APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 1978, p. 747-751 0099-2240/78/0036-0747$02.00/0 Copyright © 1978 American Society for Microbiology
Vol. 36, No. 5 Printed in U.S.A.
Comparison of Four Agar Plating Media with and Without Added Novobiocin for Isolation of Salmonellae from Beef and Deboned Poultry Meat W. A. MOATS Agricultural Research Center, U.S. Department of Agriculture, Beltsville, Maryland 20705
Received for publication 11 September 1978
Four plating media, Hektoen enteric (HE), xylose-lysine deoxycholate (XLD), tryptic soy-xylose-lysine (TSXL), and tryptic soy-brillant green (TSBG) agars with and without 10 mg of added novobiocin per ml, were evaluated for recovery of Salmonella from roast beef and deboned turkey. Colonies producing a reaction typical of H2S-positive salmonellae (alkaline with black centers) were picked. On the media without novobiocin, from 109 determinations on 75 samples, number of salmonellae found and false-positives were, respectively: HE- 13, 58; XLD- 17, 18; TSXL-23, 0; TSBG-22, 7. When novobiocin was present the corresponding results were: HE- 17, 24; XLD-21, 2; TSXL-23, 3; TSBG-20, 7. A total of 25 determinations were positive on one or more agars. False-positives on HE and XLD without novobiocin were predominantly Proteus, which were almost totally eliminated by addition of 10 mg of novobiocin per liter. If alkaline H2S-negative colonies had been considered, many more false-positives would have been found on HE and XLD but not on TSBG or TSXL. Addition of novobiocin markedly improved isolations of salmonellae from XLD and HE and reduced the number of false-positives. Addition of novobiocin did not improve performance of TSXL and slightly impaired differentiation of salmonellae from Citrobacter on TSBG. XLD with novobiocin and TSXL are highly specific for H2S-positive salmonellae, and the appearance of Salmonella-like colonies on these media can be considered a presumptive test for H2S-positive salmonellae.
Several improved plating media for isolation of salmonellae have been developed in recent years. These include the Hektoen enteric (HE) agar of King and Metzger (5), the xylose-lysine agars of Taylor (10), and two improved brilliant green formulations described by Moats and Kinner (6). All these contain indicators of H2S production so that salmonellae can be identified by the production of black-centered colonies as well as by the pH reaction. Taylor (10) also included xylose and lysine in the media to improve differentiation of salmonellae from organisms such as Citrobacter and Proteus which do not produce lysine decarboxylase. Taylor (10) described two formulations-xylose-lysine deoxycholate
(XLD), which is only mildly selective for salmonellae, and xylose-lysine-brilliant green, which is more selective. Moats and Kinner (6) developed an improved brilliant green formulation with tryptic soy agar as a base (TSBG) and also a modification which included xylose and lysine (TSXL), as described by Taylor (10). Moats and Kinner (6) found that Taylor's xylose-lysine-brilliant green agar prepared from xylose-lysine agar base was inhibitory to many
salmonellae. However, use of tryptic soy agar as a base greatly improved growth of salmonellae while retaining the desirable selective and differential properties of the medium. There have been several recent reports (4, 6-9) of the addition of novobiocin to plating media to improve selectivity for salmonellae. Novobiocin at 5 to 10 ,ug/ml has been found to totally suppress growth of Proteus strains (4, 6-9). Varying degrees of inhibition of Escherichia coli (4, 6, 8), Citrobacter (4, 6, 8), and Pseudomonas (7) have also been reported. In the present study, I compared the effectiveness of four plating media, HE, XLD, TSXL, and TSBG agars, with and without 10 mg of added novobiocin per liter for isolating salmonellae. Meat samples were used for the comparison since they have been commonly found to contain large numbers of interfering organisms. Addition of novobiocin to brilliant green agars has not been previously reported. MATERIALS AND METHODS Media. HE agar (Difco, Detroit, Mich.) was prepared in accordance with the manufacturer's instruc747
748
APPL. ENVIRON. MICROBIOL.
MOATS
34) and 10 deboned turkey samples were obtained as tetrathionate-brilliant green enrichment broths, and plates were streaked directly from these. Frozen samples (n = 34) of deboned turkey meat were tested with lactose broth preenrichment and selenite-cystine and tetrathionate enrichments. Plates were incubated for 20 to 24 h at 37°C and examined for the presence of Salmonella-like colonies. One colony was picked from each plate showing suspect colonies and inoculated onto triple sugar iron and lysine agars. All picks were further characterized biochemically whether or not they gave typical Salmonella-like reactions on triple sugar iron and lysine
tions. XLD agar was prepared from xylose-lysine agar base (Difco) with 2.50 g of sodium deoxycholate, 0.80 g of ferric ammonium citrate, and 6.80 g of sodium thiosulfate .5H20 added per 1,000 ml of medium. The dry ingredients were dissolved in water, heated to boiling, cooled to 50°C, and poured into plates. TSBG and TSXL agars were prepared as previously described (6). Formulas used were, per 1,000 ml: TSBG-tryptic soy agar (Difco), 40 g; lactose, 8 g; sucrose, 8 g; sulfanilamide, 1 g; ferric ammonium citrate, 1.5 g; sodium thiosulfate * 5H20, 5.0 g; phenol red, 80 mg; brilliant green, 5 mg; TSXL-tryptic soy agar, 40 g; sucrose, 8 g; sulfanilamide, 1 g; ferric ammonium citrate, 1.5 g; sodium thiosulfate * 5H20, 5.0 g; agar, 5.0 g; phenol red, 80 mg; brilliant green, 5 mg. The dry ingredients except brilliant green were suspended in water, and the pH was adjusted as necessary to 6.8 to 6.9 with 3 N HCl or NaOH. The media were then heated to dissolve the ingredients, sterilized for 15 min at 15 lb/in2, and cooled in a water bath at 50°C. Brilliant green was added from a filter-sterilized stock solution containing 1 mg/ml, and the medium was immediately poured into plates. The media were also prepared as above with 10 mg of sodium novobiocin (925 ytg/mg)/liter added to the other dry ingredients. The novobiocin was a gift from The Upjohn Co., Kalamazoo, Mich. Other media used were lactose broth, selenite-cystine broth, tetrathionate broth, Simmons citrate agar, triple sugar iron agar, and lysine agar, purchased from Baltimore Biological Laboratory (BBL), Cockeysville, Md. Carbohydrate test media were prepared in purple broth base (BBL). Other biochemical reagents were prepared according to Edwards and Ewing (2), using the Falkow formulations for decarboxylase test media. Test procedure. Test samples were obtained from the Microbiology Laboratory, FSQS, U.S. Department of Agriculture, Beltsville, Md., and were selected from lots of roast beef and deboned turkey suspected of containing Salmonella. The roast beef samples (n =
agars.
