Understanding and Teaching the Most Probable Number Technique1

Oblinger, J. L.; Koburger, J. A.

doi:10.4315/0022-2747-38.9.540

Cited by 175 publications

(86 citation statements)

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“…The microtitre plates were then incubated for five days and each well was scored for growth or no growth. The most probable number (MPN) technique was then used to determine the number of viable cells in the overnight culture, as described by Oblinger & Koburger (1975). This technique was found to be much more reproducible than plate counts for P. gingivalis cells.…”

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confidence: 99%

The nucleoid-associated protein HUβ affects global gene expression in Porphyromonas gingivalis

et al. 2013

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HU is a non-sequence-specific DNA-binding protein and one of the most abundant nucleoidassociated proteins in the bacterial cell. Like Escherichia coli, the genome of Porphyromonas gingivalis is predicted to encode both the HUa (PG1258) and the HUb (PG0121) subunit. We have previously reported that PG0121 encodes a non-specific DNA-binding protein and that PG0121 is co-transcribed with the K-antigen capsule synthesis operon. We also reported that deletion of PG0121 resulted in downregulation of capsule operon expression and produced a P. gingivalis strain that is phenotypically deficient in surface polysaccharide production. Here, we show through complementation experiments in an E. coli MG1655 hupAB double mutant strain that PG0121 encodes a functional HU homologue. Microarray and quantitative RT-PCR analysis were used to further investigate global transcriptional regulation by HUb using comparative expression profiling of the PG0121 (HUb) mutant strain to the parent strain, W83. Our analysis determined that expression of genes encoding proteins involved in a variety of biological functions, including iron acquisition, cell division and translation, as well as a number of predicted nucleoid associated proteins were altered in the PG0121 mutant. Phenotypic and quantitative real-time-PCR (qRT-PCR) analyses determined that under iron-limiting growth conditions, cell division and viability were defective in the PG0121 mutant. Collectively, our studies show that PG0121 does indeed encode a functional HU homologue, and HUb has global regulatory functions in P. gingivalis; it affects not only production of capsular polysaccharides but also expression of genes involved in basic functions, such as cell wall synthesis, cell division and iron uptake. INTRODUCTIONPorphyromonas gingivalis is a Gram-negative obligate anaerobe belonging to the family Bacteroidaceae that persists as a natural member of the human oral microbiota. A shift in the microbial community leading to outgrowth of this anaerobe is directly linked to periodontitis, a chronic inflammatory disease that leads to destruction of the tissues supporting the gums and ultimately, exfoliation of the teeth (Choil et al., 1990;Dzink et al., 1988;Grossi et al., 1994;Lamont & Jenkinson, 2000;Moore et al., 1991). This commensal can colonize, invade and multiply within gingival epithelial cells, as well as penetrate into deeper epithelial cell layers, potentially releasing the whole organism and/or virulence factors into the bloodstream (reviewed by Yilmaz, 2008). In addition to its ability to cause disease in the oral cavity, there are data indicating a role in systemic disease, including its ability to invade vascular endothelial cells (Dorn et al., 2000(Dorn et al., , 2002Jandik et al., 2008) and to cause aggregation of platelets (Pham et al., 2002). For many pathogenic bacteria, surface polysaccharides play a key role in immune modulation et al., 1996). HU typically acts as an accessory protein in virtually all types of nucleoprotein-mediated processes (reviewed by...

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confidence: 99%

The nucleoid-associated protein HUβ affects global gene expression in Porphyromonas gingivalis

et al. 2013

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“…The putative Campylobacter colonies selected based on morphology and Gram staining reaction were confirmed by genus-and species-specific PCR assays. The density of Campylobacter cells (MPN, 100 ml Ϫ1 ) was calculated according to the method described by Oblinger and Koburger (38) and subsequently converted to a load identical to the method described previously for the Bacteroidales source markers.…”

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confidence: 99%

Long-Term Monitoring of Waterborne Pathogens and Microbial Source Tracking Markers in Paired Agricultural Watersheds under Controlled and Conventional Tile Drainage Management

