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Xylose-Lysine-Tergitol 4: An Improved Selective Agar Medium for the Isolation of Salmonella
Xylose-Lysine-Tergitol 4: An Improved Selective Agar Medium for the Isolation of Salmonella RUSSELL G. MILLER1 and C. R. TATE USDA, Food Safety Inspection Service, Building 322, Agricultural Research Center-East, Beltsville, Maryland 20705 E. T. MALLINSON and J. A. SCHERRER University of Maryland, College Park, Maryland 20742
ABSTRACT A study was conducted to evaluate a new selective plating medium for isolating Salmonella using pure bacterial cultures, and poultry environmental specimens containing high numbers of competing enteric bacteria. Xylose-lysine-tergitol 4 (XLT4) agar was found to strongly inhibit Proteus, Pseudomonas, Providencia, and many other nonsalmonellae and to provide good differentiation between Salmonella and Citrobacter. The XLT4 medium significantly improved Salmonella isolation from chicken farm environmental drag-swab samples over the other selective plating media evaluated. (Key words: xylose-lysine-tergitol 4, Salmonella, drag-swab, poultry, environment) 1991 Poultry Science 70:2429-2432 INTRODUCTION
Most agar media used for the selection of salmonellae are not formulated for their isolation alone. Many are designed as multipurpose media for public health interests, where it is important to look for a broader spectrum of enteric pathogens. Some of the media still used today were developed near the beginning of this century. For example, MacConkey's agar was described at the turn of the century (MacConkey, 1905). A more recent medium, xylose-lysine-desoxycholate agar (XLD) (Taylor, 1965), still allows the growth of a wide variety of enteric pathogens due to the choice of inhibitors used. The more a medium is designed to isolate wider varieties of pathogens, the more other competing bacteria are able to grow, resulting in decreased selectivity (Tate et al., 1990). The aforementioned agar media, like most other media in the authors' experience for isolating non-typhimurium Salmonella, depend heavily on differentiation of colony types and much less on selectivity. Too often low or even moderate numbers of Salmonella colonies
'To whom correspondence should be addressed. XL agar, Difco, Inc., Detroit, MI 48232.
2
may be totally masked by the varied, high numbers of competing gram-negative bacteria commonly found in fecal-contaminated farm or other environmental specimens (Mallinson et al, 1989; Tate et al, 1990). Although there have been many new or modified media formulated during the 20th century, there has been relatively little change in the selective inhibitors used. Up to now, bile salts, sulfonamides, brilliant green, and other dyes alone or in combination have been the most common inhibitors used in these media. Inclusion of sodium novobiocin in agar media (Hoben et al, 1973; Restaino et al, 1977; Moats, 1978; Tate et al, 1990) used for Salmonella isolation is an improvement over identical media lacking novobiocin. However, even on novobiocin-supplemented plates. Pseudomonas, Providencia, and an occasional novobiocin-resistant strain of Proteus are still able to grow and interfere with Salmonella isolation. Because of these issues and the current concern about the prevalence of Salmonella in uncooked animal products, the present authors investigated alternative selective inhibitors to add to xylose lysine agar base.2 From past experience, it was believed that the most favorable results for this investigation would be obtained by using as the base medium plain
2429
Downloaded from http://ps.oxfordjournals.org/ at University of Sydney Library on May 3, 2015
(Received for publication May 10, 1991)
2430
MILLER ET AL.
