A 6×6 drop plate method for simultaneous colony counting and MPN enumeration of Campylobacter jejuni, Listeria monocytogenes, and Escherichia coli

Chen, Chin Yi; Nace, Gary W.; Irwin, Peter L.

doi:10.1016/s0167-7012(03)00194-5

Cited by 357 publications

(286 citation statements)

References 13 publications

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“…Briefly, 10 ml of the worm lysate was spotted on Ashdown agar supplemented with 100 mg/ml Cm using the drop plate method with modifications. 49 Colonies were counted after incubating the plates at 37°C for 48 h. Average colony numbers obtained from visually separate colonies were used for statistical analysis. Bacterial CFU per worm was calculated using the formula: (average colony number ¾ dilution factor ¾ 55 ml worm lysate)/(10 ml worm lysates plated ¾ number of worms).…”

Section: Methodsmentioning

confidence: 99%

Burkholderia pseudomalleikillsCaenorhabditis elegansthrough virulence mechanisms distinct from intestinal lumen colonization

et al. 2012

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The nematode Caenorhabditis elegans is hypersusceptible to Burkholderia pseudomallei infection. However, the virulence mechanisms underlying rapid lethality of C. elegans upon B. pseudomallei infection remain poorly defined. To probe the host-pathogen interaction, we constructed GFP-tagged B. pseudomallei and followed bacterial accumulation within the C. elegans intestinal lumen. Contrary to slow-killing by most bacterial pathogens, B. pseudomallei caused fairly limited intestinal lumen colonization throughout the period of observation. Using grinder-defective mutant worms that allow the entry of intact bacteria also did not result in full intestinal lumen colonization. In addition, we observed a significant decline in C. elegans defecation and pharyngeal pumping rates upon B. pseudomallei infection. The decline in defecation rates ruled out the contribution of defecation to the limited B. pseudomallei colonization. We also demonstrated that the limited intestinal lumen colonization was not attributed to slowed host feeding as bacterial loads did not change significantly when feeding was stimulated by exogenous serotonin. Both these observations confirm that B. pseudomallei is a poor colonizer of the C. elegans intestine. To explore the possibility of toxin-mediated killing, we examined the transcription of the C. elegans ABC transporter gene, pgp-5, upon B. pseudomallei infection of the ppgp-5::gfp reporter strain. Expression of pgp-5 was highly induced, notably in the pharynx and intestine, compared with Escherichia coli-fed worms, suggesting that the host actively thwarted the pathogenic assaults during infection. Collectively, our findings propose that B. pseudomallei specifically and continuously secretes toxins to overcome C. elegans immune responses.

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Section: Methodsmentioning

confidence: 99%

Burkholderia pseudomalleikillsCaenorhabditis elegansthrough virulence mechanisms distinct from intestinal lumen colonization

et al. 2012

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“…Serial dilutions were prepared in sterile 0.85% sodium chloride solution and plated according to the method previously described. 14 The plates were incubated at 371C for 18 to 22 h, and the number of colonies was determined. The detection level by this plating method was 1Â10 2 CFU ml À1 .…”

Section: Time-kill Studiesmentioning

confidence: 99%

Bacteriostatic versus bactericidal activity of ciprofloxacin in Escherichia coli assessed by flow cytometry using a novel far-red dye

et al. 2011

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As common microbiological methods for the assessment of bacteriostatic or bactericidal activities are very time-consuming, in this work we describe that the use of a novel far-red fluorescent stain, Vybrant DyeCycle Ruby (DCR) for the flow cytometric analysis of fluoroquinolone (ciprofloxacin) bacteriostatic and bactericidal activities in Escherichia coli proved to be specific for bacterial DNA and, after ciprofloxacin exposure, DNA distribution analysis was achieved using a 7.5 lM DCR concentration to stain 5Â10 5 ethanol-fixed bacterial cells. The analysis of the bacterial DNA histograms obtained from the ciprofloxacin concentrations tested, enabled the distinction between ciprofloxacin bacteriostatic and bactericidal activities.

