Requirements of Salmonella typhimurium for Recovery from Thermal Injury

The heating of Salmonella typhimurium 7136 at 48 C for 30 min produces a population of cells that are incompetent at division on Levine Eosin Methylene Blue Agar containing 2.0% NaCl (EMB-NaCl). When these injured cells were placed in fresh citrate salts medium they recovered, and regained their tolerance to the EMB-NaCl medium and grew out. The addition of the selective inhibitors rifamycin, 5-fluorouracil, 2,4-dinitrophenol, chlorotetracycline, chloramphenicol, and 5-methyl-tryptophan to the recovery medium showed that the recovery process was dependent on ribosomal ribonucleic acid (RNA) synthesis, adenosine triphosphate synthesis, and the synthesis of new protein. These results were substantiated by incorporation experiments, which demonstrated that during recovery no deoxyribonucleic acid synthesis, and hence no cell division, occurred. Ribosomal RNA was synthesized during recovery, but its synthesis was not the rate-limiting step. A small but significant amount of protein was also formed during the latter part of the recovery period.     

Evaluation of Culture Media for the Isolation of Salmonellas from Sewage Sludge

Ten samples of sewage sludge were examined by various methods for the isolation of salmonellas using three types of enrichment broth: Muller-Kauffmann Tetrathionate Broth (MKTB), Selenite F Broth (SFB), and Brilliant Green Broth (BGB), two temperatures (37°C and 43°C) and three selective media: Deoxycholate Citrate Agar incubated aerobically (DCA), and anaerobically (DCA(N)), Brilliant Green Agar (BGA), and Bismuth Sulphite Agar (BSA). The results suggest that a combination of pre-enriched MKTB incubated at 37°C and plated on to BGA at 24 and 48 h was the best method, but when examining contaminated material such as sewage sludge, it appears unwise to rely on one single method.     

Efficiency of different enrichment and isolation procedures for the detection of Salmonella serotypes in edible offal

Rapid detection systems for Salmonella in foodstuffs are currently being developed. However, existing standards still call for application of traditional methods employing pre-enrichment followed by selective enrichment and isolation. The efficacy of various methods was tested using 264 chicken and lamb organ meats. Pre-enrichment was carried out in Tryptone Soy Broth (TSB) and enrichment in Tetrathionate Brilliant Green Broth (TTB) at 37°C, Selenite Broth with Brilliant Green and Sulphapyridine at 37°C and 43°C, and Rappaport-Vassiliadis Broth (RV 10) at 42°C. The isolation media were Brilliant Green Agar (BGA), Deoxycholate Citrate Agar, Hektoen Enteric Agar (HEA) and Salmonella-Shigella Agar.
Enrichment in RV/42°C followed by isolation on BGA as recommended by ISO standard no. 6579 and enrichment in TTB/37°C followed by isolation in HEA, no longer recommended by that standard, produced the best results. Low percentages of positive samples and difficulties in detecting Salmonella are the result of interference by competing organisms (Enterobacteriaceae) and the number of salmonellas present after enrichment.
A total of 528 samples (TSB, eggs, lamb liver and chicken liver) were inoculated with Salm. enteritidis, Salm. kapemba and Salm. virchow , and the preceding experiment was repeated. All the TSB and egg samples tested positive, but the percentage of positive samples from the lamb and chicken liver was only 81–92%. Recovery of the salmonellas did not depend upon the method employed or the serotype inoculated but instead on interference by competing flora and the numbers of Salmonella present in the samples.     

Isolation of Shigellae. VII. Comparison of Gram-Negative Broth with Rappaport''s Enrichment Broth

Efficacies of Gram Negative Broth (GN) and Rappaport's Enrichment Broth (RE) were compared for detection of enteric pathogens from clinical specimens. Whereas direct streaking on four plating media found 57% of the salmonellae, GN found 80% and RE found 92% of the 157 isolates. By contrast, direct streaking found 87% of the shigellae whereas GN detected 93%, but RE found only 20%. RE produced 35% more Salmonella-positive plates than GN did, which resulted in a 15% increase in isolates. Statistically, RE proved to be significantly better than GN. The unsuitability of RE for shigellae, however, dictates that, if only one enrichment broth is to be used, GN must be that choice, but that maximal isolations of all enteric pathogens should result from the use of both RE and GN. Of the four plating media, Xylose Lysine Deoxycholate Agar detected 90% of the salmonellae and 85% of the shigellae. Salmonella-Shigella Agar detected 72 and 43%, respectively, and Levine Eosin Methylene Blue Agar found 45 and 55%. Bismuth Sulfite Agar detected only 22% of the salmonellae and found no shigellae. The performance of all of the plating media was enhanced by enrichment, but RE was especially effective for salmonellae when compared to GN.     