MATERIALS AND METHODS
The formulation for XLT4 agar was as follows. To each liter of distilled water, 4.6 mL of Tergitol 4 (Sigma, Catalog No. T8256)3 was added. To this, 47 g XL agar base (Difco Catalog No. 0555-01-8),2 .8 g ferric ammonium citrate, 6.8 g sodium thiosulfate, and 3 g Bacto agar2 were added and mixed for 5 rnin. The mixture was then isothermed in flowing steam for 10 min (or alternatively heated on a hot plate to near boiling), after which it was mixed again for 5 min. The formulation was then autoclaved at 118 C for 12 min. After cooling to 45 to 50 C, the formulation was mixed a third time for 5 min prior to pouring the plates. The XLT4 agar had a final pH of 7.3 ± .2. The plates sometimes were dark at first but lightened in color after cooling. Neither XLD agar nor Tergitol 7 can be substituted for plain XL agar or Tergitol 4, respectively. Poured XLT4 plates have a shelf life of at least 3 mo when stored refrigerated in sealed plastic bags. Gram-negative bacteria, mostly from the family Enterobacteriaceae were streaked onto
3 Sigma Chemical Co., St. Louis, MO 63178-9916. "Wkville, MD 20852. 5 Jobnson and Johnson, New Brunswick, NJ 08903. ^ B L , Inc., Cockeysville, MD 21030. 7 Analytab Products Inc., Plainview, NY 11803.
XLT4 agar plates. These included a number of wild strains commonly isolated from poultry farm environments, and seven strains obtained from the American Type Culture Collection (ATCC).4 Their growth characteristics were observed after overnight incubation at 35 C. In addition, 97 manure pit drag-swab samples from seven chicken layer flocks known to harbor Salmonella enteritidis serotype enteritidis (SE) were analyzed. The dragswabs were similar to those described by Kingston (1980) and Mallinson et al. (1989). Briefly, each drag-swab was composed of a 8 x 8 cm sterile gauze pad,5 premoistened with 7 to 8 mL of sterile, double-strength, skim milk2 to which a 2- to 3-m length of heavy string was attached (using a staple) for dragging across the manure pits beneath the caged chickens. After sampling, the drag-swabs were held at 3 to 5 C until the day of culturing (within 5 days of sampling). Each drag-swab was then incubated 22 to 24 h at 41 C in 60 to 80 mL of Hajna's tetrathionate broth,2 then streaked to XLT4, XLD, XLD containing 20 mcg/mL sodium novobiocin (XLDN), brilliant green agar (BGA),6 and BGA containing 20 mcg/mL sodium novobiocin (BGAN). All plates were examined after 24 and 48 h at 35 to 37 C. Up to three representative colonies from each plating medium were selected and transferred to slants of triple sugar iron agar2 and of lysine iron agar.2 Slide agglutination serologies for somatic 0 groupings (A-I)2 were performed on those culture slants that biochemically indicated salmonellae. The API20E Identification strips7 were used to identify suspect colonies when the serologies showed negative reactions. A representative number of the Salmonella-positive cultures were submitted to the Veterinary Services Laboratory (Ames, IA 50010) for serotyping. Statistical Analysis Because each drag-swab sample broth was streaked onto each of the different media, samples are matched, increasing the precision of comparison. However, when matching occurs, samples are correlated and the ordinary Pearson chi-square statistic is no longer valid. McNemar (1947) developed a test statistic for comparing two matched samples when the response is a dichotomy, i.e., positive or negative. Cochran (1950) generalized the McNemar statistic to the case of several two matched samples.
Downloaded from http://ps.oxfordjournals.org/ at University of Sydney Library on May 3, 2015
XL agar containing the H2S indicators sodium thiosulfate and ferric ammonium citrate (at the same concentrations found in XLD agar). Preliminary studies mainly employed anionic, cationic, and nonpolar surfactants at varying concentrations between 1 and 10 mL/L (data not shown). These included Tergitol Types 4, 7, 8, and NP-10 3 . Of all the potential inhibitors assessed, Tergitol 4 (7-ethyl-2methyl-4-undecanol-hydrogen sulphate, sodium salt) added to XL agar at a concentration of 4.6 mL/L (XLT4) proved superior to all other formulations tested (unpublished data). The present study was conducted to demonstrate the growth characteristics of Salmonella and other gram-negative bacteria on XLT4 plating medium, and to assess the isolation efficiency of XLT4 as compared with other commonly used selective plating media used to isolate Salmonella from highly contaminated samples.
