InternationalJournalofFtuJdMicn,bioloh~.'. 16 (1~2) 41-50 © 1992 El~vier Science Publishers B.V. All rights reserved 0168-16115/t~2/$115.111)
41
FOOD 1111498
A comparison of standard cultural methods for the detection of foodborne Salmonella J.-Y. D'Aoust, A.M. Sewell and D.W. Warburton l"~xM Directorate. Heahh Protection Branch. Heahh and Welfare (~nada, Ottawa. Olllario. (.'canacha
(Received 17 December 1991; revision received and accepted 3 April 1992)
The ~nsitivity of the standard cultural method of the International Organization for Standardization (ISO 6579 and ISO 3565 combined) was compared to that of the lleallh Protection Branch (I-IPB) procedure for the detection of fi~vdborne SabncmeUa. Of 195 fl~ds tested. 84 (43.1~;') were found to contain salmonellae by one or more cultural conditions. Of these, 75 (89.3%) and 68 (SI.0r/E) were identified by the ISO and ItPB methods, respectively. The apparent lack of agreement between both methods likely stemmed from the low indigenous numbers of salmonellae in several f [ ~ homogenatcs, and unequal transfer of the target micr(~)rganism into homologous ISO and HPB pre-cnrichment broths. The ~nsitivities of the commercially available Mullcr-Kauffmann tetrathionate broth (MKTBG.,~, Oxoid CM343). and a clo.~ly-related medium prepared with Oxoid CM29 tetrathionate ba~ varied from 86.9 to 89.3%, and were deemed equivalent to that obtained with the ISO fi)rmulation of MKTBG4.a (89.3~). Comparatively fewer contaminated samples were identified from .~lenite cystine (SC.~.O and selenite brilliant green (SBG.~s) enrichment cultures (82.1-83.3¢'~). The high ,selectivity and saccharidc-independent resl~)nse of the bismuth sulfitc agar medium warrants its consideration as a mandatory plating medium in ISO methodologies for the cffcclive detection of typical and atypical biotypes of fimdl~rne Sabnonella spp. Key words: Sahmmella: R ~ s ; ISO method: Sensitivity: Enrichment broth
Introduction F o o d m i c r o b i o l o g i s t s have t r a d i t i o n a l l y r e l i e d on s t a n d a r d c u l t u r a l m e t h o d s for t h e d e t e c t i o n o f f o o d b o r n c S a b n o n e l l a ( D ' A o u s t , 1981). Such d i a g n o s t i c p r o c e d u r e s g e n e r a l l y i n c l u d e p r e - e n r i c h m e n t o f a f o o d s a m p l e in a n o n - s e l e c t i v e b r o t h m e d i u m to f a c i l i t a t e t h e r e p a i r o f i n j u r e d o r s t r e s s e d s a l m o n e l l a e f r e q u e n t l y e n c o u n t e r e d in raw a n d p r o c e s s e d foods. S t u d i e s t h a t have r e p o r t e d g o o d r e c o v e r ies with s h o r t ( 6 - 8 h) p e r i o d s o f p r e - e n r i c h m e n t (Sadovski, 1977; S u r d y a n d H a a s , 1981) a r e o v e r s h a d o w e d by t h e c o m p e l l i n g e v i d e n c e t h a t o v e r n i g h t ( 1 6 - 2 4 h) i n c u b a t i o n is a p r e r e q u i s i t e for o p t i m a l m e t h o d sensitivity ( D ' A o u s t a n d M a i s h m e n t , 1979: M u l i n d w a a n d Pietzsch, 1979; A n d r e w s , 1986). D i r e c t selective e n r i c h -
Correspondence address: J.-Y, D'Aoust, F~x~l Directorate Itcallh Protectitm Branch. Health and Welfare Canada, Tunney's Pasture, Ottawa, Ontario, Canada KIA OL2,
ment (no pre-enrichment) of food materials suspected or known to contain high levels of endogenous microorganisms can be counterproductive, and is gradually falling into disfavor (Andrews, 1986; D'Aoust, 1989). Following pre-enrichment, replicate portions of the broth culture are generally inoculated into two selective enrichment broths. The successful recovery of foodborne salmonellae hinges on the ability of enrichment conditions to reduce the high ratio of non-salmonellae to Salmonella established during the non-selective pre-enrichment of foods. Such a change in microbial populations potentiates the isolation of salmonellae on plating media. It is important to realize that in addition to the judicious choice of enrichment media, the corresponding times and temperatures of incubation can dramatically affect method performance. For example, the diagnostic value of selective enrichment at elevated (41-43°C) temperatures, and the counterproductivity of short (6-8 h) periods of enrichment in standard cultural procedures is well established (Andrews, 1989; D'Aoust et al, 1990). The differential capacity but limited selectivity of common Salmonella plating media are regularly challenged by the large numbers and diversity of competitive non-salmonellae found in enrichment cultures. The increasing prevalence of lactose- and sucrose-positive Salmonella biotypes is equally problematic. Such diagnostic constraints have heightened the awareness of food analysts to the benefits of saccharide-independent media such as bismuth sulfite agar (Fricker, 1987; Andrews, 1989; D'Aoust, 1989). The final steps of standard methods include the biochemical screening of suspect Salmonella colonies using conventional tube media or commercial diagnostic kits (Cox et al., 1984). Polyvalent and single grouping serological reactions, thereafter, provide for the confirmation and final identification of the isolate. Although standard cultural procedures share many analytical features, differences in their use of determinant bacteriological media raise questions of method equivalency. The present study compares the sensitivity of the standard method of the International Organization for Standardization (ISO) with that of the Health Protection Branch (HPB) for the detection of Salmonella in naturally contaminated foods and animal feeds. It should be noted that, during the course of our experimental work, the ISO 6579 method (1981) was modified in 1990 by replacing the Muller-Kauffmann tetrathionate broth with the Rappaport-Vassiliadis medium (Morinigo et al., 1986).