Isolation of Shigellae. VII. Comparison of Gram-Negative Broth with Rappaport's Enrichment Broth

Efficacies of Gram Negative Broth (GN) and Rappaport''s Enrichment Broth (RE) were compared for detection of enteric pathogens from clinical specimens. Whereas direct streaking on four plating media found 57% of the salmonellae, GN found 80% and RE found 92% of the 157 isolates. By contrast, direct streaking found 87% of the shigellae whereas GN detected 93%, but RE found only 20%. RE produced 35% more Salmonella-positive plates than GN did, which resulted in a 15% increase in isolates. Statistically, RE proved to be significantly better than GN. The unsuitability of RE for shigellae, however, dictates that, if only one enrichment broth is to be used, GN must be that choice, but that maximal isolations of all enteric pathogens should result from the use of both RE and GN. Of the four plating media, Xylose Lysine Deoxycholate Agar detected 90% of the salmonellae and 85% of the shigellae. Salmonella-Shigella Agar detected 72 and 43%, respectively, and Levine Eosin Methylene Blue Agar found 45 and 55%. Bismuth Sulfite Agar detected only 22% of the salmonellae and found no shigellae. The performance of all of the plating media was enhanced by enrichment, but RE was especially effective for salmonellae when compared to GN.     

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.     

Thermal Injury and Recovery of Streptococcus faecalis

Exposure of Streptococcus faecalis R57 to sublethal heating produced a temporary change in the salt tolerance and growth of the organism. After sublethal heat treatment at 60 C for 15 min, greater than 99.0% of the viable population was unable to reproduce on media containing 6% NaCl. In addition, the heated cells displayed a sensitivity to incubation temperature, pH, and 0.01% methylene blue. When the injured cells were placed in a synthetic medium, recovery occurred at a much slower rate than in a complex medium. However, both media supported comparable growth of the uninjured organism. Various media used for the enrichment of streptococci also provided a suitable environment for the recovery of the injured cells. Generally, as more selective agents were present in the medium, the rates of recovery decreased. Metabolic inhibitor studies with chloramphenicol, penicillin, and actinomycin D substantiated the fact that the process involved was recovery and not growth, and that this recovery was linked to ribonucleic acid synthesis.     

Precursor Ribosomal Ribonucleic Acid and Ribosome Accumulation In Vivo During the Recovery of Salmonella typhimurium from Thermal Injury

When cells of S. typhimurium were heated at 48 C for 30 min in phosphate buffer (pH 6.0), they became sensitive to Levine Eosin Methylene Blue Agar containing 2% NaCl (EMB-NaCl). The inoculation of injured cells into fresh growth medium supported the return of their normal tolerance to EMB-NaCl within 6 hr. The fractionation of ribosomal ribonucleic acid (rRNA) from unheated and heat-injured cells by polyacrylamide gel electrophoresis demonstrated that after injury the 16S RNA species was totally degraded and the 23S RNA was partially degraded. Sucrose gradient analysis demonstrated that after injury the 30S ribosomal subunit was totally destroyed and the sedimentation coefficient of the 50S particle was decreased to 47S. During the recovery of cells from thermal injury, four species of rRNA accumulated which were demonstrated to have the following sedimentation coefficients: 16, 17, 23, and 24S. Under identical recovery conditions, 22, 26, and 28S precursors of the 30S ribosomal subunit and 31 and 48S precursors of the 50S ribosomal subunit accumulated along with both the 30 and 50S mature particles. The addition of chloramphenicol to the recovery medium inhibited both the maturation of 17S RNA and the production of mature 30S ribosomal subunits, but permitted the accumulation of a single 22S precursor particle. Chloramphenicol did not affect either the maturation of 24S RNA or the mechanism of formation of 50S ribosomal subunits during recovery. Very little old ribosomal protein was associated with the new rRNA synthesized during recovery. New ribosomal proteins were synthesized during recovery and they were found associated with the new rRNA in ribosomal particles. The rate-limiting step in the recovery of S. typhimurium from thermal injury was in the maturation of the newly synthesized rRNA.     

