m T a b l e 6 ( D ' A o u s t ,

1 98 9). H o w e v e r , b a s e d o n t h e o v e r a ll i n f o r m a t i o n p r o v i d e d b y th is T a b l e B I S ,

B G S , H E K a n d X L D a r e fa ir ly c o m p a r a b l e i n t h e i r r a n k in g .

B G S , H E K a n d X L D a r e s i m i l a r i n t h e i r r e s p o n s e t o c a r b o h y d r a t e ( l a c t o s e a n d

s u c r o s e ) u t il i z a ti o n , h a v e a m e d i u m t o lo w se l e c ti v it y , a n d c a n c o n t a i n n u m e r o u s

f a l s e- p o s i ti v e s ( D ' A o u s t , 1 9 89 ; T a b l e 5 ) . T h e s e a g a r s, a s w e l l a s R A M a n d E F - 1 8 ,

d o n o t r e a d i l y i d e n t i f y a t y p i c a l l a c t o s e - p o s i t i v e ( l a c + ) a n d s u c r o s e - p o s i t i v e ( su c + )

s a l m o n e l l a e ( T a b l e 7 ). T h i s f a ct s u p p o r t s t h e u s e o f B I S i n s ta n d a r d m e t h o d s d u e

t o it s n o n s a c c h a r i d e d i f f e r e n t i a l s y s t e m ( D ' A o u s t , 1 9 89 ). H o w e v e r , B I S is n o t

w i t h o u t i t s p r o b l e m s ( T a b l e s 4 a n d 5 ) a n d is u n a b l e t o d e t e c t s o m e s t r a in s o f

H 2 S - Salmonella ( H E K a n d X L D m a y ha ve p ro b l em s d e t e c t in g H 2 S -

Salmonella a s w e ll ). P r o b l e m s w i t h p r e p a r a t i o n , s t o r a g e a n d a g i ng o f B I S b e f o r e

u s e h a v e b e e n n o t e d ( D ' A o u s t , 1 98 9). A f t e r c o m p l e t i o n o f th i s s tu d y , th e p o t e n t i a l

u s e o f N o v o b i o c i n - B r il l ia n t G r e e n - G l u c o s e A g a r ( D e v e n i s h e t a l . , 1 98 6 ) t o d e t e c t


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11/13

D.W. Wa rburton e t a l . / In tern at ional Journ al o f Food Microbio logy 22 1994) 27 7-2 89 287

l a c t o s e - p o s i t i v e Salmonella w a s n o t e d . F u r t h e r c o m p a r i s o n s o f th i s a g a r t o B I S

w o u l d b e b e n e f i c i a l .

D e s p i t e t h e s e p ro b l e m s , v a r io u s c o m b i n a ti o n s o f B I S , B G S , H E K a n d X L D a r e

r e c o m m e n d e d f o r u s e b y v a r i o us s t a n d a r d m e t h o d s o r g a n i z a ti o n s 1 in C a n a d a a n d

t h e U n i t e d S t a te s ( F lo w e r s et al . , 19 92 ): A O A C , A P H A a n d F D A r e c o m m e n d B I S ,

H E K a n d X L D ; H P B r e c o m m e n d s B IS a nd B G S ; I C M S F a n d I S O r e c o m m e n d

B I S a n d b r i ll ia n t g r e e n a g a r ( B G A ) w i t h a t h i rd o p t i o n a l a g a r; N A S r e c o m m e n d s

B I S a n d B G A ; w h i l e t h e U S D A r e c o m m e n d s B I S , B G S a n d X L D .

N o s tu d ie s h a v e b e e n p u b l is h e d c o m p a r i n g e it h e r E F - 1 8 o r R A M t o o t h e r

s t a n d a r d m e d i a i n t h e i r a b i l i t y t o i s o l a t e s a l m o n e l l a e f r o m f o o d s , h o w e v e r , t h e r e

a r e a fe w p a p e r s t h a t d i s c u ss t h e i s o l a t i o n o f s a l m o n e l l a e f r o m s t o o ls a n d m a r i n e

w a t e r s o n R A M . R a m b a c h a n d c o - w o r k er s ( L a u d a t a n d R a m b a c h , 1 99 1; P r e r e e t

a l. , 19 9 2) h a v e c o m p a r e d R A M t o H E K in th e i s o l at io n o f Salmonella f r o m s t o o l

s a m p l e s i n s e v e ra l s tu d i e s . T h e y f o u n d t h a t R A M h a d a s en s it iv i ty o f 9 8 % a n d a

s p e c i fi c i ty c l o s e t o 1 0 0 % , w a s a b l e t o i n c r e a s e t h e n u m b e r o f

Salmonella

i s o l a t e s ,

a n d h a d l es s f a ls e -p o s it iv e s . D u s c h a n d A l t w e g g ( 1 99 3 ) c o m p a r e d R A M t o H E K

( a n d a n o t h e r m e d i a n o t u s e d i n th i s s tu d y ) i n t h e i r t e s t in g o f 5 04 s t o o l s a m p l e s v i a

d i r e c t p l a t i n g f o r Salmonella. T h e s e n s it iv i ti e s a n d s p e c i f ic i t ie s w e r e 6 9 a n d 9 8 %

f o r R A M a n d 1 0 0 a n d 7 9 % f o r H E K . T h e y c o n c lu d e d t h a t R A M s h o u ld n o t b e

u s e d f o r p r i m a r y p l a t i n g o f s t o o l s a m p l e s b e c a u s e o f i ts l a ck o f s e n s it iv i ty .