RESULTS AND DISCUSSION For the present study novobiocin was used at a concentration of 10 mg/liter in the agars. This concentration has been consistently found to totally suppress growth of Proteus without affecting growth of salmonellae. Higher concentrations which have been used in some cases were only partially effective in suppressing growth of other interfering organisms (4, 8) and have been reported to retard growth of salmonellae (4). The typical characteristics of various types of bacterial colonies likely to be found on the four plating media studied without added novobiocin are summarized in Table 1. HE agar is a relatively nonselective medium on which Proteus and Citrobacter freundii strains form colonies indistinguishable from salmonellae. Also, Pseudomonas and those Enterobacter hafniae strains which do not ferment sucrose, lactose, or salicin form green colonies resembling those of H2S-negative samonellae. XLD agar is also relatively nonselective. On it, Citrobacter and many Proteus strains ordinarily form yellow col-
TABLE 1. Appearance of bacterial colonies on plating media after 24-h growth at 37°C Appearance of colonies on:
Organism Salmonella S. cholerae-suis, H2S negative Salmonella, lactose positive Escherichia coli Proteus vulgaris Proteus mirabilis Klebsiella-Enterobacter Enterobacter hafniae (some) Citrobacter freundii
Pseudomonas
HE XLD Green, black Pink, black centers centers Green
Pink
Orange
Yellow
TSXL TSBG Green to pink, large black Pink, black centers centers Green or greenish pink Pink Green to pink, large black Green centers No growth No growth No growth No growth
Orange Yellow Green, black Yellow centers Green, black Yellow or pink, black No growth or tiny green No growth or tiny pink centers centers colonies; rarely, pink colonies; rarely, pink with black centers with black centers Orange Yellow Green Green Green Pink Usually no growth Usually no growth Green, black Yellow; rarely, pink with Green, occasional small Green, green with black centers black centers black centers centers; occasionally pink with black centers Green Pink No growth or tiny green- No growth or tiny pink pink colonies colonies
VOL. 36, 1978
COMPARISON OF MEDIA FOR SALMONELLAE ISOLATION
onies which are clearly distinguishable from salmonellae. However, Pseudomonas and E. hafniae strains which do not ferment lactose or sucrose form pink colonies resembling those of H2S-negative salmoneflae. TSBG and TSXL are more selective. Most Proteus strains are strongly inhibited and show little growth. C. freundii strains form green to occasionally pink colonies with black centers on TSBG. On TSXL Citrobacter colonies are green, occasionally with small black centers, which are easily distinguishable from the almost totally black Salmonella colonies. TSXL does not contain lactose, and so lactose-fermenting salmonellae appear the same as other salmonellae. Salmonellae do not give a very clear-cut alkaline reaction on TSXL, and H2S-negative types are therefore not clearly distinguishable from coliforms. The roast beef samples used were obtained as tetrathionate-brilliant green enrichments from the FSQS Microbiology Laboratory. Addition of novobiocin to XLD and HE agars improved recoveries of salmonellae from roast beef samples and virtually eliminated false-positives (Table 2). Novobiocin had little effect on results with TSBG and TSXL agars. Some of the deboned turkey samples were also streaked from tetrathionate-brilliant green broth enrichments. The remainder were carried through conventional lactose preenrichment followed by enrichment in tetrathionate and selenite-cystine broths. The tetrathionate and selenite-cystine enrichments were tabulated as separate determinations. On this sample series (Table 3), no one agar recovered salmonellae from all determinations which were positive on one or more agars. The most recoveries and the fewest false-positives were on TSXL with or without novobiocin. Fewer salmonellae were recovered on TSBG with novobiocin than on that without it. It was observed that Salmonella colonies TABLE 2. Isolations of salmonellae from roast beef samples enriched in tetrathionate brilliant green brotha No. of:
Agar
False-positives Samples positive 13 10 HE 0 14 HE-Nb 8 12 XLD 0 14 XLD-N 0 14 TSXL 1 14 TSXL-N 0 14 TSBG 0 14 TSBG-N a on for Salmonella 14 positive Thirty-one samples, one or more agars. b N, Contains 10 gg of novobiocin per ml.
