Lysine-Mannitol-Glycerol Agar, a Medium for the Isolation of

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ENVIRONMENTAL MICROBIOLOGY, Aug. 1993, p. 2602-2606

Vol. 59, No. 8

0099-2240/93/082602-05$02.00/0 Copyright © 1993, American Society for Microbiology

Lysine-Mannitol-Glycerol Agar, a Medium for the Isolation of Salmonella spp., Including S. typhi and Atypical Strains JULIAN M. COXt

Alliance Laboratory Services, P.O. Box 41, Red Hill, Queensland 4059, Australia Received 27 January 1993/Accepted 15 May 1993

An agar medium for the isolation of SalmoneUla spp. is described. The medium, lysine-mannitol-glycerol agar, has features of both xylose-lysine-deoxycholate agar and mannitol-lysine-crystal violet-brilliant green agar, but glycerol is added for the differentiation of SalmoneUla and Citrobacter spp. The medium facilitates the detection of strains having atypical fermentation patterns, such as the lactose- or sucrose-positive salmonellae. The medium also detects SalmoneUla typhi after enrichment.

The isolation of Salmonella spp. in both food and clinical settings is critical to the control of disease. In order to achieve this aim, numerous plating media have been described. These include a range of brilliant green agars, several deoxycholate-citrate-based agars, xylose-lysine-deoxycholate agar (XLD), Hektoen enteric agar, MacConkey agar, salmonella-shigella agar, and one of the most widely used media, bismuth sulfite agar (BS). BS is the medium of choice for the isolation of Salmonella typhi, particularly in medical microbiology laboratories, and it is used for the isolation of atypical, commonly lactose-fermenting salmonellae in food microbiology laboratories (7). BS does suffer from several disadvantages: isolation of S. typhi is best achieved by using freshly prepared medium, which is inhibitory to other Salmonella serotypes, as well as to contaminant organisms; because of the highly selective nature of the medium, incubation for 48 h is usually necessary for growth and development of the characteristic colony form; and the reactions of salmonellae on BS vary considerably with strain, medium batch, age, and manufacturer (10). A medium capable of detecting atypical strains is still required when workers test products in which a high proportion of strains exhibit unusual fermentation patterns, such as lactose-positive strains in dried milk powders (2). An alternative to BS for this purpose is highly desirable. New media for the isolation of salmonellae, including Rambach agar (RA) (12) and novobiocin-brilliant greenlactose agar (11), have been described. Although these media were designed to distinguish between Salmonella spp. and Proteus and Citrobacter spp., respectively, neither is likely to detect atypical strains, particularly lactose fermenters, and these media are unsuitable for the isolation of S. typhi. The primary aim of this study was to develop an inexpensive, easily interpretable medium that can be used for the isolation of atypical salmonellae, particularly lactose-fermenting strains. A secondary aim was to produce a medium suitable for the isolation of S. typhi. The medium developed was lysine-mannitol-glycerol agar (LMG). (J. M. Cox and K. Stallard originally reported the development of LMG at a meeting of the Australian Society for Microbiology [3].)

t Present address: Department of Microbiology, The University of Queensland, St. Lucia, Queensland 4072, Australia. 2602