2431
SALMONELLA PLATING MEDIUM TABLE 1. Growth characteristics of various gram-negative bacteria on xylose-lysine-tergitol 4 agar after 24 h at 35 C Organism
Source
Growth2
Appearance
Salmonella typhimurium Salmonella spp. (numerous serotypes) Salmonella enteritidis Salmonella enteritidis (r^S-positive strains) Salmonella enteritidis (r^S-negative strains) Proteus mirabilis Proteus mirabilis Proteus vulgaris Pseudomonas aeruginosa
ATCC (14028) Drag-swabs ATCC (13076) Drag-swabs
+++ +++ +++ +++
Black Black Black Black
Drag-swabs
+++
Pinkish-yellow colonies
ATCC (7002) Drag-swabs ATCC (13315) ATCC (27853)
0 0 0
Pseudomonas aeruginosa
Drag-swabs
±
Altermonas putrefaciens Providencia stuartii Providencia rettgeri
Drag-swabs Drag-swabs Drag-swabs
0 0
Citrobacter freundii Citrobacter spp. Hafnia alvei Morganella morganii Enterobacter aerogenes
Drag-swabs Drag-swabs Drag-swabs Drag-swabs ATCC (13048)
++ ++ 0 +
±
Escherichia coli
ATCC (25922)
+
Yersinia enterocolitica Acinetobacter calcoaceticus
ATCC (23715) Drag-swabs
0 0
±
Haze of pink growth at point of inoculation Haze of pink growth at point of inoculation Haze of pink growth at point of inoculation No H2S production, yellow colonies No H2S production yellow colonies Pinpoint yellow colonies Haze of yellow growth at point of inoculation Scarce yellow colonies at point of inoculation
'ATCC = American Type Culture Collection, Rockville, MD 20852; drag-swabs = various strains isolated from chicken farm environmental drag-swab samples and identified using the API20E identification system (Analytab Products, Inc., Plainview, NY 11803). All wild-type salmonellae were confirmed and serotyped at the National Veterinary Services Laboratory in Ames, IA 50010. jfhe reference medium was xylose-lysine agar base. 4++ = good growth, no detectable inhibition of colonies; ++ = fair growth, slightly inhibited colonies; + = poor growth, very inhibited colonies; ± = trace growth, barely any growth evident; 0 = no visible growth.
Cochran's (1950) Q statistic was used to determine whether there exist significant differences in the proportion of positive responses among the five different agar media. Comparisons between XLT4 and each of the other plating media were made using McNemar's (1947) test, adjusted according to the Yates (1934) correction for continuity, which is recommended in cases where df = 1. RESULTS AND DISCUSSION
As shown in Table 1, XLT4 strongly or completely inhibits Proteus, Pseudomonas, Providencia, and many other nonsalmoneUae. Although there is some growth of Citrobacter freundii, the colonies do not produce black centers on XLT4 plates in 24 h. Typical (lactose-positive) Citrobacter colonies remain yellow on XLT4 and are sticky when tested with an inoculating needle. After 20 to 24 h at
35 to 37 C, typical (H2S-positive) Salmonella colonies on XLT4 are smooth and creamy in texture and appear black or black-centered with a yellow (acid) periphery that changes to pink (alkaline) as the xylose is depleted (20 to 24 h). It was concluded that essentially the only genus capable of forming black colonies within 24 h on XLT4 was Salmonella, allowing easy differentiation from other organisms. The H2S-negative Salmonella strains appear as pinkish-yellow colonies on XLT4. These observations are very important because a highly selective plating medium can change the false-negative status of a specimen to the true result of Sa/mone/Za-positive. This is vital in the case of the egg industry, where successful detection of SE from among other Salmonella serotypes is critical in keeping eggs of SE-contaminated flocks from reaching the marketplace. Obviously, the more Salmo-
Downloaded from http://ps.oxfordjournals.org/ at University of Sydney Library on May 3, 2015
±
colonies or black-centered colonies colonies or black-centered colonies
2432
MILLER ET AL.