Materials and Methods
Naturally contaminated high moisture (121) and low moisture (74) foods and animal feeds were obtained from local retail outlets, or as a result of monitoring or compliance activities by federal government agencies. Although samples were generally obtained from domestic areas of production, most fish and shellfish were imported from Asia, Africa and Central and South America. Several low moisture foods including chocolate, spices, coconut and tea were also of imported origin. The sensitivities of the ISO and HPB standard cultural procedures were compared in terms of their ability to recover Salmonella spp. from a common food
homogenate. Test samples were prepared by blending a 200 g sample of food with a sufficient volume of sterile water (< 300 ml) to produce a viscous mixture. However, the hydrophilicity of some animal feeds required greater amounts of water (< 500 ml) to prepare homogeneous mixtures. Poultry carcasses were thoroughly rinsed in 300 ml of sterile water. Equal portions of each food homogenate or poultry rinse fluid were then analyzed in parallel by each of the following standard cultural procedures. In "',e ISO method, food homogenates (or rinse fluids)were tested according to the International Standard ISO 6579 (International Organization for Standardization, 1981) except meat and meat products which were analyzed according to the International Standard ISO 3565 (International Organization for Standardization, 1975). Both procedures are identical except that the former utilizes selenite cystine and the latter selenite brilliant green enrichment broths under identical time-temperature conditions of incubation. A replicate portion of sample homogenate (100 g) or poultry rinse (100 ml) was pre-enriched in 9 volumes of buffered peptone water (BPW) for 16-20 h at 35°C. Replicate portions (10 mi) of the resulting pre-enrichment culture were selectively enriched in 10 volumes of each of the following enrichment broths: freshly prepared Muller-Kauffmann tetrathionate brilliant green (MKTBG43); MKTBG43 stored for 1 week at 4°C (International Organization for Standardization, 1975); selenite cystine (SC35) for low moisture foods or selenite brilliant green (SBG3.0 for meat and meat products; CM343 (Oxoid Ltd) formulation of MKTBG43; related TBG43 prepared with the Oxoid CM29 tetrathionate broth base (Edel and Kampelmacher, 1969). The tetrathionate and selenite broths were incubated for up to 48 h at 43°C and 35°C, respectively. Each enrichment culture was streaked onto bismuth sulfite (BSA) and brilliant green (BGA) agar media after 24 and 48 h of incubation. All plates were examined for suspect colonies after 20-24 h of incubation at 35°C, and after 48 h of incubation (BSA only). Presumptive Salmonella isolates were screened biochemicaily on triple sugar iron (TSI) and lysine iron (Ll) agars, and confirmed serologically using polyvalent and single grouping somatic (O) antisera. All bacteriological media were obtained from Difco Laboratories (Detroit) except MKTBG43 (CM 343), TBG43 (CM 29), and Ll which were supplied by Oxoid Limited. The food homogenates and poultry rinses used in ISO cultural analyses also served as test materials for the determination of SalmoneUa by the HPB method (Health Protection Branch, 1978). In this procedure, portions (100 g or ml) of homogenates or rinse fluids were preenriched for 16-20 h at 35°C in 9 volumes of nutrient broth (NB) except chocolate which was pre-enriched in reconstituted nonfat dry milk (10% w/v) with added brilliant green (0.002% w/v). Replicate portions (1.0 ml) of each pre-enrichment culture were then inoculated into 9 volumes of tetrathionate brilliant green (TBG43) and selenite cystine (SC35), and incubated for 18-24 h at 43°C and 35°C, respectively. One additional tube of SC broth (9 mi) was similarly inoculated, and incubated overnight at 43°C (SC43). Each enrichment culture was then streaked onto BSA and brilliant green sulfa (BGS) agar media. Presumptive Salmonella colonies were screened biochemically and confirmed serologically as previously de~ribed.
The incident levels of Salmonella spp. in a limited number of foods were estimated by the three-tube Most Probable Number (MPN) technique using replicate 10.0, 1.0 and 0.1 g samples. The latter were analyzed by the HPB cultural method as de~ribed above.
Results and Discussion
Of 195 samples tested, 38 (31.4%) of 121 high moisture and 46 (62.2%) of 74 low moisture foods and animal feeds harbored salmonellae (Table 1). The latter level of contamination is unduly high because low moisture food samples were derived from lots previously found to be contaminated. With few exceptions, isolated Salmonella strains belonged to common serovars within the somatic serogroups B-E (data not shown). The sensitivity of both standard cultural methods compared favourably where the ISO and HPB procedures identified 75 (89.3%) and 68 (81.0%) positive samples, respectively, (Table !). These findings undoubtedly reflect the similar diagnostic approach and time-temperature conditions used in each method. However, it is noteworthy that these standard methods differ in at least four aspects. One of these differences lies in the use of BPW (ISO) and NB (HPB) as the principal ore-enrichment broth media. The reported equivalence of non-selective media of varying nutritive capacities for the effective resuscitation of injured or stressed salmonellae frequently encountered in stored and/or processed foods suggests that the variance in ISO and HPB pre-enrichment media would be inconsequential (van Schothorst and van Leusden, 1975; D'Aoust and Maishment, 1979; D'Aoust, 1981; Andrews, 1989). Moreover, studies indicate that the temporal and temperature conditions applied at the ore-enrichment stage of analysis are more critical than the choice of a non-selective ore-enrichment medium. The adverse effects of short (6-8 h) ore-enrichment, and incubation of pre-enrichment cultures at an elevated (41-43°C) temperature on method sensitivity are well documented (Mulindwa and Pietzsch, 1979; Wilson et al, 1980; D'Aoust, 1981; McGibbon et al., 1984; Andrews, 1986). A second instance of method divergence arises from the use of different ore-enrichment to enrichment transfer volumes in the ISO (10/100 ml) and HPB (!.0/9.0 ml) methods. The need for an unusually large (10 ml) transfer volume in the ISO method is somewhat obscure, and contrasts with the previously demonstrated equivalence of 10.0 and 1.0 mi inocula at a 1 : 10 transfer volume ratio (van Schothorst and van Leusden, 1975; D'Aoust and Maishment, 1979). Furthermore, this ISO practice is not cost-effective because it requires 10-fold greater volumes of enrichment broths compared to that used in standard methods that utilize a 1.0 ml transfer volume (Health Protection Branch, 1978; U.S. Food and Drug Administration, 1984). A third difference between both standard methods rests in the use of MKTBG43 and either SC35 (ISO 6579) or SBG3.~ (ISO 3565) in ISO methodologies, and of TBG43 and SC35 in the HPB procedure. Lack of identity in the nutritive, selective and buffering capacities of the MKTBG43 and TBG43 media could justifiably question their equivalence. However, our recent examination of the productivity of these