Detection and Incidence of Escherichia coli on Storage Pen Surfaces of Fishing Trawlers

Six methods for the detection and enumeration of Escherichia coli on the storage pen surfaces of commercial fishing trawlers and in harbor wash water were evaluated. E. coli was found consistently present in Boston harbor water used for washing vessel holds and was detected either in small numbers or not at all on storage pen surfaces. Violet Red Bile Agar as a primary enumeration medium was found ineffective for detection of coliforms because of the nonselective development of large numbers of other gram-negative organisms. The use of E. coli broth at 44.5 C for primary most-probable-number determinations, followed by confirmation of E. coli on Levine Eosine Methylene Blue Agar, appears to offer numerous advantages over more conventional methods of detecting E. coli for survey studies of the fishing industry, where coliform-like organisms result in many false-positive presumptives with other methods.     

Decolorization of industrial dyes by a Brazilian strain of Pleurotus pulmonarius producing laccase as the sole phenol-oxidizing enzyme

The ability of a Brazilian strain ofPleurotus pulmonarius to decolorize structurally different synthetic dyes (including azo, triphenylmethane, heterocyclic and polymeric dyes) was investigated in solid and submerged cultures. Both were able to decolorize completely or partially 8 of 10 dyes (Amido Black, Congo Red, Trypan Blue, Methyl Green, Remazol Brilliant Blue R, Methyl Violet, Ethyl Violet, Brilliant Cresyl Blue). No decolorization of Methylene Blue and Poly R 478 was observed. Of the four phenol-oxidizing enzymes tested in culture filtrates (lignin peroxidase, manganese peroxidase, aryl alcohol oxidase, laccase),P. pulmonarius produced only laccase. Both laccase activity and dye decolorization were related to glucose and ammonium starvation or to induction by ferulic acid. The decolorizationin vivo was tested using three dyes — Remazol Brilliant Blue R, Trypan Blue and Methyl Green. All of them were completely decolorized by crude extracellular extracts. Decolorization and laccase activity were equally affected by pH and temperature. Laccase can thus be considered to be the major enzyme involved in the ability ofP. pulmonarius to decolorize industrial dyes.     

Evaluation of SMID agar for identification of salmonella in naturally contaminated veterinary samples

Five hundred naturally contaminated samples were examined in a trial where SMID agar was compared with either Brilliant Green agar, Rambach agar or Xylose, Lysine, Desoxycholate agar. There were no significant differences in sensitivity but SMID agar detected slightly more salmonella isolates than BGA agar following RVS enrichment and slightly less following Selenite enrichment. There was, however, a marked reduction in the level of false-positives when SMID agar was used.     

Rapid Quantitative Method for Salmonella Detection in Polluted Waters

A procedure has been developed for the enumeration of salmonellae in polluted waters using several modifications of existing techniques. Confirmation of salmonellae is achieved within 48 hr. This procedure includes selective enrichment in m-Tetrathionate Broth (22 +/- 1 hr), plating on Brilliant Green Sulfa Agar (20 +/- 1 hr), and confirmation by flagellar (H) agglutination of the growth in a mannosecontaining medium (6 +/- 1 hr). An incubation temperature of 41.5 C was used throughout this procedure. Dilution to extinction techniques (most probable number) were employed to enumerate salmonellae. Large sample volumes were concentrated through the use of membrane filters. This technique proved to be rapid and reliable for the enumeration of salmonellae in water, waste water, and waste-water sludges.     