TABLE 2. Salmonella isolation rates and the average purity of Salmonella colonies observed on each medium examined from 91 SaXmoaeMn-positive drag-swab samples collected from seven table egg layer flocks Medium Variable
XLT4
XLD
XLDN
BGA
BGAN
Number positive out of 91 Percentage positive Average purity of Salmonella colonies Probability of difference from XLT43
89 98% 3+ to 4+
27 30% 1+ <.001
76 84% 2+ .002
65 71% 1+ to 2+ <001
77 85% 2+ to 3+ .002
X
XLT4 = xylose-lysine-tergitol 4 agar, XLD = xylose-lysine-desoxycholate agar, XLDN = XLD with 20 mcg/mL of sodium novobiocin; BGA = plain brilliant green agar, BGAN = BGA with 20 mcg/mL of sodium novobiocin. 2 1+ = 1 to 25% of the colonies were Salmonella; 2+ = 25 to 50%; 3+ = 51 to 75%; 4+ = 76 to 100%. Using Yates correction for continuity.
ACKNOWLEDGMENTS
The authors gratefully acknowledge Larry Shipman (USDA Animal Plant Health Inspec-
tion Service, Harrisonburg, PA 17110) for providing the drag-swab samples used in this study, and Larry Douglass (University of Maryland, College Park, MD 20742) for his contributions toward the statistical analysis. REFERENCES Cochran, W. G., 1950. The comparison of percentages in matched samples. Biometrika 37:256-266. 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. Kingston, D. J., 1980. A comparison of culturing drag swabs and litter for identification of infections with Salmonella spp. in commercial chicken flocks. Avian Dis. 25:513-516. MacConkey, A., 1905. Lactose-fermenting bacteria in feces. J. Hyg. 5:333-379. Mallinson, E. T., C. R. Tate, R. G. Miller, B. Bennett, and E. Russek-Cohen, 1989. Monitoring poultry farms for salmonella by drag-swab sampling and antigencapture immunoassay. Avian Dis. 33:684-690. McNemar, O., 1947. Note on the sampling error of the difference between corrected proportions or percentages. Psychometrika 12:153-157. Moats, W. A., 1978. Comparison of four agar plating media with and without added novobiocin for isolation of salmonella from beef and deboned poultry meat. Appl. Environ. Microbiol. 36:747-751. 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. Tate, C. R., R. G. Miller, E. T. Mallinson, L. W. Douglass, and R. W. Johnston, 1990. The isolation of salmonellae from poultry farm environmental samples by several enrichment procedures using plating media with and without novobiocin. Poultry Sci. 69: 721-726. Taylor, W. L., 1965. Xylose lysine agars; new media for isolation of enteric pathogens. Am. J. Clin. Pathol. 44:471^75. Yates, F., 1934. Contingency tables involving small numbers and the chi-square test. J. R. Stat. Soc. Suppl. 1:217-235.
Downloaded from http://ps.oxfordjournals.org/ at University of Sydney Library on May 3, 2015
nella colonies there are to select, the greater the chances of detecting the particular serotype of interest. In the present study, salmonellae were isolated from 91 of the 97 drag swab samples collected from the seven SE-positive chicken layer flocks. Many different Salmonella serotypes were isolated, including SE from numerous samples. For these 91 positive samples, XLT4 was found to significantly improve the rate of Salmonella isolations over the other plating media (Table 2). In only two cases was Salmonella isolated on one of the other plating media but not on the XLT4 medium. The Salmonella isolation rate and the average purity of Salmonella colonies on each plating medium for the 91 Sa/mone/Za-positive dragswab specimens are described in Table 2. The authors recommend using XLT4 plating medium to isolate nan-typhimurium Salmonella from fecal-contaminated farm or similar samples containing high numbers of competing bacteria. In addition, XLT4 appears promising for use in clinical applications (C. Wills, personal communication, Maryland Department of Agriculture, Animal Health Laboratory, Salisbury, MD 21802). It is suggested that two different types of plating media be inoculated to further reduce the possibility of a false-negative finding that could occur if a particular strain of Salmonella were sensitive to an inhibitor used in one of the two media. The agar BGAN (Tate et ai, 1990) is recommended as a second medium, as BGAN was found to be second only to XLT4 in selectivity (Table 2).