MATERIALS AND METHODS

Chemicals, media, and medium constituents. Lysine, mannitol, cellobiose, sodium deoxycholate, and phenol red were obtained from Sigma. Sodium chloride, sodium thiosulfate, and ferric ammonium citrate were obtained from BDH Chemicals. Proteose peptone (catalog no. L85), yeast extract (catalog no. L21), tryptone soya agar (TSA) (catalog no. CM131), XLD (catalog no. CM469), MLCB agar (catalog no. CM783), and modified brilliant green-sulfamandelate agar (BGS) (catalog no. CM329 and SR87) were obtained from Oxoid. Agar (technical) was obtained from Davis Gelatin. A Microbact 24E identification system was obtained from Disposable Products. Bacterial strains. The strains used in this study are listed in Table 1. Strains were obtained from The Australian Collection of Microorganisms, Department of Microbiology, The University of Queensland, St. Lucia, Queensland, Australia; the Institute of Medical and Veterinary Sciences, Adelaide, Australia;andAllianceLaboratoryServices,RedHill,Queensland, Australia. Strains were cultured on TSA at 37°C for 24 h and were maintained on TSA slopes at room temperature. Prototype media. The prototype media contained a base (3.0 g of proteose peptone, 5.0 g of yeast extract, 5.0 g of lysine, 3.0 g of mannitol, 5.0 g of sodium chloride, 1.0 g of sodium deoxycholate, 0.1 g of phenol red, 15 g of agar, 1,000 ml of distilled water) to which an H2S detection system was added. The H2S detection system was either the system used in XLD (6.8 g of sodium thiosulfate per liter, 0.8 g of ferric ammonium citrate per liter) or the system used in MLCB agar (4.0 g of sodium thiosulfate per liter, 1.0 g of ferric ammonium citrate per liter). Initially, the XLD system was used. To this base was added either glycerol or cellobiose at a concentration of 1.0, 2.0, 5.0, or 10.0 g/liter. The medium was mixed to dissolve most ingredients, and the pH was adjusted to 7.4 with 0.1 M sodium hydroxide. The medium was boiled to completely dissolve all components and then cooled to 50°C and poured into petri dishes. Evaluation of prototype media. A plate of each medium was streaked with a 24-h TSA culture of either Salmonella sp. serotype salford (typical biotype) strain ACM 3762 (= IMVS 1710), Salmonella sp. serotype anatum (lactose-positive biotype) strain ACM 3538, or Citrobacterfreundii ACM 2197 (= ATCC 8090). The Salmonella sp. serotype salford strain is the positive control strain currently recommended by the Australian Standard method for isolation of Salmonella spp. (13). The plates were incubated at 37°C and were

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TABLE 1. Strains of Salmonella (biotypes and serotypes) and nonsalmonellae used to evaluate the final formulation of LMG Serotype

Typical biotype Aberdeen .............

Abony .............

Adelaide ............. Anatum ............. Arizonae .............

Ballerup ............. Bareilly .............

Berta ............. Birkenhead ............. Bovismorbificans ...........

Bredeny .............

Breukelen ............. Cerro .............

Charity .............

Chester ............. Choleraesuis ............. Cubana .............

Derby .............

Enteritidis ............. Give ............. Havana ............. Hirschfeldii ............. Infantis .............

Johannesburg ............. Kottbus .............

Lexington ............. a

Serotype

No. of strains

1 1 8 5 4 1 1 1 1 3 1 1 5 2 1 1 1 2 3 4 1 1 3 1 2 1

No. of strains

Serotype or species

Typical biotype Lille .................. London ..................

IL

1 1 Meleagridis .................. 2 Minnesota .................. 1 Monschauii .................. 2 Montevideo ................. 1 Muenchen .................. 2 2 Newport .................. Ohio .................. 2 Oranienberg ................. 2 Orion .................. 1 Paratyphi A ................. 1 Paratyphi.B ................. 2 Potsdam .................. 1 Pullorum .................. 1 1 Saintpaul .................. Salford .................. 1 Schwarzengrund ........... 1 Senftenberg ................. 6 Singapore .................. 5 2 Stanley .................. 1 Taksony .................. Tennessee .................. 1 3 Typhi .................. Typhimurium ............... 22 Virchow .................. 1

No. of strains

Typical biotype Wandsworth ........................... Zanzibar ................................

1 3

Lactose-positive biotype Anatum.................................. Derby .................................... Havana ..................................

1 1 1

Sucrose-positive biotype Infantis ..................................

1

H2S-negative biotype Berta ..................................... Choleraesuis ...........................

1 1

LDC-negative biotypea Derby ....................................

1

Citrobacter diversus

....................

4

.....................

22

Proteus mirabilis ........................ Proteus vulgaris .........................

2 6

Citrobacter freundii

Escherichia

coli

.........................

Enterobacter spp

. .......................

1

2

L

R

LDC, lysine decarboxylase.

