Isolation of Shigellae: VIII. Comparison of Xylose Lysine Deoxycholate Agar, Hektoen Enteric Agar, Salmonella-Shigella Agar, and Eosin Methylene Blue Agar with Stool Specimens

Two enrichment broths and four plating media were compared for efficiency of detection of enteric pathogens from 1,597 stool specimens. Of 170 salmonellae isolated from the composite of all methods, direct streaking yielded but 54%, whereas enrichment in gram-negative broth found 87% and Selenite-F broth 97%. By contrast, gram-negative broth produced 100% of the 17 shigellae, Selenite-F broth but 77%, and direct streaking only 59%. Thus, enrichment methods produced almost twice the number of both pathogens as direct streaking. Comparison of the plating media revealed xylose lysine deoxycholate agar (XLD) and Hektoen enteric agar to be equal in their abilities to find both pathogens. Both were moderately better than Salmonella-Shigella agar and markedly superior to eosin methylene blue agar. XLD fround 83% of salmonellae produced by the composite of four media and 90% of the shigellae. Hektoen enteric agar found 80% of both. Salmonella-Shigella agar detected 74 and 68%, respectively, and eosin methylene blue agar only 42 and 63%. The numbers of false positives accruing to each medium, however, showed Hektoen enteric and Salmonella-Shigella agars to produce more than twice as many false-positive plates as XLD. Similarly, Selenite-F broth resulted in many more false-positives for all plating media than did gram-negative broth. Consequently, the index of validity, which equates successful isolation of pathogens with total pickings, favored XLD and gram-negative broth as the media of choice, with direct streaking the poorest method by all counts.     

Factors Affecting Selectivity of Brilliant Green-Phenol Red Agar for Salmonellae

Commercial brilliant green (BG)-sulfa agar was found to be nonselective toward a test series of Enterobacteriaceae. Various formulations of BG were prepared by using Trypticase soy agar (BBL) as a base. Results were more reproducible when BG dye was added after sterilization than before. Sulfonamides improved selectivity as compared with brilliant green alone. Sulfanilamide (SN) was slightly more selective for salmonellae than other sulfonamides tested. Bile salts and sodium dodecyl sulfate markedly reduced the toxicity of BG to all the test bacteria. Enterobacter strains were most difficult to inhibit. A combination of 5 mg of BG and 1 g of SN/liter prevented growth of Proteus mirabilis and Escherichia coli and retarded growth of Enterobacter strains. The BG-SN agars were superior in selectivity to a series of commercial agars tested, and numbers of salmonellae recovered on BG-SN agar and Trypticase soy agar (BBL) were the same. Brilliant green agars with various degrees of selectivity are described.     

Clinical Evaluation of Enteric Media in the Primary Isolation of Salmonella and Shigella

Over a 1-year period, media for the isolation of enteric pathogens were compared on 455 stool specimens. Fifty-three pathogens were isolated, of which 56% were Shigella sonnei and 13% were Sh. flexneri. Of these isolates, 90% were found on xylose-lysine-desoxycholate agar, 87% on Hekton enteric agar, and 80% on MacConkey without crystal violet with 2% agar and 0.007% neutral red, but only 28% were recovered on Salmonella-Shigella agar. Less than one-half of the shigellae were recovered after Selenite-F enrichment. On the other hand, enrichment was the most helpful method for isolating salmonellae. Studies on cultures from which mixed isolates were obtained indicated that numbers and chance distribution have an effect on the results obtained. The performance of Salmonella-Shigella agar in the isolation of enteric pathogens was inferior, and the effort involved to obtain those isolates was greater than for Hekton enteric and xylose-lysine-desoxycholate agars.     

A New Plating Medium for the Isolation of Enteric Pathogens: II. Comparison of Hektoen Enteric Agar with S S and E M B Agar

During this study, 2,855 stool specimens from patients at Cook County Hospital were cultured for enteric pathogens. Hektoen Enteric Agar (HE) was compared with E M B and S S Agars by replicate samplings with both direct and indirect methods. Shigella species were recovered more than twice as often on HE Agar as on S S Agar by both methods. With the direct method only, out of 98 Shigella isolated, 97 were isolated from HE Agar, 74 were recovered from E M B Agar, and 40 were found on S S Agar. In addition, HE yielded better isolation of Salmonella strains than did S S or E M B by either direct or indirect methods. The greater efficiency of HE medium is discussed with respect to colonial recognition of enteric pathogens.     