ABSTRACT A study was conducted to evaluate a new selective plating medium for isolating Salmonella using pure bacterial cultures, and poultry environmental specimens containing high numbers of competing enteric bacteria. Xylose-lysine-tergitol 4 (XLT4) agar was found to strongly inhibit Proteus, Pseudomonas, Providencia, and many other nonsalmonellae and to provide good differentiation between Salmonella and Citrobacter. The XLT4 medium significantly improved Salmonella isolation from chicken farm environmental drag-swab samples over the other selective plating media evaluated. (Key words: xylose-lysine-tergitol 4, Salmonella, drag-swab, poultry, environment) 1991 Poultry Science 70:2429-2432 INTRODUCTION
Most agar media used for the selection of salmonellae are not formulated for their isolation alone. Many are designed as multipurpose media for public health interests, where it is important to look for a broader spectrum of enteric pathogens. Some of the media still used today were developed near the beginning of this century. For example, MacConkey's agar was described at the turn of the century (MacConkey, 1905). A more recent medium, xylose-lysine-desoxycholate agar (XLD) (Taylor, 1965), still allows the growth of a wide variety of enteric pathogens due to the choice of inhibitors used. The more a medium is designed to isolate wider varieties of pathogens, the more other competing bacteria are able to grow, resulting in decreased selectivity (Tate et al., 1990). The aforementioned agar media, like most other media in the authors' experience for isolating non-typhimurium Salmonella, depend heavily on differentiation of colony types and much less on selectivity. Too often low or even moderate numbers of Salmonella colonies
'To whom correspondence should be addressed. XL agar, Difco, Inc., Detroit, MI 48232.
2
may be totally masked by the varied, high numbers of competing gram-negative bacteria commonly found in fecal-contaminated farm or other environmental specimens (Mallinson et al, 1989; Tate et al, 1990). Although there have been many new or modified media formulated during the 20th century, there has been relatively little change in the selective inhibitors used. Up to now, bile salts, sulfonamides, brilliant green, and other dyes alone or in combination have been the most common inhibitors used in these media. Inclusion of sodium novobiocin in agar media (Hoben et al, 1973; Restaino et al, 1977; Moats, 1978; Tate et al, 1990) used for Salmonella isolation is an improvement over identical media lacking novobiocin. However, even on novobiocin-supplemented plates. Pseudomonas, Providencia, and an occasional novobiocin-resistant strain of Proteus are still able to grow and interfere with Salmonella isolation. Because of these issues and the current concern about the prevalence of Salmonella in uncooked animal products, the present authors investigated alternative selective inhibitors to add to xylose lysine agar base.2 From past experience, it was believed that the most favorable results for this investigation would be obtained by using as the base medium plain
2429
Downloaded from http://ps.oxfordjournals.org/ at University of Sydney Library on May 3, 2015
(Received for publication May 10, 1991)
2430
MILLER ET AL.
MATERIALS AND METHODS
The formulation for XLT4 agar was as follows. To each liter of distilled water, 4.6 mL of Tergitol 4 (Sigma, Catalog No. T8256)3 was added. To this, 47 g XL agar base (Difco Catalog No. 0555-01-8),2 .8 g ferric ammonium citrate, 6.8 g sodium thiosulfate, and 3 g Bacto agar2 were added and mixed for 5 rnin. The mixture was then isothermed in flowing steam for 10 min (or alternatively heated on a hot plate to near boiling), after which it was mixed again for 5 min. The formulation was then autoclaved at 118 C for 12 min. After cooling to 45 to 50 C, the formulation was mixed a third time for 5 min prior to pouring the plates. The XLT4 agar had a final pH of 7.3 ± .2. The plates sometimes were dark at first but lightened in color after cooling. Neither XLD agar nor Tergitol 7 can be substituted for plain XL agar or Tergitol 4, respectively. Poured XLT4 plates have a shelf life of at least 3 mo when stored refrigerated in sealed plastic bags. Gram-negative bacteria, mostly from the family Enterobacteriaceae were streaked onto
3 Sigma Chemical Co., St. Louis, MO 63178-9916. "Wkville, MD 20852. 5 Jobnson and Johnson, New Brunswick, NJ 08903. ^ B L , Inc., Cockeysville, MD 21030. 7 Analytab Products Inc., Plainview, NY 11803.