examined after 24 and 48 h. The colony morphology was recorded at both times. Evaluation of final formulation. The colony morphologies of 133 Salmonella strains, representing 54 serotypes, as well as a number of other enterobacteria (Table 1), were assessed by streaking a 24-h TSA culture of each organism onto a plate containing LMG. Each LMG culture was incubated at 37°C and examined after 24 and 48 h. The colony morphology was recorded at both times. LMG was then evaluated for its ability to detect salmonellae in food samples and to discriminate between salmonellae and nonsalmonellae compared with other media that are used. The resuscitation and enrichment steps were performed essentially in accordance with the Australian Standard method (13). A sample of product was emulsified in 10 volumes of buffered peptone water and incubated at 37°C for 18 to 24 h. Aliquots of the buffered peptone water enrichment were then inoculated into selective broth media; 1 ml was inoculated into 10 ml of mannitol-selenite-cystine broth (catalog no. CM399; Oxoid) (4 g of sodium biselenite per liter, 0.01 g of L-cystine per liter), and 0.1 ml was inoculated into Rappaport-Vassiliadis broth (catalog no. CM669; Oxoid). These broth media were incubated for 18 to 24 h at 37°C (mannitol-selenite-cystine broth) or 42°C. The selective enrichment broth media were then streaked onto plates containing LMG, XLD, MLCB, and BGS agar media. MLCB and BGS were the plating media in use at Alliance Laboratory Services during the performance of this study. XLD was included for comparative purposes, given the similarity between LMG and XLD. Suspect colonies from each medium were then restreaked onto the medium from which they were taken. Any isolates still displaying a Salmonella-like colony form were then tested biochemically by using the Microbact 24E system according to the manufac-

turer's instructions. Chase rates of nonsalmonellae from each medium were determined. Chase rates were defined as the proportions of samples tested from which nonsalmonellae were restreaked (stage 1) or tested biochemically (stage 2) and were expressed as percentages. LMG was then used in combination with BGS to test a wide range and large number of food products (see Table 3). MLCB agar was not used because of the high number of false-positive isolates obtained with this medium. Resuscitation and selective enrichment steps were performed as described above. The number and biotypes of salmonellae isolated from each type of product on each medium were recorded.

RESULTS The prototype media were evaluated by using a typical Salmonella strain, a lactose-positive Salmonella strain, and a strain of C. freundii. The media were examined for colony forms similar to the colony forms observed on XLD. Salmonella spp. were expected to produce initial acidification of the medium (a yellow color in the medium) because of fermentation of mannitol. The salmonellae were not expected to ferment the other carbon sources used (glycerol and cellobiose). The salmonellae were then expected to produce alkaline reversion in the medium (a change in medium color from yellow, through orange, to pink) primarily because of decarboxylation of lysine and secondarily because of utilization of the proteinaceous substrates (proteose peptone and yeast extract) in the medium. Under alkaline conditions, the salmonella colonies were expected to blacken because of the production of hydrogen sulfide and the deposition of black iron sulfide in the colonies. C. freundii was expected to ferment both mannitol and glycerol

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LMG after incubation at 370Ca Colony morphology

TABLE 2. Colony morphologies of different Salmonella biotypes and other enterobacteria Organism

on

Typical, lactose-positive and sucrose-positive Salmonella spp .Colonies have black centers, ranging in size from small bull's-eyes to zones extending almost to the edges of the colonies; each colony is surrounded by a zone varying in color from yellow-orange to pink; after 48 h, the colonies are almost entirely black, and each colony is surrounded by a pink zone S. typhi .Yellow colonies are surrounded by yellow zones; after 48 h, a small black center in each colony is surrounded by a yellow zone; typical colonies occur after 24 h following selenite enrichment H2S-negative Salmonella spp .Colonies are pink or colorless and are surrounded by pink zones in the medium LDC-negative Salmonella sp. serotype derbyb.Colonies are yellow with yellow zones in the medium; after more than 48 h of incubation, slight alkaline reversion in the medium occurs and the colonies have small black centers Citrobacter spp. (most strains) .Yellow colonies occur with yellow zones in the medium; no alkaline reversion or blackening occurs after prolonged incubation (up to 7 days) Proteus spp .Colonies have dark centers; colonies are surrounded by pink zones; dark centers are generally smaller than those of Salmonella spp. and greenish grey or black a The information is for colonies growing after incubation for 24 h, unless specified otherwise. b LDC, lysine decarboxylase.