TABLE I Detection of Salmonella in naturally contaminated foods Food
Number of samples Tested
Positive
ISO ~
HPB b
Total c
24 10 4
II 13 2
10 II I
12 13 3
sausages giblets
26 7
4 I
2 0
4 I
minced retail cuts ~ef
5 3
tl 0
II 0
0 0
giblels
8 8
I l) 0
0 tl 0
I 0 0
I l) 3 (I tl
I 0 3
I 0 3 0 0
High moisture
P~mhry carcas~s/cut-up giblets others d
Pork
minced retail cuts
I
Other J'oc~s fish/shellfish lamb fermented sausages frog legs raw milk Subtotal Low moisture Animal feeds Chocolate Black pepper Pasta Egg powder Coconut Turmeric Tea Subtotal Total
7 4 3 I
I 121
l) 0
36 (Ill)"
28 (2) c
38
49 7 7 4 3 2 I I
27 0 6 3 0 I I I
29 2 5 3 () 0 I 0
32 2 6 3 0 I I i
74
39 (6) c
40 (7) c
46
75 (16) ~
68 (9) c
84
195
a Preenrichment culture selectively enriched in MKTBG4.~, and either SBG3,~ (meat and meat products) or SC3s (low moisture). b Preenrichment culture selectively enriched in TBG4,~ and SC35. c Based on combined results of all test conditions. d Cornish hens (2). chicken nugget'; ( I ). and turkey burgers ( I ). Denote the number of positive samples detected by only one of the two standard methods. and other enrichment media underlined the ability of both media to facilitate the i s o l a t i o n o f Salmonella o n p l a t i n g m e d i a ( D ' A o u s t e t al., 1990). T h e i m p o r t a n c e o f selective enrichment at an elevated temperature (41-43°C) for optimal recovery of
foodborne salmonellae cannot be over-emphasized (Rengel and Mendoza, 1984; Morinigo et al, 1986; D'Aoust, 1989; Machado and Bernado, 1990). The remaining difference between the ISO and HPB methods relates to differential plating media. Although both standard procedures stipulate the use of brilliant green agar, analyst selection of a second saccharide-dependent medium, as permitted by an ISO procedural option, would likely undermine method sensitivity by failing to recognize the increasing occurrence of lactose- a n d / o r sucrose-positive Salmonella biotypes (Blackburn and Ellis, 1973; Threlfall et al., 1983; Poisson et al., 1988). The effectiveness of such plating media is further challenged by their limited selectivities which commonly engender false-positive reactions (Fagerberg and Avens, 1976; D'Aoust, 1989; Andrews, 1989). The performance of both standard cultural methods was assessed from common food homogenates and poultry rinses. However, our effort to maintain the integrity of each analytical procedure resulted in the unique identification of 16 and 9 positive samples by the ISO and HPB-methods, respectively (Table i). Retrospective quantitative analysis of a limited number of raw meats associated with such discrepant ISO and HPB results showed levels of < 3 salmonellae per 100 g of sample (data not shown). Such low numbers of Salmonella spp. likely resulted in the inoculation of the target microorganism into only one of the homologous BPW (lSO) and NB (HPB) pre-enrichment broths.. The ISO 6579 method recommends selective enrichment in MKTBG43 and SC3.~ for the facilitated recovery of foodborne SalmoneUa spp. on plating media (International Organization for Standardization, 1981). it is noteworthy that the ISO 3565 method relates to enrichment of meats and meat products in MKTBG43 and SBGa5 rather than SC35 (International Organization for Standardization, 1975). To more accurately represent standard ISO methodology, it was arbitrarily decided to combine both protocols to provide for the enrichment of all foods in MKTBG43, and in either SC.~5 (low moisture) or SBGx~ (meat and meat products). Identification of, respectively, 75 (89.3%) and 69 (82.1%) positive samples from MKTBG43 and SC35/SBG35 enrichment broths incubated for 24 h was not unexpected (Table ll). In contrast to the low selectivity of SC35/SBG35, and their incubation at a growth permissive temperature (35°C), selective enrichment in MKTBG4.~ at a growth limiting temperature (43°C) likely provided for the effective repression of competitive microflora and recovery of Salmonella on plating media. Our concerns on the possible deterioration of prepared MKTBG43 during storage in the refrigerator for 1 week proved to be unfounded as evidenced by the identical productivity of freshly-prepared and stored MKTBG43 (data not shown). A performance assessment of the Oxoid (CM343) MKTBG43 was included in this study because of its reported non-equivalence with the formulation described in standard ISO methodology (ISO Secretariat, personal communication). Our interest in the Oxoid (CM29) TBG4.~ medium stemmed from its compositional similarity with the ISO formulation of MKTBG43, and its standardized application for the detection of feodborne Salmonella in nine European laboratories (Edel and Kampelmacher, 1969). The ability of MKTBG43 (CM343) and TBG43 (CM29) broths to identify, respectively, 7a. (88.1%) and 73 (86.9%) positive samples after 24
TABLE II Productivity of selective enrichment conditions Food
Number of positive samples ~
HPB c
ISO b MKTBG4~
SC/SBG35
Others
TBG43
SC~s
MKTBG4~ b (CM343)
TBG4.~ h (CM29)
SC4.~ c
24 d
48
24
48
24
24
24
48
24
48
24
High moisture (38) c Low moisture (46) ~
36 39
36 39
32 37
32 38
28 40
26 39
36 38
36 39
36 37
36 38
27 38
Total (84) c
75
75
69
70
68
65
74
75
73
74
65
a Enrichment cultures were each plated on BSA and BGA. b BPW preenrichment cultures were selectively enriched in MKTBG4~. TBG43. (CM29) and in eilher SC~ s (low moisture) or SBG~s (meat and meat products). c Nutrient broth (or other) preenrichment cultures were selectively enriched in TBG4~, SC~s and SC4.~, d Period (h) of selective enrichment. c Number of positive samples by all test conditions.
48 TABLE !11 The efficacyof enrichment-plating conditions for the isolation of foodborne salmonellae ~ Plating medium
Number of positive samples ISO b ltPB ~
MKTBG43 SC/ SBG.~s High moisture (38) 'J BSA 34 31 BGA/BGS ~ 36 22
Others
T B G 4 3 SC35 MKTBG4.~t, (CM343)
TBG43h
SC.t~
27 28
23 20
32 34
34 36
27 21
(CM29)
Low moisture (46) d
BSA BGA/BGS ~
39 38
37 31
411 40
38 32
39 37
38 37
38 32
73 74
68 53
67 68
61 52
71 71
72 73
65 53
Total (84) d
BSA BGA/BGS
Data relate to recoveries after 24 h of selective enrichment only.
b.~ See corresponding footnotes in Table II. d Number of positive samples by all test conditions. c ISO and other MKTBG43 enrichment cultures were plated on BGA whereas HPB and SC43 enrichment cultures were plated on BGS.