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“…One microliter of an overnight culture grown in LB was spotted in the center of each plate section and was dried for 5 min in a flow hood with the lids removed. To ensure consistent inoculum densities, cell counts were determined by the drop plate method (16) and swarm plates were accepted only if the inoculum size started within 4 mm in diameter. Plates were placed on a tray covered in Saran Wrap to maintain humidity and incubated at 37°C before time point photos were taken with the same imaging system.…”

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confidence: 99%

“…The bacterial survival assay was modified from previous methods (13,39,53); details are provided in the supplemental material. At various time points, macrophages were washed 3 times with phosphate-buffered saline (PBS) and lysed with 1 ml 0.5% deoxycholate-PBS, and surviving bacteria were enumerated by serial dilutions using the drop plate method (16).…”

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confidence: 99%

Cysteine Catabolism and Cysteine Desulfhydrase (CdsH/STM0458) in Salmonella enterica Serovar Typhimurium

2012

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Cysteine is potentially toxic and can affect diverse functions such as oxidative stress, antibiotic resistance, and swarming motility. The contribution of cysteine catabolism in modulating responses to cysteine has not been examined, in part because the genes have not been identified and mutants lacking these genes have not been isolated or characterized. We identified the gene for a previously described cysteine desulfhydrase, which we designated cdsH (formerly STM0458). We also identified a divergently transcribed gene that regulates cdsH expression, which we designated cutR (formerly ybaO, or STM0459). CdsH appears to be the major cysteine-degrading and sulfide-producing enzyme aerobically but not anaerobically. Mutants with deletions of cdsH and ybaO exhibited increased sensitivity to cysteine toxicity and altered swarming motility but unaltered cysteine-enhanced antibiotic resistance and survival in macrophages. Cysteine desulfhydrase (CDS) degrades cysteine to pyruvate, ammonia, and sulfide (19,26,43). The major CDS in Escherichia coli appears to be tryptophanase (TnaA), which has an apparent K m for cysteine of 11 mM (56). TnaA primarily catabolizes tryptophan, which appears to be abundant in the intestinal tract (29). In E. coli, TnaA can become 10% of soluble protein, which suggests the potential to also degrade cysteine in vivo, despite an unfavorable K m (56). Several factors regulate TnaA expression. TnaA is repressed by glucose, pyruvate, and acetate (6, 9, 10, 31). Expression requires cyclic AMP and is induced by tryptophan, cysteine, indole, growth in alkaline broth, depletion of heme and phosphatidyl glycerol, and anaerobic growth with an alternate electron acceptor (9, 51, 52). From these factors, it could be speculated that TnaA contributes to energy generation by degrading tryptophan and possibly cysteine to pyruvate.Salmonella enterica does not have TnaA, yet cysteine induces a powerful CDS (19,26,43). This CDS has been purified from S. enterica, and similarly to TnaA, it contains pyridoxal-5=-phosphate (42). The k cat /K m ratio for L-cysteine, a measure of catalytic efficiency, is 10,340 mM Ϫ1 s Ϫ1 for S. enterica CDS (calculated from values reported by Kredich et al. [42,43]) and 6.7 mM Ϫ1 s Ϫ1for E. coli TnaA (calculated from values reported by Snell [56]). In other words, the S. enterica CDS appears to be more than three orders of magnitude more efficient than TnaA. To explore the function of the S. enterica CDS and cysteine catabolism, we identified the gene for the major CDS and characterized its regulation and the phenotype of mutants lacking this enzyme. MATERIALS AND METHODSStrains and plasmids. Table 1 lists the strains and plasmids used in this study. All gene deletions and replacements were performed originally into the TT22971 background strain as described previously (21), followed by P22 transduction into the appropriate genetic background, and the products were ensured to be phage free by cross-streaking against P22-H5 on green plate agar (15). The following codons were delet...

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A 6×6 drop plate method for simultaneous colony counting and MPN enumeration of Campylobacter jejuni, Listeria monocytogenes, and Escherichia coli

Cited by 357 publications

References 13 publications

Burkholderia pseudomalleikillsCaenorhabditis elegansthrough virulence mechanisms distinct from intestinal lumen colonization

Burkholderia pseudomalleikillsCaenorhabditis elegansthrough virulence mechanisms distinct from intestinal lumen colonization

Bacteriostatic versus bactericidal activity of ciprofloxacin in Escherichia coli assessed by flow cytometry using a novel far-red dye

Cysteine Catabolism and Cysteine Desulfhydrase (CdsH/STM0458) in Salmonella enterica Serovar Typhimurium

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