Examination of Feces from Food Handlers for Salmonellae, Shigellae, Enteropathogenic Escherichia coli, and Clostridium perfringens

Duplicate fecal specimens from food handlers were collected in Louisiana. One set of specimens was examined immediately for salmonellae and shigellae by the Central Laboratory of the Louisiana State Board of Health in New Orleans; the other set was shipped to the Food Microbiology Unit at the Robert A. Taft Sanitary Engineering Center in Cincinnati, Ohio, where it was examined for enteropathogenic Escherichia coli (EEC) and Clostridium perfringens. A total of 219 specimens were examined by both laboratories. None yielded salmonellae or shigellae; 171 (78.1%) yielded C. perfringens; 175 (79.9%) yielded E. coli; and 14 (6.4%) yielded EEC. The 14 isolates of EEC were distributed among eight serotypes; one specimen yielded two serotypes. Multiple isolations of C. perfringens strains (two to four) were made from 64 (37.4%) of the specimens, and a total of 244 strains were isolated and studied for identifying characteristics. Of the total, only 87 (35.5%) could be identified serologically by a battery of 67 antisera; only 4 (1.6%) possessed the characteristics of the English “food-poisoning type.” The hemolytic activity on agar containing horse, ox, or sheep blood showed that 140 (57.1%) were “hemolytic,” 81 (33.1%) were “nonhemolytic,” and 23 (9.8%) gave varied results. Only 12 (4.9%) of the strains produced spores that resisted boiling for 30 min or more.     

Rapid Determination of Salmonella in Samples of Egg Noodles, Cake Mixes, and Candies

A glass apparatus system was compared with a standard enrichment broth-selective agar method to test samples of egg noodles, cake mixes, and candy for the presence or absence of salmonellae. The glass apparatus system used fermentation of mannitol, production of H(2)S, or motility, in conjunction with a serological test of flagellar antigens, to detect salmonellae. No salmonellae were detected in 173 samples of food products. Of these samples, 171 were found to be Salmonella-negative after 48 hr with the glass apparatus system. After 72 hr, the standard Salmonella procedure yielded 38 samples which produced Salmonella false-positive results on selective agars. Inoculation of samples with cultures of Salmonella showed that approximately one inoculated cell could be detected after 48 hr of incubation with the glass apparatus. The standard Salmonella test requires a minimum of 72 hr for completion. Compared with the standard Salmonella test, the glass apparatus system is a more rapid and simple system that can be used to determine the presence or absence of Salmonella in these food products.     

Comparison of Four Agar Plating Media with and Without Added Novobiocin for Isolation of Salmonellae from Beef and Deboned Poultry Meat

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.     

Effects of Varying Concentrations of Novobiocin Incorporated into Two Salmonella Plating Media on the Recovery of Four Enterobacteriaceae

Hydrogen sulfide-producing strains of salmonellae, Escherichia coli, Citrobacter freundii, and Proteus mirabilis were isolated from fresh pork sausage. All the strains produced black-centered colonies on Hektoen enteric agar (HE). On xylose lysine deoxycholate agar (XLD), C. freundii produced yellow colonies, and the strains of the other three genera formed black-centered colonies. The selectivity of HE and XLD for salmonellae was improved by the addition of novobiocin to both media. With increasing concentrations of novobiocin, the degree of growth inhibition for the four genera was less on HE than on XLD. Novobiocin concentrations of 80 μg/ml in HE and 5 μg/ml in XLD did not affect the growth or colonial morphology of salmonellae. When 80 μg of novobiocin per ml was incorporated into HE, P. mirabilis strains were not recovered, 40% of C. freundii strains failed to form black-centered colonies, and growth of E. coli strains was not affected but colonies were altered without eliminating the black centers. When novobiocin at 5 μg/ml was incorporated into XLD, colonies of P. mirabilis strains were not recovered, C. freundii formed yellow colonies, and the colonies of the H₂S-producing E. coli strains were unaffected.     

A new selective medium for isolating Listeria spp. from heavily contaminated material

Food-associated outbreaks of human listeriosis have emphasized the importance and necessity of screening food for the presence of Listeria isolates-selective agar medium combining acriflavine (10 mg/liter) with ceftazidime (50 mg/liter) was developed. A total of 1,099 cheese production specimens were cultured, from which 157 Listeria isolates. (14.3%) grew. When compared with modified McBride agar, the acriflavine-ceftazidime agar recovered more Listeria isolates (98 versus 65%, P less than 0.001) more rapidly (57% after 48 h of incubation of the enrichment broth versus 35%, P less than 0.01) and in greater amounts. Acriflavine-ceftazidime selective agar medium proved to be a highly sensitive medium to recover Listeria spp. from heavily contaminated food products.     

A new selective medium for isolating Listeria spp. from heavily contaminated material.