h of incubation, and one additional sample each after 48 h of selective enrichment, compared favorably with the 75 (89.3%) positive samples recognized by the ISO formulation of MKTBG43 under homologous conditions of analysis (Table 11). Such lack of marked differences in recovery after 24 and 48 h of selective enrichment corroborates earlier findings on the mitigated success of prolonged ( > 48 h) enrichment for increased method sensitivity (Andrews, 1989; D'Aoust et ai., 1992). In P " H P B method study, synergism between the high selectivity of T B G and incubation at a repressive (43°C) temperature likely played a determinant role in the observed productivities of homologous TBG43, SC35 and SC43 enrichment cultures (Table !1). Although incubation of SC at 43°C reportedly inhibits the growth of Salmonella spp. (Greenfield and Bigland, 1970), this could not be substantiated in the present study. The efficacies of BSA and B G A / B G S agar media compared favorably when plates were inoculated from tetrathionate enrichment cultures (Tabi~ i11). in contrast, the efficacy of BSA greatly exceeded that of B G A / B G S in combination with SC35 and SBG35 cultures. T h e high selectivity of the BSA plating medium seemingly compensated for the limited ability of SC and SBG to inhibit the growth of competitive microorganisms. T h e diagnostic value of the saccharide-independent BSA medium in detecting typical and atypical foodborne Salmonella biotypes is reflected in its wide application in food analyses (D'Aoust, 1989). Identification of BSA as a mandatory plating medium in relevant ISO methodologies would not
only favor a g r e a t e r international standardization o f Salmonella analytical procedures, but would also e n h a n c e m e t h o d sensitivity and proactively a c c o m m o d a t e the c h a n g i n g biochemical p a t t e r n s o f f o o d b o r n c salmonellae. T h e p u r p o r t e d inability o f B S A to detect H2S-negativc Salmonella strains (as classically defined f r o m T S ! reactions) is a non-issue because o f the high sensitivity o f the H 2 S detection system in B S A which provides for the devel(~pment o f distinct black colonies with classical H 2 S - n c g a t i v e biotypcs ( D ' A o u s t , u:lpublished data). U n d e r the limiting experimental conditions o f the present study, the sensitivities o f the I S O and H P B s t a n d a r d m e t h o d s were seemingly c o m p a r a b l e for the detection o f f o o d b o r n e Salmonel'a spp. T h e MKTBG43 (CM343) a n d TBG43 (CM29) c o m m e r c i a l p r o d u c t s were also f o u n d to be equivalent to the I S O formulation o f MKTBG43. T h e ability o f B S A to detect typical and atypical Salmonella biotypes, c o u p l e d with an intrinsically high level o f selectivity, w a r r a n t s its consideration as a m a n d a t o r y plating m e d i u m in I S O m e t h o d o l o g y .
References
Andrews, W.H. (1986) Resuscitation of injured Sabnonclla spp. and coliforms from foods. J. Food Prot. 49, 62-75. Andrews. W.H. (1989) Methods for recovering injured "classical"enteric pathogenic bacteria (Salmonella, Shigel/a, and enteropathogenic Escherichia coli) from foods. In: B. Ray (Ed.), Injured Index and Pathogenic Bacteria: Occurrence and Detection in Foods, Water and Feeds, CRC Press inc., Boca Raton, pp. 55-113. Blackburn, B.O. and Ellis, E.M. (1973) Lactose-fermenting Sahnonella from dried milk and milk-drying plants. Appl. Microbiol. 26, 672-674. Cox, N.A., Fung, D.Y.C., Goldschmidt, M.C., Bailey, J.S. and Thomson, J.E. (1984) Selecting a miniaturized system for identification of Enterobacteriaceae. J. Food Prot. 47, 74-77. D'Aoust, J.-Y. (1981) Update on pre-enrichment and selective enrichment conditions for detection of Salmonella in foods. J. Food Prof. 44, 369-374. D'Aoust, J.-Y. (1989) Salmonella. In: M.P. Doyle (Ed.), Foodbornc Bacterial Pathogens, Marcel l::)¢kkcr Inc., New York, NY, pp. 327-445. D'Aoust, J.-Y. and Maishment, C. (19791 Preenrichment conditions for effective recovery of Sahnonella in foods and feed ingredients. J. F[md Prof. 42, 153-157. D'Aoust, J.-Y., Sewell, A. and .lean, A. (1990) Limited sensitivity of short (6 h) selective enrichment for detection of foodborne Sabnom, lla. J. Food Prot. 53, 562-565. D'Aoust, J.-Y., Sewell, A.M. and Jean, A. (1992) Efficacy of prolong¢d (48 h) selective enrichment for the detection of foodborne Salmonella Int. J. F(x)d Microbiol. 15, 121-130. Edel, W. and Kampelmacher, E.H. (1969) Salmonella isolation in nine European laboratories using a standardized technique. Bull. WHO 41,297-306, Fagerberg, D.J. and Avens, J.S. (1976) Enrichment and plating methodology for Salmonella detection in food. A review. J. Milk Food Technol. 39, 628-646. Fricker, C.R. (1987)The isolation of salmonellas and campylobacters. J. Appl. Bacteriol. 63, 99-116. Greenfield, J. and Bigland, C.H. (1970) Selective inhibition of certain enteric bacteria by selenite media incubated at 35 and 43°C. Can. J. Microbiol. 16, 1267-1271. Health Protection Branch (1978) Methods for the isolation and identification of Salmonella from foods. HPB Method MFHPB-20, Health and Welfare Canada, Ottawa. International Organization for Standardization (1975) Meat and meat products--detection of salmonellae (Reference Method). International Standard ISO 3565, Geneva.
International Organization for Standardization (1981) Microbiology--General guidance on methods for the detection of Salmonella. International Standard ISO 6579. Geneva. Machado, J. and Bernado, F. (1990) Prevalence of Salmonella in chicken carcasses in Portugal. J. Appl. Bacteriol. 69, 477-480. McGibbon, L., Ouail, E. and Fricker, C.R. (1984) Isolation of salmonellae using two forms of Rappaport-Vassiliadis medium and brilliant green agar. Int. J. Food Microbiol. !, 171-177. Morinigo, M.A., Borrego, J.J. and Romero, P. (1986) Comparative study of different methods for dctcction and enumeration of Salmonella spp. in natural waters. J. Appl. Bacteriol. 61, 169-176. Mulindwa, D.K. and Pietzsch, O. (1979) Studies on the influence of competitive Enterobaeteriaceae flora on Salmonella isolation. Zentralb. Bakt. Mikrobiol. Hyg. Abt. I. Orig. A. 243, 336-348. Poisson, D.M., Bertin, N.D., Pariente, E.A. and Calamy, G. (1988) Salmonella agona saccharose-positive: ~tude bact6riologigue succinte el 6pid6miologique r6trospective h propos de I0 souches isol6es au CHR d'Orl6ans. M~d. Maladies Infect. 10, 433-439. Rengel, A. and Mendoza, S. (1984) Isolation of Salmonella from raw chicken in Venezuela. J. Food Prot. 47, 213-216. Sadovski, A.Y. (1977) Technical note: Acid sensitivity of freeze injured salmonellae in relation to their isolation from frozen vegetables by pre-enriehment procedure. J. Food Technol. 12, 85-91. Surdy, T.E. and Haas, S.O. (1981) Modified enrichment-serology procedure for detection of salmonellae in soy products. Appl. Environ. Microbiol. 42, 704-707. Threlfall, E.J., Hall, M.L.M. and Rowe, B. (1983) Lactose-fermenting salmonellae in Britain. FEMS Microbiol. Lctt. 17, 127-130. U.S. Food and Drug Administration (1984) Bacteriological Analytical Manual (6th edition). Association of Official Analytical Chemists, Arlington. van Schothorst, M. and van Leusden, F.M. (1975)Comparison of several methods for the isolation of salmonellae from egg products. Can. J. Microbiol. 21, 1041-1045. Wilson, C.R., Andrews, W.H. and Poelma, P.L. (1980) Recovery of Salmonella from milk chocolate using a chemically defined medium and five nondefined broths. J. Food Sci. 45, 310-313; 316.