749
were slightly less alkaline and Citrobacter were slightly less acid on TSBG with novobiocin than on that without novobiocin, and these effects resulted in confusion of the two. False-positives on XLD were almost eliminated by addition of novobiocin, but slightly fewer salmonellae were recovered than on TSXL or TSBG. Recovery of salmonellae on HE agar was not improved by addition of novobiocin. The number of falsepositives was reduced but was still far higher than on the other agars. The combined data for beef and turkey are summarized in Table 4. The classification of types of colonies picked as Salmonella-like is summarized in Table 5. Organisms producing false-positive colonies on HE and XLD agars were predominantly Proteus mirabilis, with a few Proteus vulgaris from HE agar. Addition of novobiocin, which eliminated Proteus strains, was therefore highly advantageous with these agars. With HE agar, a substantial number of false-positive colonies from Citrobacter strains remained after elimination of Proteus. Citrobacter strains rarely form falsepositive colonies on XLD agar, so, with the elimination of Proteus, this medium is virtually TABLE 3. Isolations of salmonellae from 44 samples of turkey meat No. of: Agar
Salmo-
nella isolated 3 1 4 6 7 7 7 5
S. ari- Total' Salzonae iso- monella
lated 0 2 0 1 2 2 1 1
False-positives
45 22 4 10 7 2 9 0 2 9 8 7 6 6 aSeventy-eight determinations, 11 positive for salmonellae on one or more agars. b N, Contains 10 mg of novobiocin per liter.
HE HE-N b XLD XLD-N TSXL TSXL-N TSBG TSBG-N
3 3
TABLE 4. Recovery of salmonellae from all testsa No. false-positive No. positive Agar 58 13 HE 24 17 HE-Nb 18 17 XLD 2 21 XLD-N 0 23 TSXL 3 23 TSXL-N 7 22 TSBG 7 20 TSBG-N One hundred nine determinations, 25 positive for on one or more agars. salmonellae b N, Contains 10 mg of novobiocin per liter. a
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MOATS
TABLE 5. Classification of Salmonella-like colonies Agar
Proteus vulgaris Proteus mirabilis
HE HE-Na XLD XLD-N TSXL TSXL-N TSBG TSBG-N a N, Contains
3 44 1 0 0 17 0 0 0 0 0 0 0 1 0 0 10 mg of novobiocin per liter.
freundoc
Salmonella
S. arizonae
Total picks
11
13 15 17
0 2 0 1 2 2 1 1
71 41 35 23 23 26 28 28
23 1 2 0 3 6 7
specific for H2S-positive salmonellae. Since Proteus strains rarely produce false-positive colonies on TSBG and TSXL, addition of novobiocin is of marginal value. In the present sample series it appears that novobiocin may have slightly impaired differentiation of sahnonellae from Citrobacter as shown by false-positives on TSXL with novobiocin (10 mg/liter) and poorer recoveries of salmonellae on TSBG with novobiocin (10 mg/liter). False-positive colonies picked from TSXL with novobiocin (10 mg/liter) were rather atypical of Salmonella and would not have been picked if typical Salmonella colonies had been present on the same plate. Working with stool samples, Taylor and Schelhart (11) found that P. mirabilis was the predominant H2S-producing organism producing false-positives on XLD and that far more C. freundii produced false-positives on HE than on XLD. Restaino et al. (8) found that on HE and XLD the predominant H2S-producing false-positives from pork sausage were C. freundii and P. mirabilis, respectively. They also observed that H2S-producing E. coli, which appear to be quite rare, could also produce false-positives on HE and XLD and were not inhibited by novobiocin. For the present study, only H2S-positive colonies were picked. However, H2S-negative salmonellae do occur occasionally, and some alkaline H2S-negative colonies were observed on HE and XLD agars both with and without novobiocin. A few of these were picked and identified as either Pseudomonas or E. hafniae. If these were included in the data, the number of false-positives on HE and XLD agars would have been increased. No colonies of this type were found on TSBG or TSXL agars, and the isolates from HE and XLD showed little if any growth on TSBG. It would therefore appear that TSBG would be more satisfactory than HE or XLD for detection of H2S-negative salmonellae. As previously discussed, TSXL agar does not clearly differentiate H2S-negative sahnonellae from coliforms. Taylor and Schelhart (11) found substantial numbers of H2S-negative organisms in
20 21 21 21 19
stool samples which produced false-positives on HE and XLD agars. These were predominantly Pseudomonas and slow-fermenting strains of Escherichia, Klebsiella, Enterobacter, Serratia, and Aeromonas. Proteus rettgeri and P. morganii and assorted nonfermnentative gramnegative organisms also produced small numbers of false-positives. The reasons why salmonellae were not isolated from plating mddia where one or more plates from the enrichment were positive are summarized in Table 6. In most cases where salmonellae were not found, no Salmonella-like colonies were present. Occasionally, especially on HE agar, Salmonella-like colonies were present, but Salmonella, if present, were not picked. Possibly, if more colonies had been picked, salmonellae would have been found. However, the difficulty in picking salmonellae in the presence of other organisms producing Salmonella-like colonies is obvious. The absence of Salmonellalike colonies may result from overgrowth of salmonellae by other organisms, poor differentiation of salmonellae from other organisms, or failure of salmonellae to grow on the plates. Where only one or two Salmonella-like colonies were found on positive plates, which occurred several times, other plates streaked from the same enrichment may have been negative purely by chance. Assuming that salmonellae grew equally well on all the media tested, more recoveries would be expected on the more selective media (TSXL and TSBG) since overgrowth by other organisms is less likely; this is what was found. Biochemical confirmation of salmonellae suspect colonies from any of the four agars studied is relatively simple since considerable biochemical differentiation is accomplished on the agars. All picks from HE and XLD agars and most from TSXL and TSBG also gave Salmonellalike reactions on triple sugar iron agar. Triple sugar iron agar was therefore not used in the last half of the study. Lysine agar, on the other hand, proved extremely useful because most picks
COMPARISON OF MEDIA FOR SALMONELLAE ISOLATION
VOL. 36, 1978
TABLE 6. Reasons why salmonellae were not found on plating media False-negatives
Agar
No. posi-
tive
No.