H o w e v e r , th e y al so c o n c l u d e d t h a t R A M m i g h t b e u s ed f o r su b c u l t u ri n g o f

e n r i c h m e n t b r o t h c u l t u r es b e c a u s e o f i t s h i g h s p ec i fi ci ty ( c o m p a r e d w i t h H E K ) a n d

t h a t t h i s c o u l d s u b s t a n t i a l l y r e d u c e t h e w o r k l o a d i n a l a b o r a t o r y . D u r i n g t h i s

s t u d y , i t w a s f o u n d t h a t R A M w a s o n e o f t h e e a s i e s t m e d i a t o u s e in p u r i f y i n g

i s o l a t e s a n d s t o c k c u l t u r e s .

A l o n s o e t al . ( 1 99 2 ) f o u n d t h a t, i n g e n e r a l, R A M p e r f o r m e d a s w e l l a s H E K in

t h e i s o l a t i o n o f

Salmonella

w h i l e t e s t i n g m a r i n e w a t e r s a n d t h a t t h e r e w a s n o

s i g n i f i c a n t d i f f e r e n c e i n Salmonella r e c o v e ry b e t w e e n t h e t w o m e d ia . R A M w a s

m o r e e f f i c ie n t t h a n H E K w h e n t h e r e w a s lo w fe c a l c o n t a m i n a t i o n b u t , i n t h e

p r e s e n c e o f h ig h l e v el s o f c o m p e t i n g b a c t e r ia , H E K s h o w e d g r e a t e r s e l ec ti vi ty .

D u s c h a n d A l tw e g g ( 1 99 3 ) f o u n d R A M t o b e v e r y s p ec if ic , h av i n g o b t a i n e d o n l y

1 0 f a l s e - p o s i t i v e s f r o m 5 0 4 s t o o l s a m p l e s . T h e s e i n c l u d e d

Escherichia coli

f o u r

Citrobacter freundii i s o l a t e s , t h r e e Citrobacter amalonaticus i s o l a t e s , Klebsiella

oxytoca

a n d

E. agglomerans.

F r e y d i e r e a n d G i l l e ( 1 9 9 1 ) f o u n d t h a t

Pseudomonas

a n d

Acinetobacter

g a v e f a ls e -p o s it iv e s r e a c ti o n s o n R A M . M a n y o f th e s e n o n -

Salmonella

g i v e f a l se - p o s i t iv e r e a c t i o n s o n t h e o t h e r m e d i a a s w e l l ( T a b l e 4 ) .

F l o w e r s e t a l. (1 9 9 2 ) s t a t e t h a t n o n e o f t h e g e n e r a l l y u s e d a g a r s ( B I S , B G S ,

H E K , X L D a n d o t h e r s ) a r e i d e a l f o r all s i t u a ti o n s, ju s ti fy i n g t h e r e c o m m e n d a t i o n

in m a n y r e f e r e n c e m e t h o d s f o r t h e u s e o f t w o o r m o r e a g a r m e d i a . B a s e d u p o n o u r

1 A O A C , A s s o c i a t i o n o f O f f ic i a l A n a l y t i c a l Ch e m i s t s ; A PH A , A m e r i c a n Pu b l i c H e a l t h A s s o c i a t i o n ;

F D A , U S F o o d a n d D r u g A d m i n i s tr a t i o n ; H P B , H e a l t h P r o t e c t io n B r a n c h ; I C M S F , I n t e r n a t i o n a l

Co m mi s s i o n o n M i c r o b io l o g i c a l Sp e c i f i c a ti o n s f o r Fo o d ; I SO , I n t e r n a t i o n a l O r g a n i z a t i o n f o r S t a n d a r d -

i z a t i o n ; N A S, N a t i o n a l A c a d e my o f Sc i e n c e s ( U S) ; U SD A , U S D e p a r t me n t o f A g r i c u l t u r e ) .


12/13

288 D.W. Warburton et al. /Internat ional Journal of Food Microbiology 22 1994) 277-289

r e s u l t s a n d t h o s e f r o m o t h e r s t u d i e s , a n d t h e f o l l o w i n g f a c t s : ( 1 ) B I S is c u r r e n t l y a

m e d i u m o f c h o i c e in C a n a d i a n s t a n d a r d m e t h o d s d u e t o i ts n o n - s a c c h a r i d e

d i f f e r e n t i a l s y s t e m ; a n d ( 2) n e w l y d e v e l o p e d o r t e s t e d m e d i a m u s t b e e x t e n s iv e l y

f i e l d t e s t e d b e f o r e i n c o r p o r a t i o n i n t o C a n a d i a n s t a n d a r d m e t h o d s u s e d in c o m p l i -

a n c e a c ti v it y , o u r r e c o m m e n d a t i o n f o r n e w l y d e v e l o p e d l a b o r a t o r y p r o c e d u r e s is

t h a t u s e o f B I S b e c o m p u l s o r y a n d a t l e a s t o n e o t h e r a g a r ( B G S , H E K , X L D ,

R A M a n d E F - 1 8 ) b e u s e d a s w e l l .

Ackn ow l e d g em en t s

A t h a n k y o u i s e x t e n d e d t o a ll s t a f f w h o a i d e d i n t h e p r e p a r a t i o n o f m e d i a a n d

t h e s u b c u l t u r i n g o f s a m p l e s t o a d d i t i o n a l t e s t m e d i a .

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