41
FOOD 1111498
A comparison of standard cultural methods for the detection of foodborne Salmonella J.-Y. D'Aoust, A.M. Sewell and D.W. Warburton l"~xM Directorate. Heahh Protection Branch. Heahh and Welfare (~nada, Ottawa. Olllario. (.'canacha
(Received 17 December 1991; revision received and accepted 3 April 1992)
The ~nsitivity of the standard cultural method of the International Organization for Standardization (ISO 6579 and ISO 3565 combined) was compared to that of the lleallh Protection Branch (I-IPB) procedure for the detection of fi~vdborne SabncmeUa. Of 195 fl~ds tested. 84 (43.1~;') were found to contain salmonellae by one or more cultural conditions. Of these, 75 (89.3%) and 68 (SI.0r/E) were identified by the ISO and ItPB methods, respectively. The apparent lack of agreement between both methods likely stemmed from the low indigenous numbers of salmonellae in several f [ ~ homogenatcs, and unequal transfer of the target micr(~)rganism into homologous ISO and HPB pre-cnrichment broths. The ~nsitivities of the commercially available Mullcr-Kauffmann tetrathionate broth (MKTBG.,~, Oxoid CM343). and a clo.~ly-related medium prepared with Oxoid CM29 tetrathionate ba~ varied from 86.9 to 89.3%, and were deemed equivalent to that obtained with the ISO fi)rmulation of MKTBG4.a (89.3~). Comparatively fewer contaminated samples were identified from .~lenite cystine (SC.~.O and selenite brilliant green (SBG.~s) enrichment cultures (82.1-83.3¢'~). The high ,selectivity and saccharidc-independent resl~)nse of the bismuth sulfitc agar medium warrants its consideration as a mandatory plating medium in ISO methodologies for the cffcclive detection of typical and atypical biotypes of fimdl~rne Sabnonella spp. Key words: Sahmmella: R ~ s ; ISO method: Sensitivity: Enrichment broth
Introduction F o o d m i c r o b i o l o g i s t s have t r a d i t i o n a l l y r e l i e d on s t a n d a r d c u l t u r a l m e t h o d s for t h e d e t e c t i o n o f f o o d b o r n c S a b n o n e l l a ( D ' A o u s t , 1981). Such d i a g n o s t i c p r o c e d u r e s g e n e r a l l y i n c l u d e p r e - e n r i c h m e n t o f a f o o d s a m p l e in a n o n - s e l e c t i v e b r o t h m e d i u m to f a c i l i t a t e t h e r e p a i r o f i n j u r e d o r s t r e s s e d s a l m o n e l l a e f r e q u e n t l y e n c o u n t e r e d in raw a n d p r o c e s s e d foods. S t u d i e s t h a t have r e p o r t e d g o o d r e c o v e r ies with s h o r t ( 6 - 8 h) p e r i o d s o f p r e - e n r i c h m e n t (Sadovski, 1977; S u r d y a n d H a a s , 1981) a r e o v e r s h a d o w e d by t h e c o m p e l l i n g e v i d e n c e t h a t o v e r n i g h t ( 1 6 - 2 4 h) i n c u b a t i o n is a p r e r e q u i s i t e for o p t i m a l m e t h o d sensitivity ( D ' A o u s t a n d M a i s h m e n t , 1979: M u l i n d w a a n d Pietzsch, 1979; A n d r e w s , 1986). D i r e c t selective e n r i c h -
Correspondence address: J.-Y, D'Aoust, F~x~l Directorate Itcallh Protectitm Branch. Health and Welfare Canada, Tunney's Pasture, Ottawa, Ontario, Canada KIA OL2,
ment (no pre-enrichment) of food materials suspected or known to contain high levels of endogenous microorganisms can be counterproductive, and is gradually falling into disfavor (Andrews, 1986; D'Aoust, 1989). Following pre-enrichment, replicate portions of the broth culture are generally inoculated into two selective enrichment broths. The successful recovery of foodborne salmonellae hinges on the ability of enrichment conditions to reduce the high ratio of non-salmonellae to Salmonella established during the non-selective pre-enrichment of foods. Such a change in microbial populations potentiates the isolation of salmonellae on plating media. It is important to realize that in addition to the judicious choice of enrichment media, the corresponding times and temperatures of incubation can dramatically affect method performance. For example, the diagnostic value of selective enrichment at elevated (41-43°C) temperatures, and the counterproductivity of short (6-8 h) periods of enrichment in standard cultural procedures is well established (Andrews, 1989; D'Aoust et al, 1990). The differential capacity but limited selectivity of common Salmonella plating media are regularly challenged by the large numbers and diversity of competitive non-salmonellae found in enrichment cultures. The increasing prevalence of lactose- and sucrose-positive Salmonella biotypes is equally problematic. Such diagnostic constraints have heightened the awareness of food analysts to the benefits of saccharide-independent media such as bismuth sulfite agar (Fricker, 1987; Andrews, 1989; D'Aoust, 1989). The final steps of standard methods include the biochemical screening of suspect Salmonella colonies using conventional tube media or commercial diagnostic kits (Cox et al., 1984). Polyvalent and single grouping serological reactions, thereafter, provide for the confirmation and final identification of the isolate. Although standard cultural procedures share many analytical features, differences in their use of determinant bacteriological media raise questions of method equivalency. The present study compares the sensitivity of the standard method of the International Organization for Standardization (ISO) with that of the Health Protection Branch (HPB) for the detection of Salmonella in naturally contaminated foods and animal feeds. It should be noted that, during the course of our experimental work, the ISO 6579 method (1981) was modified in 1990 by replacing the Muller-Kauffmann tetrathionate broth with the Rappaport-Vassiliadis medium (Morinigo et al., 1986).