Salmonella like colonies absent
Salmonellalike colonies
picked-not SalmoneUa
HE 13 12 1 11 HE-N a 17 8 5 3 XLD 11 9 7 2 XLD-N 21 4 4 0 TSXL 23 2 2 0 TSXL-N 23 2 2 0 22 TSBG 3 3 0 TSBG-N 20 5 4 1 N, Contains 10 mg of novobiocin per liter. a
correctly identified on it. Proteus strains easily identified from the characteristic red slants, and most Citrobacter strains gave an acid butt. Of other biochemical tests, lysine and ornithine decarboxylases, mannitol and dulcitol fermentation, and malonate utilization ordinarily differentiated the H2S-positive picks satisfactorily. Simmons citrate and urease provided additional confirmation. Lactose fermentation was also tested with picks from TSXL, which does not contain lactose. Of non-H2S-producing picks, Pseudomonas strains could be readily differentiated by the oxidase test or oxidation-fermentation of glucose. E. hafniae strains picked from XLD with novobiocin (10 mg/liter) did not ferment lactose or sucrose and were otherwise quite similar biochemically to salmonellae, but could be distinguished by failure to ferment sorbitol. Bisciello and Schrade (1) tested over 2,000 assorted foods and found that HE agar gave more isolations of salmonellae with fewer falsepositives than bismuth sulfite, salmonella-shigella, or brilliant green agars. On the other hand, Goo et al. (3), in analyzing over 11,000 food samples, obtained better recovery of salmonellae on brilliant green than on HE agar, although more false-positives were found on brilliant green. Taylor and Schelhart (11) found that HE and XLD agars gave equivalent recoveries of salmonellae and shigellae from stool specimens but that HE agar gave twice as many false-
isfactory with these samples than the other three agars tested, even with added novobiocin, both from the standpoint of salmonellae recovered and false/positives. TSBG agar produced slightly more false-positives than TSXL but was nearly comparable for the isolation of salmonellae. Growth of organisms which might interfere with detection of H2S-negative salmonellae was largely suppressed on TSBG. In our laboratory all enrichments are routinely streaked on both TSBG and TSXL agars. This allows the possibility of recovering both lactose-fermenting and H2S-negative salmonellae as well as typical types. Citrobacter strains which produce falsepositives on TSBG are immediately identified by their failure to form Salmonella-like colonies on TSXL.
were were
ACKNOWLEDGMENTS I thank Alice Moran, U.S. Department of Agriculture Food Safety and Quality Service Microbiology Laboratory, for providing the samples used and for helpful discussions; The Upjohn Co., Kalamazoo, Mich., for providing the novobiocin used; and T. M. Brennan and J. I. Shultz, Jr., for technical assistance.
LITERATURE CITED 1. Bisciello, N. B., and T. P. Schrade. 1974. Evaluation of 2. 3.
4.
5. 6.
7.
8.
positives.
XLD with novobiocin and TSXL are highly specific for H2S-positive samonellae, and the occurrence of salmonellae-like colonies on these media can be considered a presumptive test for H2S-positive salmonellae. TSXL gave slightly higher recoveries of salmonellae than XLD with novobiocin in the present study and has the additional capability of detecting lactose-fermenting salmonellae. HE was distinctly less sat-
751
9. 10. 11.
Hektoen enteric agar for the detection of Salmonella in foods and feeds. J. Assoc. Off. Anal. Chem. 57:992-996. Edwards, P. R., and W. H. Ewing. 1972. Identification of Enterobacteriaceae, 3rd ed. Burgess Publishing Co., Minneapolis. Goo, V. Y. L., G. Q. L. Ching, and J. M. Gooch. 1973. Comparison of brilliant green agar and Hektoen enteric agar media in the isolation of salmonellae from food products. Appl. Microbiol. 26:288-292. Hoben, D. A., D. H. Ashton, and A. C. Peterson. 1973. Some observations on the incorporation of novobiocin into Hektoen enteric agar for improved Salmonella isolation. Appl. Microbiol. 26:126-127. King, S., and W. I. Metzger. 1969. A new plating medium for the isolation of enteric pathogens. I. Hektoen enteric agar. Appl. Microbiol. 16:577-578. Moats, W. A., and J. A. Kinner. 1976. Observations on brilliant green agar with an H2S indicator. Appl. Environ. Microbiol. 31:380-384. Reamer, R. H., R. E. Hargrove, and F. E. McDonough. 1974. A selective plating agar for direct enumeration of Salmonella in artificially contaminated dairy products. J. Milk Food Technol. 37:441-444. Restaino, L., G. S. Grauman, W. A. McCall, and W. M. Hill. 1977. Effects of varying concentrations of novobiocin incorporated into two Salmonella plating media on the recovery of four Enterobacteriaceae. Appl. Environ. Microbiol. 33:585-589. Shanson, D. C. 1975. A new selective medium for the isolation of salmonellae other than Salmonella typhi. J. Med. Microbiol. 8:357-364. Taylor, W. I. 1965. Isolation of Shigellae. I. Xylose lysine agars; new media for the isolation of enteric pathogens. Am. J. Clin. Pathol. 44:471-475. Taylor, W. I., and D. Schelhart. 1971. Isolation of shigellae. VIII. Comparison of xylose lysine desoxycholate agar, Hektoen enteric agar, Salmonella-Shigella agar, and eosin methylene blue agar with stool specimens. Appl. Microbiol. 21:32-37.