Novel media for detection of microbial producers of cellulase and xylanase

Abstract Agar nutrient media containing 0.2% soluble hydroxyethylcellulose covalently dyed with Ostazin Brilliant Red H-3B or soluble beechwood 4-O-methyl- d -glucurono- d -xylan dyed with Remazol Brilliant Blue R were used for sensitive detection of microorganisms producing and secreting into the surrounding medium endo-1,4-β-glucanase and/or endo-1,4-β-xylanase. Pale clearing zones formed around the colonies grown on such media indicated the production of corresponding polysaccharide-hydrolases.     

白腐真菌落叶松锈迷孔菌产漆酶液体培养基的优化及其对染料的脱色作用

以白腐真菌落叶松锈迷孔菌(Porodaedalea laricis)胞外漆酶为响应值,通过将Plackett-Burman设计、最陡爬坡设计和Box-Behnken设计相结合,获得了P.laricis产胞外漆酶的最适培养基为:去皮马铃薯365.61 g/L、蛋白胨5.0 g/L、葡萄糖20.0 g/L、KH2PO41.0 g/L、MgSO47H2O 0.5 g/L、MnSO4 H2O 0.15 g/L、CaCl22H2O 0.03 g/L、酒石酸铵6.68 g/L、琥珀酸钠1.5 g/L、吐温800.48 mL/L、玉米芯46.43 g/L、维生素B10.01 g/L。在该条件下,P.laricis漆酶活性为3.29 U/mL,相比于优化前提高了2.81倍,与理论值3.32 U/mL相近,说明该模型准确可靠。此外,将漆酶应用于降解多种合成染料包括活性亮蓝X-BR、雷马素亮蓝R、酸性黑172、刚果红、亚甲基蓝、中性红、靛蓝、萘酚绿B和结晶紫,反应168 h后脱色率分别可达到95.64%、97.21%、36.11%、91.63%、61.42%、74.65%、48.60%、25.13%和68.80%。     

The Giemsa-11 technique for species-specific chromosome differentiation

Summary Oxidizing Methylene Blue and adding the reaction products to Eosin Y and Azure B makes possible a highly reliable Giemsa-11 technique for discrimination of chromosomes in hybrid cells according to their parental origin. This staining can be combined in a sequential procedure with a fluorescent banding technique allowing the exact identification of the chromosomes.     

Microspectrofluorometry of ultraviolet-inducible fluorescence in supravitally-stained ascites tumour cells

Synopsis Ultraviolet irradiation of tumour cells (Ehrlich tetraploid ascites tumour of mice, TO strain), supravitally stained with thiazine dyes (Azure II, Azure A, Methylene Blue, Toluidine Blue) or an oxazine dye (Brilliant Cresyl Blue), induces blue fluorescence in cytoplasmic bodies believed to be lipid droplets or lysosome-like bodies. Microspectrofluorometry of the inducible fluorescence in Ehrlich tumour cells gives bimodal excitation (340/394 nm) and emission (443/700 nm) curves.     

A Modification of Brilliant Green Agar for Improved Isolation of Salmonella

Five organisms commonly found to be capable of growth on commercial Brilliant Green Agar (BGA) after enrichment in Muller-Kauffman Tetrathionate broth (MKT) were tested for sensitivity to 18 antimicrobial agents. The sensitivities of two Salmonella serotypes to these agents were also tested. A combination of sulphacetamide (at 1.0 mg/ml) and mandelic acid (at 0.25 mg/ml) incorporated into BGA was found to give maximum recovery of salmonellas from MKT broth enrichment whilst giving maximum suppression of contaminating organisms. More importantly, this Antibiotic-enriched Brilliant Green Agar (ABG) gave a lower incidence of false positive results when compared with commercial BGA. Increasing the incubation temperature from 35 to 43°C was found to accentuate the selectivity of ABG without inhibiting the growth of salmonellas. A total of 31 Salmonella serotypes were tested for their ability to grow on ABG at 43°C; all produced typical colonies.     