cellobiose, prolonging acid conditions in the medium and inhibiting H2S production. Cellobiose proved to be less effective than glycerol as a carbon source for the differentiation of Salmonella and Citrobacter spp. At all of the concentrations of cellobiose tested, alkaline reversion by both of the Salmonella strains tested was retarded. Blackening of the colonies was poor after 24 h but obvious after 48 h. C. freundii colonies did not blacken on the cellobiose media, which remained acid (yellow) after prolonged incubation. Both Salmonella and Citrobacter strains produced alkaline reversion in the glycerolcontaining media, with blackening of the colonies at the lower concentrations (1 and 2 g/liter) of glycerol. At a glycerol concentration of 5 g/liter Salmonella strains produced alkaline reversion in the medium, and the colonies became black within 24 h. This medium inoculated with C. freundii remained acid, and no blackening was observed after prolonged incubation (up to 7 days). The highest concentration of glycerol delayed alkaline reversion by the salmonellae. Detection of H2S production was found to be stronger and more rapid on media when the MLCB agar detection system was used, as blackening of the Salmonella colonies was more complete after 24 h compared with colonies on media containing the XLD system. The final formulation of LMG contained (per liter) 3.0 g of proteose peptone, 5.0 g of yeast extract, 5.0 g of lysine, 5.0 g of mannitol, 5.0 g of glycerol, 5.0 g of sodium chloride, 1.0 g of sodium deoxycholate, 4.0 g of sodium thiosulfate, 1.0 g of ferric ammonium citrate, 0.1 g of phenol red, and 15 g of or

LMG, XLD, and MLCB agar. Over a period of 3 months, 317 food samples were tested. These included predominantly egg and dairy products, as well as prepared meals, fresh and fermented meats, seafood, animal feeds, and feed components. During this period, two lactose-positive Salmonella strains were isolated from LMG and MLCB cultures but not from XLD or BGS cultures. The chase rates (see above) are shown in Table 3. The chase rates for LMG and XLD were similar for both stages of testing. Considerably more falsepositive cultures were detected on MLCB agar, and a high proportion required biochemical testing. The most common (97%) false-positive colonies on LMG were cultures of Citrobacter spp. In most cases, as the chase rates indicate, restreaking of the colonies on LMG was sufficient to reveal these colonies as false-positive colonies without the need for biochemical testing. The suspect colony morphology of Citrobacter spp. was also different from the colony morphology produced by salmonellae, as the black colonies were shiny in appearance while Salmonella colonies were matte or dull in appearance. Salmonella colonies were generally larger than colonies of Citrobacter spp. Proteus spp. produced similar colony morphologies in pure culture and on primary isolation plates. With experience, differentiation of Citrobacter and Proteus spp. from Salmonella spp. was straightforward. TABLE 3. Chase rates, expressed as percentages, for LMG, XLD, and MLCB agar'

agar.

The final formulation was evaluated by using a large set of strains (Table 1). The colony morphologies on LMG of different serotypes or biotypes of Salmonella spp., most strains of Citrobacter spp., and Proteus spp. are shown in Table 2. Most salmonellae produced a colony morphology on LMG similar to that observed on XLD. Two strains of Citrobacter diversus, which were lysine decarboxylase positive, produced a colony form similar to the salmonella colony form. The efficiency of Salmonella isolation and discrimination between salmonellae and nonsalmonellae were compared for

Chase rates (%)

Stage of testing

1 (restreak) 2 (Microbact 24E identification system)

LMG

XLD

MLCB MC agar

16.7 (53)b 3.2 (10)

11.7 (37) 3.5 (11)

42.6 (135) 22.7 (72)

a The chase rate was the proportion of samples tested (on each of the agar media) from which nonsalmonellae producing a suspect colony form were restreaked (stage 1) or were tested biochemically by using the Microbact 24E identification system (stage 2). A total of 317 samples were tested. b The numbers in parentheses are the numbers of retests performed.

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LMG AGAR FOR ISOLATION OF SALMONELLA SPP.

TABLE 4. Food products tested and number of Salmonellapositive samples as determined by using LMG No. of

Type of sample

No. of

samples

Egg products Whole eggs Yolk Albumen Peeled boiled eggs Other Dairy products Dried milk powders Ice cream Chocolate or cocoa Meat or meat products Raw meat Fermented meat Meat meal Chicken (cooked) Seafood Salad Prepared meals or mixed foods

Salmonella-

Total no. of

positive samples on.

samplesa

positive

LMG

BGS

192 105 101 304 241

3

2

1 1

1 1

3 0 1 1 0

541

10

0

10

83 11

1

1

0 1

2

2

14

15

3

3

29 39 23 2 11 5 267

2 0 15 0 0 0 3

a The total number of positive samples is the total number of samples positive for SalmoneUa spp. rather than the sum of the numbers of salmonel-

lae isolated

on

the two media.