XLT4 agar plates. These included a number of wild strains commonly isolated from poultry farm environments, and seven strains obtained from the American Type Culture Collection (ATCC).4 Their growth characteristics were observed after overnight incubation at 35 C. In addition, 97 manure pit drag-swab samples from seven chicken layer flocks known to harbor Salmonella enteritidis serotype enteritidis (SE) were analyzed. The dragswabs were similar to those described by Kingston (1980) and Mallinson et al. (1989). Briefly, each drag-swab was composed of a 8 x 8 cm sterile gauze pad,5 premoistened with 7 to 8 mL of sterile, double-strength, skim milk2 to which a 2- to 3-m length of heavy string was attached (using a staple) for dragging across the manure pits beneath the caged chickens. After sampling, the drag-swabs were held at 3 to 5 C until the day of culturing (within 5 days of sampling). Each drag-swab was then incubated 22 to 24 h at 41 C in 60 to 80 mL of Hajna's tetrathionate broth,2 then streaked to XLT4, XLD, XLD containing 20 mcg/mL sodium novobiocin (XLDN), brilliant green agar (BGA),6 and BGA containing 20 mcg/mL sodium novobiocin (BGAN). All plates were examined after 24 and 48 h at 35 to 37 C. Up to three representative colonies from each plating medium were selected and transferred to slants of triple sugar iron agar2 and of lysine iron agar.2 Slide agglutination serologies for somatic 0 groupings (A-I)2 were performed on those culture slants that biochemically indicated salmonellae. The API20E Identification strips7 were used to identify suspect colonies when the serologies showed negative reactions. A representative number of the Salmonella-positive cultures were submitted to the Veterinary Services Laboratory (Ames, IA 50010) for serotyping. Statistical Analysis Because each drag-swab sample broth was streaked onto each of the different media, samples are matched, increasing the precision of comparison. However, when matching occurs, samples are correlated and the ordinary Pearson chi-square statistic is no longer valid. McNemar (1947) developed a test statistic for comparing two matched samples when the response is a dichotomy, i.e., positive or negative. Cochran (1950) generalized the McNemar statistic to the case of several two matched samples.
Downloaded from http://ps.oxfordjournals.org/ at University of Sydney Library on May 3, 2015
XL agar containing the H2S indicators sodium thiosulfate and ferric ammonium citrate (at the same concentrations found in XLD agar). Preliminary studies mainly employed anionic, cationic, and nonpolar surfactants at varying concentrations between 1 and 10 mL/L (data not shown). These included Tergitol Types 4, 7, 8, and NP-10 3 . Of all the potential inhibitors assessed, Tergitol 4 (7-ethyl-2methyl-4-undecanol-hydrogen sulphate, sodium salt) added to XL agar at a concentration of 4.6 mL/L (XLT4) proved superior to all other formulations tested (unpublished data). The present study was conducted to demonstrate the growth characteristics of Salmonella and other gram-negative bacteria on XLT4 plating medium, and to assess the isolation efficiency of XLT4 as compared with other commonly used selective plating media used to isolate Salmonella from highly contaminated samples.