Vol. 36, No. 5 Printed in U.S.A.
Comparison of Four Agar Plating Media with and Without Added Novobiocin for Isolation of Salmonellae from Beef and Deboned Poultry Meat W. A. MOATS Agricultural Research Center, U.S. Department of Agriculture, Beltsville, Maryland 20705
Received for publication 11 September 1978
Four plating media, Hektoen enteric (HE), xylose-lysine deoxycholate (XLD), tryptic soy-xylose-lysine (TSXL), and tryptic soy-brillant green (TSBG) agars with and without 10 mg of added novobiocin per ml, were evaluated for recovery of Salmonella from roast beef and deboned turkey. Colonies producing a reaction typical of H2S-positive salmonellae (alkaline with black centers) were picked. On the media without novobiocin, from 109 determinations on 75 samples, number of salmonellae found and false-positives were, respectively: HE- 13, 58; XLD- 17, 18; TSXL-23, 0; TSBG-22, 7. When novobiocin was present the corresponding results were: HE- 17, 24; XLD-21, 2; TSXL-23, 3; TSBG-20, 7. A total of 25 determinations were positive on one or more agars. False-positives on HE and XLD without novobiocin were predominantly Proteus, which were almost totally eliminated by addition of 10 mg of novobiocin per liter. If alkaline H2S-negative colonies had been considered, many more false-positives would have been found on HE and XLD but not on TSBG or TSXL. Addition of novobiocin markedly improved isolations of salmonellae from XLD and HE and reduced the number of false-positives. Addition of novobiocin did not improve performance of TSXL and slightly impaired differentiation of salmonellae from Citrobacter on TSBG. XLD with novobiocin and TSXL are highly specific for H2S-positive salmonellae, and the appearance of Salmonella-like colonies on these media can be considered a presumptive test for H2S-positive salmonellae.
Several improved plating media for isolation of salmonellae have been developed in recent years. These include the Hektoen enteric (HE) agar of King and Metzger (5), the xylose-lysine agars of Taylor (10), and two improved brilliant green formulations described by Moats and Kinner (6). All these contain indicators of H2S production so that salmonellae can be identified by the production of black-centered colonies as well as by the pH reaction. Taylor (10) also included xylose and lysine in the media to improve differentiation of salmonellae from organisms such as Citrobacter and Proteus which do not produce lysine decarboxylase. Taylor (10) described two formulations-xylose-lysine deoxycholate
(XLD), which is only mildly selective for salmonellae, and xylose-lysine-brilliant green, which is more selective. Moats and Kinner (6) developed an improved brilliant green formulation with tryptic soy agar as a base (TSBG) and also a modification which included xylose and lysine (TSXL), as described by Taylor (10). Moats and Kinner (6) found that Taylor's xylose-lysine-brilliant green agar prepared from xylose-lysine agar base was inhibitory to many
salmonellae. However, use of tryptic soy agar as a base greatly improved growth of salmonellae while retaining the desirable selective and differential properties of the medium. There have been several recent reports (4, 6-9) of the addition of novobiocin to plating media to improve selectivity for salmonellae. Novobiocin at 5 to 10 ,ug/ml has been found to totally suppress growth of Proteus strains (4, 6-9). Varying degrees of inhibition of Escherichia coli (4, 6, 8), Citrobacter (4, 6, 8), and Pseudomonas (7) have also been reported. In the present study, I compared the effectiveness of four plating media, HE, XLD, TSXL, and TSBG agars, with and without 10 mg of added novobiocin per liter for isolating salmonellae. Meat samples were used for the comparison since they have been commonly found to contain large numbers of interfering organisms. Addition of novobiocin to brilliant green agars has not been previously reported. MATERIALS AND METHODS Media. HE agar (Difco, Detroit, Mich.) was prepared in accordance with the manufacturer's instruc747
748
APPL. ENVIRON. MICROBIOL.
MOATS
34) and 10 deboned turkey samples were obtained as tetrathionate-brilliant green enrichment broths, and plates were streaked directly from these. Frozen samples (n = 34) of deboned turkey meat were tested with lactose broth preenrichment and selenite-cystine and tetrathionate enrichments. Plates were incubated for 20 to 24 h at 37°C and examined for the presence of Salmonella-like colonies. One colony was picked from each plate showing suspect colonies and inoculated onto triple sugar iron and lysine agars. All picks were further characterized biochemically whether or not they gave typical Salmonella-like reactions on triple sugar iron and lysine
tions. XLD agar was prepared from xylose-lysine agar base (Difco) with 2.50 g of sodium deoxycholate, 0.80 g of ferric ammonium citrate, and 6.80 g of sodium thiosulfate .5H20 added per 1,000 ml of medium. The dry ingredients were dissolved in water, heated to boiling, cooled to 50°C, and poured into plates. TSBG and TSXL agars were prepared as previously described (6). Formulas used were, per 1,000 ml: TSBG-tryptic soy agar (Difco), 40 g; lactose, 8 g; sucrose, 8 g; sulfanilamide, 1 g; ferric ammonium citrate, 1.5 g; sodium thiosulfate * 5H20, 5.0 g; phenol red, 80 mg; brilliant green, 5 mg; TSXL-tryptic soy agar, 40 g; sucrose, 8 g; sulfanilamide, 1 g; ferric ammonium citrate, 1.5 g; sodium thiosulfate * 5H20, 5.0 g; agar, 5.0 g; phenol red, 80 mg; brilliant green, 5 mg. The dry ingredients except brilliant green were suspended in water, and the pH was adjusted as necessary to 6.8 to 6.9 with 3 N HCl or NaOH. The media were then heated to dissolve the ingredients, sterilized for 15 min at 15 lb/in2, and cooled in a water bath at 50°C. Brilliant green was added from a filter-sterilized stock solution containing 1 mg/ml, and the medium was immediately poured into plates. The media were also prepared as above with 10 mg of sodium novobiocin (925 ytg/mg)/liter added to the other dry ingredients. The novobiocin was a gift from The Upjohn Co., Kalamazoo, Mich. Other media used were lactose broth, selenite-cystine broth, tetrathionate broth, Simmons citrate agar, triple sugar iron agar, and lysine agar, purchased from Baltimore Biological Laboratory (BBL), Cockeysville, Md. Carbohydrate test media were prepared in purple broth base (BBL). Other biochemical reagents were prepared according to Edwards and Ewing (2), using the Falkow formulations for decarboxylase test media. Test procedure. Test samples were obtained from the Microbiology Laboratory, FSQS, U.S. Department of Agriculture, Beltsville, Md., and were selected from lots of roast beef and deboned turkey suspected of containing Salmonella. The roast beef samples (n =
agars.