During 9 months of routine use, 1,954 food and feed samples were tested (Table 4) and 36 salmonellae were isolated; 24 of these salmonellae were biochemically typical Salmonella strains, and 11 were lactose-positive strains. One Salmonella sp. serotype arizonae strain was isolated from pasteurized egg pulp. The other 10 isolates were lactosepositive strains belonging to subspecies 1 serotypes (2 Salmonella sp. serotype derby strains and 8 Salmonella sp. serotype anatum strains) isolated from dried milk powder or powder residues from the production environment. An H2Snegative strain was isolated from meat meal on BGS, but not on LMG. DISCUSSION LMG was developed to provide a medium that had good differential properties and was capable of detecting atypical, particularly lactose-fermenting, salmonellae. The formulation was based on a hybrid of XLD and MLCB agar, and another fermentable carbon source was added to distinguish between Salmonella and Citrobacter spp. Glycerol proved to be superior to cellobiose as the additional substrate. Cellobiose and glycerol are two carbon sources which may differentiate Salmonella and Citrobacter spp. In Salmonella subspecies I (other than S. typhi), the percentage of glycerolor cellobiose-fermenting strains is approximately 5% (4, 5), while the percentages in C. freundii and C. diversus are 98% (glycerol) and 55 and 99%, respectively (cellobiose). There are conflicting reports as to the percentage of glycerolfermenting strains of S. typhi. Farmer et al. (5) give a figure of 20%, while Ewing (4) provided a figure of 0% after 24 to 48 h, with 94% of the strains producing a weak-positive reaction after more than 72 h of incubation. This suggests that LMG should be examined within 48 h after inoculation. In prac-

2605

tice, most salmonellae produced a suspect colony form within 24 h, so late fermentation of glycerol should not interfere with the interpretation of plates. Unlike many other members of Salmonella subspecies I, Salmonella sp. serotype typhi produces little or no H2S on media such as triple sugar iron agar or Kligler's iron agar, which are designed for detection of the product (4). Salmonella sp. serotype typhi does not produce H2S from inorganic sulfur compounds, such as thiosulfate, but rather produces H2S from organic sulfur compounds, such as amino acids (1). It is likely that the presence of proteose peptone and yeast extract in the medium provides enough such compounds to allow S. typhi to clearly express this phenotype. Of interest was the improved appearance of Salmonella sp. serotype typhi on LMG after enrichment in selenite broth compared with a subculture from a nutrient agar. No simple explanation could be found for this. Presumably, growth in the broth stimulated cellular mechanisms responsible for, or involved in, H2S production on the solid medium. Although the medium was designed to distinguish between Citrobacter and Salmonella spp., Citrobacter spp. were the most common organisms producing false-positive colony morphology from mixed cultures (i.e., from food sample enrichments). In most cases, a nonsuspect colony form was observed after the suspect colonies were streaked on LMG. Presumably, other organisms close to the non-Salmonella colonies on the primary plate were able to produce localized conditions in the medium which allowed the non-Salmonella organism to produce a suspect colony form in the mixed culture. Once isolated by restreaking, the non-Salmonella organism produced a typical, nonsuspect colony form. The rate of false-positive results due to these organisms was comparable to the rate found with XLD and significantly lower than the rate when MLCB agar was used. Although MLCB agar is suitable for the detection of atypical salmonellae (9) and has been used successfully in the analysis of food samples, particularly in combination with RappaportVassiliadis broth (14), the rates of false-positive results obtained in previous studies and in this study were considered unacceptable. MLCB agar is unsuitable for the isolation of S. typhi because of the inhibitory concentration of brilliant green. Proteus spp. also produced a suspect colony form on LMG, but with experience Proteus sp. colonies are easily differentiated from salmonella colonies. In most food microbiology laboratories, PIroteus spp. are encountered infrequently, whereas they are common contaminants in medical specimens, notably feces. Direct replacement of XLD with LMG in a given isolation protocol has advantages. As the colony forms of salmonellae are similar on the two media, operator familiarity is quickly developed. The rates of falsepositive results are comparable, and LMG is capable of detecting atypical strains. LMG provides several distinct advantages over BS, the medium currently used for detection of atypical salmonellae. The incubation time is shorter (24 versus 48 h), the distinct differential properties facilitate selection of suspect isolates, and the frequency of isolation of Salmonella spp. is increased through lower selective pressure. LMG can also be used for the isolation of S. typhi. In summary, LMG provides a suitable alternative to BS for the isolation of atypical salmonellae and S. typhi. Because BS was no longer in routine use in my laboratory at the time of development of LMG, no comparison was made between LMG and BS. While LMG was capable of distinguishing lactose- and