2431
SALMONELLA PLATING MEDIUM TABLE 1. Growth characteristics of various gram-negative bacteria on xylose-lysine-tergitol 4 agar after 24 h at 35 C Organism
Source
Growth2
Appearance
Salmonella typhimurium Salmonella spp. (numerous serotypes) Salmonella enteritidis Salmonella enteritidis (r^S-positive strains) Salmonella enteritidis (r^S-negative strains) Proteus mirabilis Proteus mirabilis Proteus vulgaris Pseudomonas aeruginosa
ATCC (14028) Drag-swabs ATCC (13076) Drag-swabs
+++ +++ +++ +++
Black Black Black Black
Drag-swabs
+++
Pinkish-yellow colonies
ATCC (7002) Drag-swabs ATCC (13315) ATCC (27853)
0 0 0
Pseudomonas aeruginosa
Drag-swabs
±
Altermonas putrefaciens Providencia stuartii Providencia rettgeri
Drag-swabs Drag-swabs Drag-swabs
0 0
Citrobacter freundii Citrobacter spp. Hafnia alvei Morganella morganii Enterobacter aerogenes
Drag-swabs Drag-swabs Drag-swabs Drag-swabs ATCC (13048)
++ ++ 0 +
±
Escherichia coli
ATCC (25922)
+
Yersinia enterocolitica Acinetobacter calcoaceticus
ATCC (23715) Drag-swabs
0 0
±
Haze of pink growth at point of inoculation Haze of pink growth at point of inoculation Haze of pink growth at point of inoculation No H2S production, yellow colonies No H2S production yellow colonies Pinpoint yellow colonies Haze of yellow growth at point of inoculation Scarce yellow colonies at point of inoculation
'ATCC = American Type Culture Collection, Rockville, MD 20852; drag-swabs = various strains isolated from chicken farm environmental drag-swab samples and identified using the API20E identification system (Analytab Products, Inc., Plainview, NY 11803). All wild-type salmonellae were confirmed and serotyped at the National Veterinary Services Laboratory in Ames, IA 50010. jfhe reference medium was xylose-lysine agar base. 4++ = good growth, no detectable inhibition of colonies; ++ = fair growth, slightly inhibited colonies; + = poor growth, very inhibited colonies; ± = trace growth, barely any growth evident; 0 = no visible growth.
Cochran's (1950) Q statistic was used to determine whether there exist significant differences in the proportion of positive responses among the five different agar media. Comparisons between XLT4 and each of the other plating media were made using McNemar's (1947) test, adjusted according to the Yates (1934) correction for continuity, which is recommended in cases where df = 1. RESULTS AND DISCUSSION
As shown in Table 1, XLT4 strongly or completely inhibits Proteus, Pseudomonas, Providencia, and many other nonsalmoneUae. Although there is some growth of Citrobacter freundii, the colonies do not produce black centers on XLT4 plates in 24 h. Typical (lactose-positive) Citrobacter colonies remain yellow on XLT4 and are sticky when tested with an inoculating needle. After 20 to 24 h at
35 to 37 C, typical (H2S-positive) Salmonella colonies on XLT4 are smooth and creamy in texture and appear black or black-centered with a yellow (acid) periphery that changes to pink (alkaline) as the xylose is depleted (20 to 24 h). It was concluded that essentially the only genus capable of forming black colonies within 24 h on XLT4 was Salmonella, allowing easy differentiation from other organisms. The H2S-negative Salmonella strains appear as pinkish-yellow colonies on XLT4. These observations are very important because a highly selective plating medium can change the false-negative status of a specimen to the true result of Sa/mone/Za-positive. This is vital in the case of the egg industry, where successful detection of SE from among other Salmonella serotypes is critical in keeping eggs of SE-contaminated flocks from reaching the marketplace. Obviously, the more Salmo-
Downloaded from http://ps.oxfordjournals.org/ at University of Sydney Library on May 3, 2015
±
colonies or black-centered colonies colonies or black-centered colonies
2432
MILLER ET AL.