Materials and Methods
Naturally contaminated high moisture (121) and low moisture (74) foods and animal feeds were obtained from local retail outlets, or as a result of monitoring or compliance activities by federal government agencies. Although samples were generally obtained from domestic areas of production, most fish and shellfish were imported from Asia, Africa and Central and South America. Several low moisture foods including chocolate, spices, coconut and tea were also of imported origin. The sensitivities of the ISO and HPB standard cultural procedures were compared in terms of their ability to recover Salmonella spp. from a common food
homogenate. Test samples were prepared by blending a 200 g sample of food with a sufficient volume of sterile water (< 300 ml) to produce a viscous mixture. However, the hydrophilicity of some animal feeds required greater amounts of water (< 500 ml) to prepare homogeneous mixtures. Poultry carcasses were thoroughly rinsed in 300 ml of sterile water. Equal portions of each food homogenate or poultry rinse fluid were then analyzed in parallel by each of the following standard cultural procedures. In "',e ISO method, food homogenates (or rinse fluids)were tested according to the International Standard ISO 6579 (International Organization for Standardization, 1981) except meat and meat products which were analyzed according to the International Standard ISO 3565 (International Organization for Standardization, 1975). Both procedures are identical except that the former utilizes selenite cystine and the latter selenite brilliant green enrichment broths under identical time-temperature conditions of incubation. A replicate portion of sample homogenate (100 g) or poultry rinse (100 ml) was pre-enriched in 9 volumes of buffered peptone water (BPW) for 16-20 h at 35°C. Replicate portions (10 mi) of the resulting pre-enrichment culture were selectively enriched in 10 volumes of each of the following enrichment broths: freshly prepared Muller-Kauffmann tetrathionate brilliant green (MKTBG43); MKTBG43 stored for 1 week at 4°C (International Organization for Standardization, 1975); selenite cystine (SC35) for low moisture foods or selenite brilliant green (SBG3.0 for meat and meat products; CM343 (Oxoid Ltd) formulation of MKTBG43; related TBG43 prepared with the Oxoid CM29 tetrathionate broth base (Edel and Kampelmacher, 1969). The tetrathionate and selenite broths were incubated for up to 48 h at 43°C and 35°C, respectively. Each enrichment culture was streaked onto bismuth sulfite (BSA) and brilliant green (BGA) agar media after 24 and 48 h of incubation. All plates were examined for suspect colonies after 20-24 h of incubation at 35°C, and after 48 h of incubation (BSA only). Presumptive Salmonella isolates were screened biochemicaily on triple sugar iron (TSI) and lysine iron (Ll) agars, and confirmed serologically using polyvalent and single grouping somatic (O) antisera. All bacteriological media were obtained from Difco Laboratories (Detroit) except MKTBG43 (CM 343), TBG43 (CM 29), and Ll which were supplied by Oxoid Limited. The food homogenates and poultry rinses used in ISO cultural analyses also served as test materials for the determination of SalmoneUa by the HPB method (Health Protection Branch, 1978). In this procedure, portions (100 g or ml) of homogenates or rinse fluids were preenriched for 16-20 h at 35°C in 9 volumes of nutrient broth (NB) except chocolate which was pre-enriched in reconstituted nonfat dry milk (10% w/v) with added brilliant green (0.002% w/v). Replicate portions (1.0 ml) of each pre-enrichment culture were then inoculated into 9 volumes of tetrathionate brilliant green (TBG43) and selenite cystine (SC35), and incubated for 18-24 h at 43°C and 35°C, respectively. One additional tube of SC broth (9 mi) was similarly inoculated, and incubated overnight at 43°C (SC43). Each enrichment culture was then streaked onto BSA and brilliant green sulfa (BGS) agar media. Presumptive Salmonella colonies were screened biochemically and confirmed serologically as previously de~ribed.
The incident levels of Salmonella spp. in a limited number of foods were estimated by the three-tube Most Probable Number (MPN) technique using replicate 10.0, 1.0 and 0.1 g samples. The latter were analyzed by the HPB cultural method as de~ribed above.
Results and Discussion
Of 195 samples tested, 38 (31.4%) of 121 high moisture and 46 (62.2%) of 74 low moisture foods and animal feeds harbored salmonellae (Table 1). The latter level of contamination is unduly high because low moisture food samples were derived from lots previously found to be contaminated. With few exceptions, isolated Salmonella strains belonged to common serovars within the somatic serogroups B-E (data not shown). The sensitivity of both standard cultural methods compared favourably where the ISO and HPB procedures identified 75 (89.3%) and 68 (81.0%) positive samples, respectively, (Table !). These findings undoubtedly reflect the similar diagnostic approach and time-temperature conditions used in each method. However, it is noteworthy that these standard methods differ in at least four aspects. One of these differences lies in the use of BPW (ISO) and NB (HPB) as the principal ore-enrichment broth media. The reported equivalence of non-selective media of varying nutritive capacities for the effective resuscitation of injured or stressed salmonellae frequently encountered in stored and/or processed foods suggests that the variance in ISO and HPB pre-enrichment media would be inconsequential (van Schothorst and van Leusden, 1975; D'Aoust and Maishment, 1979; D'Aoust, 1981; Andrews, 1989). Moreover, studies indicate that the temporal and temperature conditions applied at the ore-enrichment stage of analysis are more critical than the choice of a non-selective ore-enrichment medium. The adverse effects of short (6-8 h) ore-enrichment, and incubation of pre-enrichment cultures at an elevated (41-43°C) temperature on method sensitivity are well documented (Mulindwa and Pietzsch, 1979; Wilson et al, 1980; D'Aoust, 1981; McGibbon et al., 1984; Andrews, 1986). A second instance of method divergence arises from the use of different ore-enrichment to enrichment transfer volumes in the ISO (10/100 ml) and HPB (!.0/9.0 ml) methods. The need for an unusually large (10 ml) transfer volume in the ISO method is somewhat obscure, and contrasts with the previously demonstrated equivalence of 10.0 and 1.0 mi inocula at a 1 : 10 transfer volume ratio (van Schothorst and van Leusden, 1975; D'Aoust and Maishment, 1979). Furthermore, this ISO practice is not cost-effective because it requires 10-fold greater volumes of enrichment broths compared to that used in standard methods that utilize a 1.0 ml transfer volume (Health Protection Branch, 1978; U.S. Food and Drug Administration, 1984). A third difference between both standard methods rests in the use of MKTBG43 and either SC35 (ISO 6579) or SBG3.~ (ISO 3565) in ISO methodologies, and of TBG43 and SC35 in the HPB procedure. Lack of identity in the nutritive, selective and buffering capacities of the MKTBG43 and TBG43 media could justifiably question their equivalence. However, our recent examination of the productivity of these