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sucrose-fermenting strains, it was not suitable for the isolation of other atypical strains, such as H2S- and lysine decarboxylase-negative strains. The use of LMG with other media, such as the brilliant green agars, provides a combination which permits the isolation of most serotypes and biotypes of Salmonella spp. During the course of the trial with food samples, an H2S-negative strain was isolated on the brilliant green agar used. The probability of encountering a strain having a biotype (such as H2S negative, lactose positive) which would elude detection on such a combination of media is extremely low. The versatility of LMG compares favorably with the versatilities of other recently developed media. Rambach (12) used a novel property, the utilization of propylene glycol, to differentiate between Salmonella spp. and other enterobacteria, particularly Proteus spp. Typical salmonellae produce a crimson red color on RA. RA also contains the substrate 5-bromo-4-chloro-3-indolyl-,-D-galactopyranoside (X-Gal) for the differentiation of lactose-fermenting enterobacteria, such as Citrobacter spp. Lactosepositive salmonellae are capable of utilizing this substrate and thus produce a violet, nonsuspect colony form similar to that described for Citrobacter spp. Some salmonellae have been found to produce nontypical colonies on RA (6, 8), but the fermentation patterns of these strains were not reported. S. typhi is not detected on RA as the S. typhi serotype produces colorless colonies. Besides these limitations, RA is costly for routine commercial use, primarily because of the cost of the X-Gal substrate. Poisson (11) incorporated glycerol as a substrate in novobiocin-brilliant green-lactose agar on the basis of the same principle that was used in this study (i.e., differentiation of Salmonella and Citrobacter spp.). Novobiocin-brilliant green-lactose agar also contains lactose, which makes the medium inappropriate for the isolation of lactose-positive strains. The concentration of brilliant green was found to inhibit S. typhi. LMG has been shown to be suitable for the isolation of the S. typhi serotype, particularly following enrichment. The suitability of LMG for use in a clinical microbiology setting is currently being evaluated. This study will also compare the efficiencies of LMG and BS for the isolation of salmonellae. The use of novobiocin as an additional selective agent is also being assessed. ACKNOWLEDGMENTS I thank the technical staff of Alliance Laboratory Services, particularly Nicole McMillan, for their assistance with this study. I

also thank Lindsay Sly and Chris Murray for generously providing cultures and Linda Blackall for revision of the manuscript.

1. 2.

3. 4.

5.

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Microbiol. 21:46-76. 6. Freydiere, A.-M., and Y. Gille. 1991. Detection of salmonellae by using Rambach agar and by a C8 esterase spot test. J. Clin.

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campylobacters. J. Appl. Bacteriol. 63:99-116. 8. Gruenewald, R., R. W. Henderson, and S. Yappow. 1991. Use of Rambach propylene glycol-containing agar for identification of Salmonella spp. J. Clin. Microbiol. 29:2354-2356. 9. Inoue, T., S. Takagi, A. Onishi, K. Tamura, and A. Suzuki. 1968. Food-borne disease Salmonella isolation medium (MLCB). Jpn. J. Vet. Sci. 30(Suppl.):26. 10. Jay, L. S., and G. R. Davey. 1989. Salmonella: characteristics, identification and enumeration, p. 51-82. In K. A. Buckle, J. A. Davey, M. J. Eyles, A. D. Hocking, K. G. Newton, and E. J. Stuttard (ed.), Foodborne microorganisms of public health significance. Australian Institute of Food Science and Technology New South Wales Food Microbiology Group, Sydney, Australia. 11. Poisson, D. M. 1992. Novobiocin, brilliant green, glycerol, lactose agar: a new medium for the isolation of Salmonella strains. Res. Microbiol. 143:211-216. 12. Rambach, A. 1990. New plate medium for facilitated differentiation of Salmonella spp. from Proteus spp. and other enteric bacteria. Appl. Environ. Microbiol. 56:301-303. 13. Standards Association of Australia. 1989. Australian Standard 1766.2.5-1989. Methods for the microbiological examination of foods-salmonellae. Standards Association of Australia, Sydney, Australia. 14. van Schothorst, M., A. Renaud, and C. van Beek. 1987. Salmonella isolation using RVS broth and MLCB agar. Food Microbiol. 4:11-18.