TABLE 2. Salmonella isolation rates and the average purity of Salmonella colonies observed on each medium examined from 91 SaXmoaeMn-positive drag-swab samples collected from seven table egg layer flocks Medium Variable
XLT4
XLD
XLDN
BGA
BGAN
Number positive out of 91 Percentage positive Average purity of Salmonella colonies Probability of difference from XLT43
89 98% 3+ to 4+
27 30% 1+ <.001
76 84% 2+ .002
65 71% 1+ to 2+ <001
77 85% 2+ to 3+ .002
X
XLT4 = xylose-lysine-tergitol 4 agar, XLD = xylose-lysine-desoxycholate agar, XLDN = XLD with 20 mcg/mL of sodium novobiocin; BGA = plain brilliant green agar, BGAN = BGA with 20 mcg/mL of sodium novobiocin. 2 1+ = 1 to 25% of the colonies were Salmonella; 2+ = 25 to 50%; 3+ = 51 to 75%; 4+ = 76 to 100%. Using Yates correction for continuity.
ACKNOWLEDGMENTS
The authors gratefully acknowledge Larry Shipman (USDA Animal Plant Health Inspec-
tion Service, Harrisonburg, PA 17110) for providing the drag-swab samples used in this study, and Larry Douglass (University of Maryland, College Park, MD 20742) for his contributions toward the statistical analysis. REFERENCES Cochran, W. G., 1950. The comparison of percentages in matched samples. Biometrika 37:256-266. 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. Kingston, D. J., 1980. A comparison of culturing drag swabs and litter for identification of infections with Salmonella spp. in commercial chicken flocks. Avian Dis. 25:513-516. MacConkey, A., 1905. Lactose-fermenting bacteria in feces. J. Hyg. 5:333-379. Mallinson, E. T., C. R. Tate, R. G. Miller, B. Bennett, and E. Russek-Cohen, 1989. Monitoring poultry farms for salmonella by drag-swab sampling and antigencapture immunoassay. Avian Dis. 33:684-690. McNemar, O., 1947. Note on the sampling error of the difference between corrected proportions or percentages. Psychometrika 12:153-157. Moats, W. A., 1978. Comparison of four agar plating media with and without added novobiocin for isolation of salmonella from beef and deboned poultry meat. Appl. Environ. Microbiol. 36:747-751. 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. Tate, C. R., R. G. Miller, E. T. Mallinson, L. W. Douglass, and R. W. Johnston, 1990. The isolation of salmonellae from poultry farm environmental samples by several enrichment procedures using plating media with and without novobiocin. Poultry Sci. 69: 721-726. Taylor, W. L., 1965. Xylose lysine agars; new media for isolation of enteric pathogens. Am. J. Clin. Pathol. 44:471^75. Yates, F., 1934. Contingency tables involving small numbers and the chi-square test. J. R. Stat. Soc. Suppl. 1:217-235.
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nella colonies there are to select, the greater the chances of detecting the particular serotype of interest. In the present study, salmonellae were isolated from 91 of the 97 drag swab samples collected from the seven SE-positive chicken layer flocks. Many different Salmonella serotypes were isolated, including SE from numerous samples. For these 91 positive samples, XLT4 was found to significantly improve the rate of Salmonella isolations over the other plating media (Table 2). In only two cases was Salmonella isolated on one of the other plating media but not on the XLT4 medium. The Salmonella isolation rate and the average purity of Salmonella colonies on each plating medium for the 91 Sa/mone/Za-positive dragswab specimens are described in Table 2. The authors recommend using XLT4 plating medium to isolate nan-typhimurium Salmonella from fecal-contaminated farm or similar samples containing high numbers of competing bacteria. In addition, XLT4 appears promising for use in clinical applications (C. Wills, personal communication, Maryland Department of Agriculture, Animal Health Laboratory, Salisbury, MD 21802). It is suggested that two different types of plating media be inoculated to further reduce the possibility of a false-negative finding that could occur if a particular strain of Salmonella were sensitive to an inhibitor used in one of the two media. The agar BGAN (Tate et ai, 1990) is recommended as a second medium, as BGAN was found to be second only to XLT4 in selectivity (Table 2).