Food-associated outbreaks of human listeriosis have emphasized the importance and necessity of screening food for the presence of Listeria isolates-selective agar medium combining acriflavine (10 mg/liter) with ceftazidime (50 mg/liter) was developed. A total of 1,099 cheese production specimens were cultured, from which 157 Listeria isolates. (14.3%) grew. When compared with modified McBride agar, the acriflavine-ceftazidime agar recovered more Listeria isolates (98 versus 65%, P less than 0.001) more rapidly (57% after 48 h of incubation of the enrichment broth versus 35%, P less than 0.01) and in greater amounts. Acriflavine-ceftazidime selective agar medium proved to be a highly sensitive medium to recover Listeria spp. from heavily contaminated food products.     

Variation in Plating Efficiency of Salmonellae on Eight Lots of Brilliant Green Agar

The plating efficiency of Salmonella anatum, S. cubana, S. dublin, S. tennessee, and S. typhimurium was determined for eight lots of Brilliant Green Agar made by two manufacturers. Washed cells were used as the inoculum and cultures were incubated at 41.5 C. All lots of Brilliant Green Agar were supplemented with 12 mg of sulfadiazine per 100 ml of medium. Of the eight lots of Brilliant Green Agar tested, average recovery of the test salmonellae in three did not differ from recoveries with Trypticase Soy Agar, which was used as a control to indicate the number of viable salmonellae in the test suspension capable of growth on a nonselective medium. Two lots of Brilliant Green Agar gave salmonellae recoveries with geometric means about 25% lower than, and significantly different from, those of the control agar. The remaining three lots of Brilliant Green Agar were generally unproductive.     

Comparison of Media for Direct Isolation and Transport of Shigellae from Fecal Specimens

Xylose-lysine-deoxycholate (XLD) agar, SS agar, and MacConkey agar for isolating shigellae from fecal specimens were compared. XLD agar was superior to both SS agar and MacConkey agar for isolating Shigella sonnei, and both XLD and SS agar were superior to MacConkey agar for isolating S. flexneri. Direct plating of the fecal specimens in the field resulted in a greater yield of shigellae as compared to transporting specimens to the laboratory either in holding media or enrichment broth. Buffered glycerol saline was superior to other transport media evaluated, yielding 83% of shigella isolates when plated within 48 hr as compared to direct plating. The combination of XLD agar and SS agar is recommended for direct isolation of shigellae, and, whenever possible, these solid media should be taken to the bedside and inoculated directly.     

A System for Detecting Salmonellae in Meat and Meat Products

Leifson's selenite F broth was more selective for salmonellae when incubated at 43° instead of the traditional 37°. Different selective agar media produced different numbers of colonies from similar inocula of salmonella cells, but Difco brilliant green agar consistently gave the highest recoveries when tested in this way. Combined with 43° selenite broth enrichment it provided a useful system for isolating salmonellae from foods. In a short comparative test this system compared favourably with more classical techniques employing enrichment of each sample at 37° in two different enrichment broths, followed by streaking on two selective agars.     

Lysine mannitol glycerol agar (LMG) and LMG with sulphamandelate for isolation of Salmonella spp. from clinical specimens

Lysine mannitol glycerol agar (LMG) was compared to xylose lysine deoxycholate agar (XLD), bismuth sulphite agar (BS), and Salmonella-Shigella agar (SS) for the ability to detect Salmonella spp. in clinical specimens, primarily faeces samples. During an 8-month period, 15 salmonellae were isolated from 940 faeces on LMG, while 14 strains were obtained on XLD, 11 on SS and only 3 strains on BS. Salmonella typhi was recovered from two blood cultures in 24 h on LMG, compared to 48 h on BS. LMG was augmented by addition of a sulphacetamide/mandelic acid (sulphamandelate) selective supplement (LMGS). During a 20-month period, 43 salmonellae were isolated from 2622 faeces on LMG and LMGS. The selectivity of LMGS was superior to that of LMG with no decrease in sensitivity of detection; all salmonellae isolated on LMG were isolated on LMGS. Both LMG and LMGS were suitable for routine use in the isolation of Salmonella spp. from clinical specimens.     

Isolation of Salmonellae and Shigellae from an Artificial Mixture of Fecal Bacteria

Numerous selective media, available commercially, act by suppressing "normal" bacterial inhabitants of the intestine while permitting the growth of so-called pathogenic representatives of the family Enterobacteriaceae. This investigation attempts to evaluate the action of Salmonella-Shigella (SS) agar, xylose lysine desoxycholate (XLD) agar, and hektoen enteric (HE) agar. Salmonellae and shigellae, isolated from clinical material, were mixed in various ratios with escherichiae, Klebsiella-Enterobacter-Serratia group bacteria, and members of the tribe Proteeae, also of clinical origin. Several of the mixtures were plated in multiple dilutions on the three media. Stools in preservative were also used for evaluation of the media after the addition of definite numbers of the pathogenic bacteria. Results indicate that SS agar suppresses the shigellae along with the autochthonous members of Enterobacteriaceae. XLD and HE agars readily permit the recovery of shigellae as well as salmonellae. This recovery is not obscured by the higher yield of other species obtained with these media.     