RESULTS AND DISCUSSION For the present study novobiocin was used at a concentration of 10 mg/liter in the agars. This concentration has been consistently found to totally suppress growth of Proteus without affecting growth of salmonellae. Higher concentrations which have been used in some cases were only partially effective in suppressing growth of other interfering organisms (4, 8) and have been reported to retard growth of salmonellae (4). The typical characteristics of various types of bacterial colonies likely to be found on the four plating media studied without added novobiocin are summarized in Table 1. HE agar is a relatively nonselective medium on which Proteus and Citrobacter freundii strains form colonies indistinguishable from salmonellae. Also, Pseudomonas and those Enterobacter hafniae strains which do not ferment sucrose, lactose, or salicin form green colonies resembling those of H2S-negative samonellae. XLD agar is also relatively nonselective. On it, Citrobacter and many Proteus strains ordinarily form yellow col-
TABLE 1. Appearance of bacterial colonies on plating media after 24-h growth at 37°C Appearance of colonies on:
Organism Salmonella S. cholerae-suis, H2S negative Salmonella, lactose positive Escherichia coli Proteus vulgaris Proteus mirabilis Klebsiella-Enterobacter Enterobacter hafniae (some) Citrobacter freundii
Pseudomonas
HE XLD Green, black Pink, black centers centers Green
Pink
Orange
Yellow
TSXL TSBG Green to pink, large black Pink, black centers centers Green or greenish pink Pink Green to pink, large black Green centers No growth No growth No growth No growth
Orange Yellow Green, black Yellow centers Green, black Yellow or pink, black No growth or tiny green No growth or tiny pink centers centers colonies; rarely, pink colonies; rarely, pink with black centers with black centers Orange Yellow Green Green Green Pink Usually no growth Usually no growth Green, black Yellow; rarely, pink with Green, occasional small Green, green with black centers black centers black centers centers; occasionally pink with black centers Green Pink No growth or tiny green- No growth or tiny pink pink colonies colonies
VOL. 36, 1978
COMPARISON OF MEDIA FOR SALMONELLAE ISOLATION
onies which are clearly distinguishable from salmonellae. However, Pseudomonas and E. hafniae strains which do not ferment lactose or sucrose form pink colonies resembling those of H2S-negative salmoneflae. TSBG and TSXL are more selective. Most Proteus strains are strongly inhibited and show little growth. C. freundii strains form green to occasionally pink colonies with black centers on TSBG. On TSXL Citrobacter colonies are green, occasionally with small black centers, which are easily distinguishable from the almost totally black Salmonella colonies. TSXL does not contain lactose, and so lactose-fermenting salmonellae appear the same as other salmonellae. Salmonellae do not give a very clear-cut alkaline reaction on TSXL, and H2S-negative types are therefore not clearly distinguishable from coliforms. The roast beef samples used were obtained as tetrathionate-brilliant green enrichments from the FSQS Microbiology Laboratory. Addition of novobiocin to XLD and HE agars improved recoveries of salmonellae from roast beef samples and virtually eliminated false-positives (Table 2). Novobiocin had little effect on results with TSBG and TSXL agars. Some of the deboned turkey samples were also streaked from tetrathionate-brilliant green broth enrichments. The remainder were carried through conventional lactose preenrichment followed by enrichment in tetrathionate and selenite-cystine broths. The tetrathionate and selenite-cystine enrichments were tabulated as separate determinations. On this sample series (Table 3), no one agar recovered salmonellae from all determinations which were positive on one or more agars. The most recoveries and the fewest false-positives were on TSXL with or without novobiocin. Fewer salmonellae were recovered on TSBG with novobiocin than on that without it. It was observed that Salmonella colonies TABLE 2. Isolations of salmonellae from roast beef samples enriched in tetrathionate brilliant green brotha No. of:
Agar
False-positives Samples positive 13 10 HE 0 14 HE-Nb 8 12 XLD 0 14 XLD-N 0 14 TSXL 1 14 TSXL-N 0 14 TSBG 0 14 TSBG-N a on for Salmonella 14 positive Thirty-one samples, one or more agars. b N, Contains 10 gg of novobiocin per ml.