TABLE I Detection of Salmonella in naturally contaminated foods Food
Number of samples Tested
Positive
ISO ~
HPB b
Total c
24 10 4
II 13 2
10 II I
12 13 3
sausages giblets
26 7
4 I
2 0
4 I
minced retail cuts ~ef
5 3
tl 0
II 0
0 0
giblels
8 8
I l) 0
0 tl 0
I 0 0
I l) 3 (I tl
I 0 3
I 0 3 0 0
High moisture
P~mhry carcas~s/cut-up giblets others d
Pork
minced retail cuts
I
Other J'oc~s fish/shellfish lamb fermented sausages frog legs raw milk Subtotal Low moisture Animal feeds Chocolate Black pepper Pasta Egg powder Coconut Turmeric Tea Subtotal Total
7 4 3 I
I 121
l) 0
36 (Ill)"
28 (2) c
38
49 7 7 4 3 2 I I
27 0 6 3 0 I I I
29 2 5 3 () 0 I 0
32 2 6 3 0 I I i
74
39 (6) c
40 (7) c
46
75 (16) ~
68 (9) c
84
195
a Preenrichment culture selectively enriched in MKTBG4.~, and either SBG3,~ (meat and meat products) or SC3s (low moisture). b Preenrichment culture selectively enriched in TBG4,~ and SC35. c Based on combined results of all test conditions. d Cornish hens (2). chicken nugget'; ( I ). and turkey burgers ( I ). Denote the number of positive samples detected by only one of the two standard methods. and other enrichment media underlined the ability of both media to facilitate the i s o l a t i o n o f Salmonella o n p l a t i n g m e d i a ( D ' A o u s t e t al., 1990). T h e i m p o r t a n c e o f selective enrichment at an elevated temperature (41-43°C) for optimal recovery of
foodborne salmonellae cannot be over-emphasized (Rengel and Mendoza, 1984; Morinigo et al, 1986; D'Aoust, 1989; Machado and Bernado, 1990). The remaining difference between the ISO and HPB methods relates to differential plating media. Although both standard procedures stipulate the use of brilliant green agar, analyst selection of a second saccharide-dependent medium, as permitted by an ISO procedural option, would likely undermine method sensitivity by failing to recognize the increasing occurrence of lactose- a n d / o r sucrose-positive Salmonella biotypes (Blackburn and Ellis, 1973; Threlfall et al., 1983; Poisson et al., 1988). The effectiveness of such plating media is further challenged by their limited selectivities which commonly engender false-positive reactions (Fagerberg and Avens, 1976; D'Aoust, 1989; Andrews, 1989). The performance of both standard cultural methods was assessed from common food homogenates and poultry rinses. However, our effort to maintain the integrity of each analytical procedure resulted in the unique identification of 16 and 9 positive samples by the ISO and HPB-methods, respectively (Table i). Retrospective quantitative analysis of a limited number of raw meats associated with such discrepant ISO and HPB results showed levels of < 3 salmonellae per 100 g of sample (data not shown). Such low numbers of Salmonella spp. likely resulted in the inoculation of the target microorganism into only one of the homologous BPW (lSO) and NB (HPB) pre-enrichment broths.. The ISO 6579 method recommends selective enrichment in MKTBG43 and SC3.~ for the facilitated recovery of foodborne SalmoneUa spp. on plating media (International Organization for Standardization, 1981). it is noteworthy that the ISO 3565 method relates to enrichment of meats and meat products in MKTBG43 and SBGa5 rather than SC35 (International Organization for Standardization, 1975). To more accurately represent standard ISO methodology, it was arbitrarily decided to combine both protocols to provide for the enrichment of all foods in MKTBG43, and in either SC.~5 (low moisture) or SBGx~ (meat and meat products). Identification of, respectively, 75 (89.3%) and 69 (82.1%) positive samples from MKTBG43 and SC35/SBG35 enrichment broths incubated for 24 h was not unexpected (Table ll). In contrast to the low selectivity of SC35/SBG35, and their incubation at a growth permissive temperature (35°C), selective enrichment in MKTBG4.~ at a growth limiting temperature (43°C) likely provided for the effective repression of competitive microflora and recovery of Salmonella on plating media. Our concerns on the possible deterioration of prepared MKTBG43 during storage in the refrigerator for 1 week proved to be unfounded as evidenced by the identical productivity of freshly-prepared and stored MKTBG43 (data not shown). A performance assessment of the Oxoid (CM343) MKTBG43 was included in this study because of its reported non-equivalence with the formulation described in standard ISO methodology (ISO Secretariat, personal communication). Our interest in the Oxoid (CM29) TBG4.~ medium stemmed from its compositional similarity with the ISO formulation of MKTBG43, and its standardized application for the detection of feodborne Salmonella in nine European laboratories (Edel and Kampelmacher, 1969). The ability of MKTBG43 (CM343) and TBG43 (CM29) broths to identify, respectively, 7a. (88.1%) and 73 (86.9%) positive samples after 24
TABLE II Productivity of selective enrichment conditions Food
Number of positive samples ~
HPB c
ISO b MKTBG4~
SC/SBG35
Others
TBG43
SC~s
MKTBG4~ b (CM343)
TBG4.~ h (CM29)
SC4.~ c
24 d
48
24
48
24
24
24
48
24
48
24
High moisture (38) c Low moisture (46) ~
36 39
36 39
32 37
32 38
28 40
26 39
36 38
36 39
36 37
36 38
27 38
Total (84) c
75
75
69
70
68
65
74
75
73
74
65
a Enrichment cultures were each plated on BSA and BGA. b BPW preenrichment cultures were selectively enriched in MKTBG4~. TBG43. (CM29) and in eilher SC~ s (low moisture) or SBG~s (meat and meat products). c Nutrient broth (or other) preenrichment cultures were selectively enriched in TBG4~, SC~s and SC4.~, d Period (h) of selective enrichment. c Number of positive samples by all test conditions.
48 TABLE !11 The efficacyof enrichment-plating conditions for the isolation of foodborne salmonellae ~ Plating medium
Number of positive samples ISO b ltPB ~
MKTBG43 SC/ SBG.~s High moisture (38) 'J BSA 34 31 BGA/BGS ~ 36 22
Others
T B G 4 3 SC35 MKTBG4.~t, (CM343)
TBG43h
SC.t~
27 28
23 20
32 34
34 36
27 21
(CM29)
Low moisture (46) d
BSA BGA/BGS ~
39 38
37 31
411 40
38 32
39 37
38 37
38 32
73 74
68 53
67 68
61 52
71 71
72 73
65 53
Total (84) d
BSA BGA/BGS
Data relate to recoveries after 24 h of selective enrichment only.
b.~ See corresponding footnotes in Table II. d Number of positive samples by all test conditions. c ISO and other MKTBG43 enrichment cultures were plated on BGA whereas HPB and SC43 enrichment cultures were plated on BGS.