Brilliant Blue as an alternative dye to Fast Green for in ovo electroporation

Chick embryo electroporation is a powerful tool for the introduction of transgenes into tissues of interest for the study of developmental biology. This method often uses Fast Green to visualize the injected area by staining the solution containing DNA green. Here, we show that Fast Green fluoresces in a red color after electroporation, suggesting that researchers need to be cautious when detecting red fluorescence. Fast Green solution did not show any fluorescence before injection into chick embryos, but fluoresced red within 3 min post-injection into chick embryos. We identified Brilliant Blue as suitable alternative dye for use as an indicator of injection sites in ovo electroporation. We found that 0.2% of Brilliant Blue was sufficient to track the area of DNA injection. In addition, this chemical did not show red fluorescence after electroporation. Our findings demonstrate that Brilliant Blue can be used for detecting red fluorescent proteins introduced into chick embryos by electroporation. Our study also shows useful examples for the application of Brilliant Blue for the precise quantification of two fluorescence intensities after EGFP and mCherry co-electroporation.     

Isolation of Salmonella from environmental samples collected in the reptile department of Antwerp Zoo using different selective methods

AIMS: To evaluate the environmental spread of Salmonella strains in the reptile department of Antwerp Zoo and to compare different isolation methods for Salmonella. METHODS AND RESULTS: One hundred environmental samples were collected in the service sections and public spaces of the reptile department. After pre-enrichment in buffered peptone water (BPW), selective enrichment was performed in Rappaport Vassiliadis Single Component Enrichment Broth (RVS), Selenite Cystine Broth (SEL) and Mueller Kauffman Tetrathionate Broth (MKTTn). Subculturing on Modified Semisolid Rappaport-Vassiliadis (MSRV) Medium, and the combined use of immunomagnetic separation (IMS) and RVS was evaluated. The isolation media used were Hektoen Enteric Agar (HE), Phenol Red Brilliant Green Agar (BG) and Xylose Lysine Decarboxylase Agar (XLD). Salmonella strains were found in 47 samples (47.0%). Most isolations were made on HE after combined IMS/RVS enrichment. Sixty-six Salmonella strains were serotyped, 29 belonged to Salmonella enterica ssp. enterica (I), 3 to ssp. salamae (II), 29 to ssp. arizonae or diarizonae (IIIa/b), 4 to ssp. houtenae (IV) and 1 strain showed autoagglutination. In addition, a 10-year survey (1995-2004) of Salmonella serovars isolated from reptiles at Antwerp Zoo is presented. CONCLUSIONS: A high prevalence of Salmonella strains was noted in the service sections of the reptile department. Only a few isolations were made in the public spaces. Selective enrichment in RVS was the most efficient. In combination with IMS, this method gave an even higher isolation rate than the International Standard method (ISO 6579:2002). SIGNIFICANCE AND IMPACT OF THE STUDY: This study confirms the importance of reptiles as spreaders of Salmonella in their surroundings. The possible infectious risks for zoo personnel and visitors are evaluated. Improved laboratory protocols for the isolation of Salmonella from the environment are suggested.     

Oxytetracycline-Resistant Coliforms in Commercial Poultry Products

The presence of oxytetracycline-resistant bacteria was investigated with commercially frozen chicken thighs and drumsticks. Bacterial flora were surveyed by means of total and coliform counts with Tryptone Glucose Extract Agar and Desoxycholate Agar, respectively. After counting, the Desoxycholate Agar plates were replicated on the same medium containing 25, 50, 75, and 100 ppm of oxytetracycline. Resistant colonies were found on all samples that were replicated. Of 2613 colonies isolated on Desoxycholate Agar, 47.8% grew in the presence of 25 ppm of oxytetracycline. From 50 to 100 ppm, the number of resistant isolates remained essentially the same, near 34%. Of 812 colonies of antibiotic-resistant bacteria identified with dulcitol-lactose-iron-agar, 82.5% were paracolons, 13.7% were pseudomonads, and 3.8% were Escherichia or Aerobacter. Bacteria resistant to oxytetracycline were shown to be present on commercially processed chicken. The origin of the resistance to oxytetracycline was not established; however, since the antibiotic was not used during processing, it appeared that these antibiotic-resistant bacteria arose in the intestines of the chickens as a result of feed which contained antibiotic. This is supported by a comparison with the antibiotic resistance of coliforms from chickens raised on feed both with and without oxytetracycline, for the percentages of resistant colonies are similar in both commercial chicken and chicken raised on feed containing the antibiotic.