Delayed secondary enrichment for the isolation of salmonellae from broiler chickens and their environment.

Specimens collected from six broiler flocks were cultured for salmonellae by three methods. (i) For direct enrichment, the specimen was homogenized, and 1 ml of the homogenate was inoculated into tetrathionate-brillant green broth; (ii) for preenrichment, liquid specimens and homogenates were incubated at 37 degrees C, and on the next day 1 ml was inoculated into tetrathionate-brillant green broth; and (iii) for delayed secondary enrichment, incubated preenrichment cultures were held at room temperature for 7 to 10 days and then subcultured to fresh tetrathionate-brilliant green broth. All tetrathionate-brilliant green broth cultures were incubated at 42 degrees C for 24 to 48 h before plating. Significantly more isolations of salmonellae were obtained by delayed secondary enrichment than by direct enrichment or preenrichment. Salmonellae were isolated from 417 of 2,283 (18.3%) samples of litter, intestinal contents, and feces cultured by all three methods. Of these positive specimens, direct enrichment detected 208 (49.9%), preenrichment detected 282 (67.6%), and delayed secondary enrichment detected 373 (89.4%). Of 896 specimens of swabs and rinse fluids that were cultured by preenrichment and delayed secondary enrichment, 259 (28.9%) yielded salmonellae. Delayed secondary enrichment detected 254 (98.1%) of these, and preenrichment detected 147 (56.8%). A total of 23 serotypes of salmonellae were identified. The greater effectiveness of delayed secondary enrichment for the isolation of salmonellae was not likely due to the selection of certain serotypes or to an increased inhibition of competing flora.     

Influence of Incubation Temperature and Sodium Heptadecyl Sulfate (Tergitol No. 7) on the Isolation of Salmonellae from Pork Sausage

Cultures of 68 samples of fresh pork sausage purchased locally were incubated at 37 and 43 C, with and without Tergitol No. 7 (sodium heptadecyl sulfate) added to the tetrathionate-Brilliant Green enrichment broth. The results indicated an advantage in incubating the tetrathionate broth at 43 C rather than 37 C in attempting to isolate salmonellae from pork sausage. Without Tergitol, more samples were positive at 43 C than at 37 C, but with Tergitol there was no difference. The higher temperature suppressed the competing gram-negative bacteria and permitted Salmonella to grow in relatively pure culture, thus providing an advantage for isolating and identifying the organisms. Tergitol dispersed and emulsified the fat which improved the isolation of Salmonella when the cultures were incubated at 37 C but not at 43 C. Brilliant Green-sulfadiazine agar was superior to bismuth sulfite agar for isolating salmonellae from tetrathionate broth cultures of fresh pork sausage.     

Lysine-Iron Agar in the Detection of Arizona Cultures

A lysine-iron agar is described and recommended for the detection of Arizona strains which ferment lactose rapidly. Black colonies which appear on bismuth sulfite agar should be transferred to the medium. Salmonellae and Arizona cultures produce a distinctive reaction since they are the only recognized groups of enteric bacteria which regularly produce lysine decarboxylase rapidly and form large amounts of hydrogen sulfide. Use of the medium is particularly recommended in the examination of specimens from enteric infections in which shigellae and salmonellae are not detected.     

New selective media for isolation of clostridia from faecal specimens

Two selective media, novobiocin-colistin agar (NCA) and colistin-crystal violet agar (CCA), were developed for isolating clostridia from human and animal faeces. The basal medium was modified Eggerth-Gagnon agar. The NCA medium contains novobiocin (8 μg ml^-1) and colistin (8 μg ml^-1) and the CCA medium contains colistin (10 μg ml^-1) and crystal violet (10 μg ml^-1). Nine faecal specimens were cultured. Clostridia isolated on these media were similar to those on non-selective media, and higher than those isolated after heat treatment. However, more clostridial species were isolated on the new selective media compared with the non-selective medium. These selective agars were particularly useful for enumerating and isolating clostridia from human faeces.     

Comparison of four agar plating media with and without added novobiocin for isolation of salmonellae from beef and deboned poultry meat

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 H(2)S-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 H(2)S-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 H(2)S-positive salmonellae, and the appearance of Salmonella-like colonies on these media can be considered a presumptive test for H(2)S-positive salmonellae.