h of incubation, and one additional sample each after 48 h of selective enrichment, compared favorably with the 75 (89.3%) positive samples recognized by the ISO formulation of MKTBG43 under homologous conditions of analysis (Table 11). Such lack of marked differences in recovery after 24 and 48 h of selective enrichment corroborates earlier findings on the mitigated success of prolonged ( > 48 h) enrichment for increased method sensitivity (Andrews, 1989; D'Aoust et ai., 1992). In P " H P B method study, synergism between the high selectivity of T B G and incubation at a repressive (43°C) temperature likely played a determinant role in the observed productivities of homologous TBG43, SC35 and SC43 enrichment cultures (Table !1). Although incubation of SC at 43°C reportedly inhibits the growth of Salmonella spp. (Greenfield and Bigland, 1970), this could not be substantiated in the present study. The efficacies of BSA and B G A / B G S agar media compared favorably when plates were inoculated from tetrathionate enrichment cultures (Tabi~ i11). in contrast, the efficacy of BSA greatly exceeded that of B G A / B G S in combination with SC35 and SBG35 cultures. T h e high selectivity of the BSA plating medium seemingly compensated for the limited ability of SC and SBG to inhibit the growth of competitive microorganisms. T h e diagnostic value of the saccharide-independent BSA medium in detecting typical and atypical foodborne Salmonella biotypes is reflected in its wide application in food analyses (D'Aoust, 1989). Identification of BSA as a mandatory plating medium in relevant ISO methodologies would not
only favor a g r e a t e r international standardization o f Salmonella analytical procedures, but would also e n h a n c e m e t h o d sensitivity and proactively a c c o m m o d a t e the c h a n g i n g biochemical p a t t e r n s o f f o o d b o r n c salmonellae. T h e p u r p o r t e d inability o f B S A to detect H2S-negativc Salmonella strains (as classically defined f r o m T S ! reactions) is a non-issue because o f the high sensitivity o f the H 2 S detection system in B S A which provides for the devel(~pment o f distinct black colonies with classical H 2 S - n c g a t i v e biotypcs ( D ' A o u s t , u:lpublished data). U n d e r the limiting experimental conditions o f the present study, the sensitivities o f the I S O and H P B s t a n d a r d m e t h o d s were seemingly c o m p a r a b l e for the detection o f f o o d b o r n e Salmonel'a spp. T h e MKTBG43 (CM343) a n d TBG43 (CM29) c o m m e r c i a l p r o d u c t s were also f o u n d to be equivalent to the I S O formulation o f MKTBG43. T h e ability o f B S A to detect typical and atypical Salmonella biotypes, c o u p l e d with an intrinsically high level o f selectivity, w a r r a n t s its consideration as a m a n d a t o r y plating m e d i u m in I S O m e t h o d o l o g y .
References
Andrews, W.H. (1986) Resuscitation of injured Sabnonclla spp. and coliforms from foods. J. Food Prot. 49, 62-75. Andrews. W.H. (1989) Methods for recovering injured "classical"enteric pathogenic bacteria (Salmonella, Shigel/a, and enteropathogenic Escherichia coli) from foods. In: B. Ray (Ed.), Injured Index and Pathogenic Bacteria: Occurrence and Detection in Foods, Water and Feeds, CRC Press inc., Boca Raton, pp. 55-113. Blackburn, B.O. and Ellis, E.M. (1973) Lactose-fermenting Sahnonella from dried milk and milk-drying plants. Appl. Microbiol. 26, 672-674. Cox, N.A., Fung, D.Y.C., Goldschmidt, M.C., Bailey, J.S. and Thomson, J.E. (1984) Selecting a miniaturized system for identification of Enterobacteriaceae. J. Food Prot. 47, 74-77. D'Aoust, J.-Y. (1981) Update on pre-enrichment and selective enrichment conditions for detection of Salmonella in foods. J. Food Prof. 44, 369-374. D'Aoust, J.-Y. (1989) Salmonella. In: M.P. Doyle (Ed.), Foodbornc Bacterial Pathogens, Marcel l::)¢kkcr Inc., New York, NY, pp. 327-445. D'Aoust, J.-Y. and Maishment, C. (19791 Preenrichment conditions for effective recovery of Sahnonella in foods and feed ingredients. J. F[md Prof. 42, 153-157. D'Aoust, J.-Y., Sewell, A. and .lean, A. (1990) Limited sensitivity of short (6 h) selective enrichment for detection of foodborne Sabnom, lla. J. Food Prot. 53, 562-565. D'Aoust, J.-Y., Sewell, A.M. and Jean, A. (1992) Efficacy of prolong¢d (48 h) selective enrichment for the detection of foodborne Salmonella Int. J. F(x)d Microbiol. 15, 121-130. Edel, W. and Kampelmacher, E.H. (1969) Salmonella isolation in nine European laboratories using a standardized technique. Bull. WHO 41,297-306, Fagerberg, D.J. and Avens, J.S. (1976) Enrichment and plating methodology for Salmonella detection in food. A review. J. Milk Food Technol. 39, 628-646. Fricker, C.R. (1987)The isolation of salmonellas and campylobacters. J. Appl. Bacteriol. 63, 99-116. Greenfield, J. and Bigland, C.H. (1970) Selective inhibition of certain enteric bacteria by selenite media incubated at 35 and 43°C. Can. J. Microbiol. 16, 1267-1271. Health Protection Branch (1978) Methods for the isolation and identification of Salmonella from foods. HPB Method MFHPB-20, Health and Welfare Canada, Ottawa. International Organization for Standardization (1975) Meat and meat products--detection of salmonellae (Reference Method). International Standard ISO 3565, Geneva.
International Organization for Standardization (1981) Microbiology--General guidance on methods for the detection of Salmonella. International Standard ISO 6579. Geneva. Machado, J. and Bernado, F. (1990) Prevalence of Salmonella in chicken carcasses in Portugal. J. Appl. Bacteriol. 69, 477-480. McGibbon, L., Ouail, E. and Fricker, C.R. (1984) Isolation of salmonellae using two forms of Rappaport-Vassiliadis medium and brilliant green agar. Int. J. Food Microbiol. !, 171-177. Morinigo, M.A., Borrego, J.J. and Romero, P. (1986) Comparative study of different methods for dctcction and enumeration of Salmonella spp. in natural waters. J. Appl. Bacteriol. 61, 169-176. Mulindwa, D.K. and Pietzsch, O. (1979) Studies on the influence of competitive Enterobaeteriaceae flora on Salmonella isolation. Zentralb. Bakt. Mikrobiol. Hyg. Abt. I. Orig. A. 243, 336-348. Poisson, D.M., Bertin, N.D., Pariente, E.A. and Calamy, G. (1988) Salmonella agona saccharose-positive: ~tude bact6riologigue succinte el 6pid6miologique r6trospective h propos de I0 souches isol6es au CHR d'Orl6ans. M~d. Maladies Infect. 10, 433-439. Rengel, A. and Mendoza, S. (1984) Isolation of Salmonella from raw chicken in Venezuela. J. Food Prot. 47, 213-216. Sadovski, A.Y. (1977) Technical note: Acid sensitivity of freeze injured salmonellae in relation to their isolation from frozen vegetables by pre-enriehment procedure. J. Food Technol. 12, 85-91. Surdy, T.E. and Haas, S.O. (1981) Modified enrichment-serology procedure for detection of salmonellae in soy products. Appl. Environ. Microbiol. 42, 704-707. Threlfall, E.J., Hall, M.L.M. and Rowe, B. (1983) Lactose-fermenting salmonellae in Britain. FEMS Microbiol. Lctt. 17, 127-130. U.S. Food and Drug Administration (1984) Bacteriological Analytical Manual (6th edition). Association of Official Analytical Chemists, Arlington. van Schothorst, M. and van Leusden, F.M. (1975)Comparison of several methods for the isolation of salmonellae from egg products. Can. J. Microbiol. 21, 1041-1045. Wilson, C.R., Andrews, W.H. and Poelma, P.L. (1980) Recovery of Salmonella from milk chocolate using a chemically defined medium and five nondefined broths. J. Food Sci. 45, 310-313; 316.