749
were slightly less alkaline and Citrobacter were slightly less acid on TSBG with novobiocin than on that without novobiocin, and these effects resulted in confusion of the two. False-positives on XLD were almost eliminated by addition of novobiocin, but slightly fewer salmonellae were recovered than on TSXL or TSBG. Recovery of salmonellae on HE agar was not improved by addition of novobiocin. The number of falsepositives was reduced but was still far higher than on the other agars. The combined data for beef and turkey are summarized in Table 4. The classification of types of colonies picked as Salmonella-like is summarized in Table 5. Organisms producing false-positive colonies on HE and XLD agars were predominantly Proteus mirabilis, with a few Proteus vulgaris from HE agar. Addition of novobiocin, which eliminated Proteus strains, was therefore highly advantageous with these agars. With HE agar, a substantial number of false-positive colonies from Citrobacter strains remained after elimination of Proteus. Citrobacter strains rarely form falsepositive colonies on XLD agar, so, with the elimination of Proteus, this medium is virtually TABLE 3. Isolations of salmonellae from 44 samples of turkey meat No. of: Agar
Salmo-
nella isolated 3 1 4 6 7 7 7 5
S. ari- Total' Salzonae iso- monella
lated 0 2 0 1 2 2 1 1
False-positives
45 22 4 10 7 2 9 0 2 9 8 7 6 6 aSeventy-eight determinations, 11 positive for salmonellae on one or more agars. b N, Contains 10 mg of novobiocin per liter.
HE HE-N b XLD XLD-N TSXL TSXL-N TSBG TSBG-N
3 3
TABLE 4. Recovery of salmonellae from all testsa No. false-positive No. positive Agar 58 13 HE 24 17 HE-Nb 18 17 XLD 2 21 XLD-N 0 23 TSXL 3 23 TSXL-N 7 22 TSBG 7 20 TSBG-N One hundred nine determinations, 25 positive for on one or more agars. salmonellae b N, Contains 10 mg of novobiocin per liter. a
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APPL. ENVIRON. MICROBIOL.
MOATS
TABLE 5. Classification of Salmonella-like colonies Agar
Proteus vulgaris Proteus mirabilis
HE HE-Na XLD XLD-N TSXL TSXL-N TSBG TSBG-N a N, Contains
3 44 1 0 0 17 0 0 0 0 0 0 0 1 0 0 10 mg of novobiocin per liter.
freundoc
Salmonella
S. arizonae
Total picks
11
13 15 17
0 2 0 1 2 2 1 1
71 41 35 23 23 26 28 28
23 1 2 0 3 6 7
specific for H2S-positive salmonellae. Since Proteus strains rarely produce false-positive colonies on TSBG and TSXL, addition of novobiocin is of marginal value. In the present sample series it appears that novobiocin may have slightly impaired differentiation of sahnonellae from Citrobacter as shown by false-positives on TSXL with novobiocin (10 mg/liter) and poorer recoveries of salmonellae on TSBG with novobiocin (10 mg/liter). False-positive colonies picked from TSXL with novobiocin (10 mg/liter) were rather atypical of Salmonella and would not have been picked if typical Salmonella colonies had been present on the same plate. Working with stool samples, Taylor and Schelhart (11) found that P. mirabilis was the predominant H2S-producing organism producing false-positives on XLD and that far more C. freundii produced false-positives on HE than on XLD. Restaino et al. (8) found that on HE and XLD the predominant H2S-producing false-positives from pork sausage were C. freundii and P. mirabilis, respectively. They also observed that H2S-producing E. coli, which appear to be quite rare, could also produce false-positives on HE and XLD and were not inhibited by novobiocin. For the present study, only H2S-positive colonies were picked. However, H2S-negative salmonellae do occur occasionally, and some alkaline H2S-negative colonies were observed on HE and XLD agars both with and without novobiocin. A few of these were picked and identified as either Pseudomonas or E. hafniae. If these were included in the data, the number of false-positives on HE and XLD agars would have been increased. No colonies of this type were found on TSBG or TSXL agars, and the isolates from HE and XLD showed little if any growth on TSBG. It would therefore appear that TSBG would be more satisfactory than HE or XLD for detection of H2S-negative salmonellae. As previously discussed, TSXL agar does not clearly differentiate H2S-negative sahnonellae from coliforms. Taylor and Schelhart (11) found substantial numbers of H2S-negative organisms in
20 21 21 21 19
stool samples which produced false-positives on HE and XLD agars. These were predominantly Pseudomonas and slow-fermenting strains of Escherichia, Klebsiella, Enterobacter, Serratia, and Aeromonas. Proteus rettgeri and P. morganii and assorted nonfermnentative gramnegative organisms also produced small numbers of false-positives. The reasons why salmonellae were not isolated from plating mddia where one or more plates from the enrichment were positive are summarized in Table 6. In most cases where salmonellae were not found, no Salmonella-like colonies were present. Occasionally, especially on HE agar, Salmonella-like colonies were present, but Salmonella, if present, were not picked. Possibly, if more colonies had been picked, salmonellae would have been found. However, the difficulty in picking salmonellae in the presence of other organisms producing Salmonella-like colonies is obvious. The absence of Salmonellalike colonies may result from overgrowth of salmonellae by other organisms, poor differentiation of salmonellae from other organisms, or failure of salmonellae to grow on the plates. Where only one or two Salmonella-like colonies were found on positive plates, which occurred several times, other plates streaked from the same enrichment may have been negative purely by chance. Assuming that salmonellae grew equally well on all the media tested, more recoveries would be expected on the more selective media (TSXL and TSBG) since overgrowth by other organisms is less likely; this is what was found. Biochemical confirmation of salmonellae suspect colonies from any of the four agars studied is relatively simple since considerable biochemical differentiation is accomplished on the agars. All picks from HE and XLD agars and most from TSXL and TSBG also gave Salmonellalike reactions on triple sugar iron agar. Triple sugar iron agar was therefore not used in the last half of the study. Lysine agar, on the other hand, proved extremely useful because most picks
COMPARISON OF MEDIA FOR SALMONELLAE ISOLATION
VOL. 36, 1978
TABLE 6. Reasons why salmonellae were not found on plating media False-negatives
Agar
No. posi-
tive
No.