Wilkes

Brassard

Edge

et al. 2014

Appl Environ Microbiol

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f Surface waters from paired agricultural watersheds under controlled tile drainage (CTD) and uncontrolled tile drainage (UCTD) were monitored over 7 years in order to determine if there was an effect of CTD (imposed during the growing season) on occurrences and loadings of bacterial and viral pathogens, coliphages, and microbial source tracking markers. There were significantly lower occurrences of human, ruminant, and livestock (ruminant plus pig) Bacteroidales markers in the CTD watershed in relation to the UCTD watershed. As for pathogens, there were significantly lower occurrences of Salmonella spp. and Arcobacter spp. in the CTD watershed. There were no instances where there were significantly higher quantitative loadings of any microbial target in the CTD watershed, except for F-specific DNA (F-DNA) and F-RNA coliphages, perhaps as a result of fecal inputs from a hobby farm independent of the drainage practice treatments. There was lower loading of the ruminant marker in the CTD watershed in relation to the UCTD system, and results were significant at the level P ‫؍‬ 0.06. The odds of Salmonella spp. occurring increased when a ruminant marker was present relative to when the ruminant marker was absent, yet for Arcobacter spp., the odds of this pathogen occurring significantly decreased when a ruminant marker was present relative to when the ruminant marker was absent (but increased when a wildlife marker was present relative to when the wildlife marker was absent). Interestingly, the odds of norovirus GII (associated with human and swine) occurring in water increased significantly when a ruminant marker was present relative to when a ruminant marker was absent. Overall, this study suggests that fecal pollution from tiledrained fields to stream could be reduced by CTD utilization.

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“…Genomic DNA of all samples was extracted according Marmur (1961) for subsequent qPCR analysis. In addition, seeds were planted in plastic pots containing vermiculite and maintained in greenhouse for 28 days for nodulation efficiency and most probable number (MPN) analysis (Oblinger and Koburger 1975). All pure and inoculants samples conditions were accompanied by CFU, pH, and turbidity analysis.…”

Section: Biological Inoculants Desiccation Assay and Greenhousementioning

confidence: 99%

Detection and Enumeration of Bradyrhizobia Cells by Real-Time PCR Quantification

2015

JAES

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Bradyrhizobiumelkanii and Bradyrhizobiumjaponicum are nitrogen-fixing bacteria that establish symbiosis with soybean plants. The optimization of this symbiosis and the success of biological nitrogen fixation are reached by inoculating the seeds with strains of bradyrhizobia. The aim of this work consisted in the enumeration of bacterial cells of inoculants from different ages by real-time PCR quantification (qPCR) based on 16S rDNA and the evaluation of surviving cells rates under desiccation experiments. The results of counting by colony forming units (CFU) were correlated with those obtained by qPCR. All qPCR values were higher than those for CFU values in cell enumeration for all samples and conditions observed. Cell resistance to desiccation was shown until 24 hours onto soybean seeds, with considerable fall in all the inoculants after 48 hours under desiccation. This was probably due to biochemical and physiological changes in its metabolism, making use of defensive mechanism to the adverse conditions for its survival.Keywords: Cellular enumeration, soybean inoculants, biological nitrogen fixation, bradyrhizobia IntroductionThe main sources of nitrogen (N) for soybean crops provide from application of nitrogen fertilizers and biological nitrogen fixation (BNF) that uses atmospheric N2 through symbiotic association between legumes and rhizobia BNF is the process that involves the conversion of inert N2 gas into ammonia (NH3) through expending large amounts of energy to break triple chemical bonds N atoms (Oldroydet al., 2011). Bradyrhizobia are nitrogenfixing bacteria that possess a complex enzyme named nitrogenase that is able to realize N2 conversion to produced NH3, which in turn is assimilated into organic compounds by soybean plants able to establish symbiotic interactions with those microrganisms (Kuykendalet al., 1992). The maximization of the symbiosis events can be achieved by inoculation with selected strains of rhizobia performing the best and desirable characteristics, reducing the cost of soybean production. On the other hand, rhizobia inoculation is also a factor that contributes to reducing pollution in the environment by decreasing N fertilizers applications in the field (Abdel-Fattah I El-Shaarawiet al., 2011). Official recommendation of Bradyrhizobiumelkanii and/or Bradyrhizobiumjaponicum strains for soybean's inoculants production in Brazil is made by Ministry of Agriculture, Livestock and Food, Supply (MAPA). These bradyrhizobia are genetic and physiological different (Soares and Passaglia 2010) and can be used separately or in combination. MAPA requires a periodic control of inoculants quality and found that in some cases there is a discrepancy between the numbers of cells detected by the CFU (colony forming units) traditional method of counting performed by different laboratories. In this paper, the development and applying of a new method for inoculants cell enumeration was tested by real-time PCR quantification (qPCR) based on 16S rDNA. This technique was applied both to pure ...

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Understanding and Teaching the Most Probable Number Technique1

Cited by 175 publications

References 0 publications

The nucleoid-associated protein HUβ affects global gene expression in Porphyromonas gingivalis

The nucleoid-associated protein HUβ affects global gene expression in Porphyromonas gingivalis

Long-Term Monitoring of Waterborne Pathogens and Microbial Source Tracking Markers in Paired Agricultural Watersheds under Controlled and Conventional Tile Drainage Management

Detection and Enumeration of Bradyrhizobia Cells by Real-Time PCR Quantification

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