Salmonella like colonies absent
Salmonellalike colonies
picked-not SalmoneUa
HE 13 12 1 11 HE-N a 17 8 5 3 XLD 11 9 7 2 XLD-N 21 4 4 0 TSXL 23 2 2 0 TSXL-N 23 2 2 0 22 TSBG 3 3 0 TSBG-N 20 5 4 1 N, Contains 10 mg of novobiocin per liter. a
correctly identified on it. Proteus strains easily identified from the characteristic red slants, and most Citrobacter strains gave an acid butt. Of other biochemical tests, lysine and ornithine decarboxylases, mannitol and dulcitol fermentation, and malonate utilization ordinarily differentiated the H2S-positive picks satisfactorily. Simmons citrate and urease provided additional confirmation. Lactose fermentation was also tested with picks from TSXL, which does not contain lactose. Of non-H2S-producing picks, Pseudomonas strains could be readily differentiated by the oxidase test or oxidation-fermentation of glucose. E. hafniae strains picked from XLD with novobiocin (10 mg/liter) did not ferment lactose or sucrose and were otherwise quite similar biochemically to salmonellae, but could be distinguished by failure to ferment sorbitol. Bisciello and Schrade (1) tested over 2,000 assorted foods and found that HE agar gave more isolations of salmonellae with fewer falsepositives than bismuth sulfite, salmonella-shigella, or brilliant green agars. On the other hand, Goo et al. (3), in analyzing over 11,000 food samples, obtained better recovery of salmonellae on brilliant green than on HE agar, although more false-positives were found on brilliant green. Taylor and Schelhart (11) found that HE and XLD agars gave equivalent recoveries of salmonellae and shigellae from stool specimens but that HE agar gave twice as many false-
isfactory with these samples than the other three agars tested, even with added novobiocin, both from the standpoint of salmonellae recovered and false/positives. TSBG agar produced slightly more false-positives than TSXL but was nearly comparable for the isolation of salmonellae. Growth of organisms which might interfere with detection of H2S-negative salmonellae was largely suppressed on TSBG. In our laboratory all enrichments are routinely streaked on both TSBG and TSXL agars. This allows the possibility of recovering both lactose-fermenting and H2S-negative salmonellae as well as typical types. Citrobacter strains which produce falsepositives on TSBG are immediately identified by their failure to form Salmonella-like colonies on TSXL.
were were
ACKNOWLEDGMENTS I thank Alice Moran, U.S. Department of Agriculture Food Safety and Quality Service Microbiology Laboratory, for providing the samples used and for helpful discussions; The Upjohn Co., Kalamazoo, Mich., for providing the novobiocin used; and T. M. Brennan and J. I. Shultz, Jr., for technical assistance.
LITERATURE CITED 1. Bisciello, N. B., and T. P. Schrade. 1974. Evaluation of 2. 3.
4.
5. 6.
7.
8.
positives.
XLD with novobiocin and TSXL are highly specific for H2S-positive samonellae, and the occurrence of salmonellae-like colonies on these media can be considered a presumptive test for H2S-positive salmonellae. TSXL gave slightly higher recoveries of salmonellae than XLD with novobiocin in the present study and has the additional capability of detecting lactose-fermenting salmonellae. HE was distinctly less sat-
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9. 10. 11.
Hektoen enteric agar for the detection of Salmonella in foods and feeds. J. Assoc. Off. Anal. Chem. 57:992-996. Edwards, P. R., and W. H. Ewing. 1972. Identification of Enterobacteriaceae, 3rd ed. Burgess Publishing Co., Minneapolis. Goo, V. Y. L., G. Q. L. Ching, and J. M. Gooch. 1973. Comparison of brilliant green agar and Hektoen enteric agar media in the isolation of salmonellae from food products. Appl. Microbiol. 26:288-292. Hoben, D. A., D. H. Ashton, and A. C. Peterson. 1973. Some observations on the incorporation of novobiocin into Hektoen enteric agar for improved Salmonella isolation. Appl. Microbiol. 26:126-127. King, S., and W. I. Metzger. 1969. A new plating medium for the isolation of enteric pathogens. I. Hektoen enteric agar. Appl. Microbiol. 16:577-578. Moats, W. A., and J. A. Kinner. 1976. Observations on brilliant green agar with an H2S indicator. Appl. Environ. Microbiol. 31:380-384. Reamer, R. H., R. E. Hargrove, and F. E. McDonough. 1974. A selective plating agar for direct enumeration of Salmonella in artificially contaminated dairy products. J. Milk Food Technol. 37:441-444. Restaino, L., G. S. Grauman, W. A. McCall, and W. M. Hill. 1977. Effects of varying concentrations of novobiocin incorporated into two Salmonella plating media on the recovery of four Enterobacteriaceae. Appl. Environ. Microbiol. 33:585-589. Shanson, D. C. 1975. A new selective medium for the isolation of salmonellae other than Salmonella typhi. J. Med. Microbiol. 8:357-364. Taylor, W. I. 1965. Isolation of Shigellae. I. Xylose lysine agars; new media for the isolation of enteric pathogens. Am. J. Clin. Pathol. 44:471-475. Taylor, W. I., and D. Schelhart. 1971. Isolation of shigellae. VIII. Comparison of xylose lysine desoxycholate agar, Hektoen enteric agar, Salmonella-Shigella agar, and eosin methylene blue agar with stool specimens. Appl. Microbiol. 21:32-37.
