Estimating microbial population counts by ‘most probable number’ using Microsoft Excel®

Briones, Aurelio; Reichardt, Wolfgang

doi:10.1016/s0167-7012(98)00111-0

Cited by 101 publications

(60 citation statements)

References 6 publications

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“…The medium either contained 6.8 mM acetate for the estimation of the acetate-utilizing population or was incubated in closed glass desiccators under a headspace containing 10% (vol/vol) CH 4 for estimation of the methane-utilizing population. MPNs were calculated using an Excel-based program (6).…”

Section: Growth Experimentsmentioning

confidence: 99%

Methylocella Species Are Facultatively Methanotrophic

2005

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All aerobic methanotrophic bacteria described to date are unable to grow on substrates containing carboncarbon bonds. Here we demonstrate that members of the recently discovered genus Methylocella are an exception to this. These bacteria are able to use as their sole energy source the one-carbon compounds methane and methanol, as well as the multicarbon compounds acetate, pyruvate, succinate, malate, and ethanol. To conclusively verify facultative growth, acetate and methane were used as model substrates in growth experiments with the type strain Methylocella silvestris BL2. Quantitative real-time PCR targeting the mmoX gene, which encodes a subunit of soluble methane monooxygenase, showed that copies of this gene increased in parallel with cell counts during growth on either acetate or methane as the sole substrate. This verified that cells possessing the genetic basis of methane oxidation grew on acetate as well as methane. Cloning of 16S rRNA genes and fluorescence in situ hybridization with strain-specific and genus-specific oligonucleotide probes detected no contaminants in cultures. The growth rate and carbon conversion efficiency were higher on acetate than on methane, and when both substrates were provided in excess, acetate was preferably used and methane oxidation was shut down. Our data demonstrate that not all methanotrophic bacteria are limited to growing on one-carbon compounds. This could have major implications for understanding the factors controlling methane fluxes in the environment.Aerobic methanotrophic bacteria occupy a key position in the global methane cycle. In aerobic interfaces of flooded soils and wetlands their activity limits potential methane efflux to the atmosphere, and in well-aerated upland soils they consume atmospheric methane directly (8, 25). The 11 described genera of methanotrophic bacteria include species with diverse environmental tolerances and biochemical properties, but one trait considered common to all is the inability to grow on substrates containing carbon-carbon bonds (5,18,20). Growth is limited to methane, methanol, and in some cases formate, formaldehyde, and methylamines (5). Experiments with 14 C-labeled substrates have shown that small amounts of some organic acids can also be assimilated during exponential growth on one-carbon substrates. However, this accounts for at most 5 to 10% of the total C assimilation, no energy is gained, and no growth occurs on these compounds alone (18).Recently, the new methanotrophic species Methylocella palustris (13, 14), Methylocella silvestris (17), and Methylocella tundrae (9) were isolated from acidic peat, forest, and tundra soils, respectively. Together with Methylocapsa acidiphila (12), these species form a distinct taxonomic cluster of acidophilic, methanotrophic bacteria. These belong to the Alphaproteobacteria (type II methanotrophs) but are not monophyletic with the previously known type II methanotrophs of the genera Methylosinus and Methylocystis. Instead, Methylocella species are closely related to the nonmeth...

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Section: Growth Experimentsmentioning

confidence: 99%

Methylocella Species Are Facultatively Methanotrophic

2005

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“…The growth index for each vial was measured every week by using an automatic ion chamber (BACTEC 460; Johnston Laboratories, Towson, MD), and the numbers of M. avium subsp. paratuberculosis in the original suspension were estimated by using the most-probablenumber technique (4,35).…”

mentioning

confidence: 99%

Adsorption ofMycobacterium aviumsubsp.paratuberculosisto Soil Particles

Dhand

Toribio

Whittington

2009

Appl Environ Microbiol

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Attachment of Mycobacterium avium subsp. paratuberculosis to soil particles could increase their availability to farm animals, as well as influence the transportation of M. avium subsp. paratuberculosis to water sources. To investigate the possibility of such attachment, we passed a known quantity of M. avium subsp. paratuberculosis through chromatography columns packed with clay soil, sandy soil, pure silica, clay-silica mixture, or clay-silica complexes and measured the organisms recovered in the eluent using culture or quantitative PCR. Experiments were repeated using buffer at a range of pH levels with pure silica to investigate the effect of pH on M. avium subsp. paratuberculosis attachment. Linear mixed-model analyses were conducted to compare the proportional recovery of M. avium subsp. paratuberculosis in the eluent between different substrates and pH levels. Of the organisms added to the columns, 83 to 100% were estimated to be retained in the columns after adjustment for those retained in empty control columns. The proportions recovered were significantly different across different substrates, with the retention being significantly greater (P < 0.05) in pure substrates (silica and clay-silica complexes) than in soil substrates (clay soil and sandy soil). However, there were no significant differences in the retention of M. avium subsp. paratuberculosis between silica and clay-silica complexes or between clay soil and sandy soil. The proportion retained decreased with increasing pH in one of the experiments, indicating greater adsorption of M. avium subsp. paratuberculosis to soil particles at an acidic pH (P < 0.05). The results suggest that under experimental conditions M. avium subsp. paratuberculosis adsorbs to a range of soil particles, and this attachment is influenced by soil pH.

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“…One positive and one negative control for each NV genogroup were included for each series of samples (i.e., four controls per series). For this purpose, 10 l of stool samples of each genogroup (10 Ϫ3 to 10 Ϫ6 dilutions, depending on the viral titer of the different stools) was inoculated into 1 liter of sterile water. These spiked samples were treated in the same way as bottled water samples and used as controls for the whole procedure.…”

Section: Methodsmentioning

confidence: 99%

“…The number of PCR positive samples seen on agarose gels for given dilution triplets were used to calculate statistical DNA template population estimates by direct most-probablenumber PCR (MPN-PCR) according to Fode-Vaughan et al (14). Calculations were made with Microsoft Excel spreadsheet and its Solver tool (Frontline Systems) as described previously (10). DNA population estimates derived from MPN-PCR values were converted to PCRU per milliliter) for each sample, taking into account all of the dilution steps along the procedure.…”

Section: Methodsmentioning

confidence: 99%

Reverse Transcription-PCR Analysis of Bottled and Natural Mineral Waters for the Presence of Noroviruses

Lamothe¹,

Putallaz

Joosten

et al. 2003

Appl Environ Microbiol

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A seminested reverse transcription-PCR method coupled to membrane filtration was optimized to investigate the presence of norovirus (NV) RNA sequences in bottled and natural mineral waters. The recovery of viral particles by filtration varied between 28 and 45%, while the limit of detection of the overall method ranged from 6 to 95 viral particles. The assay was broadly reactive, as shown by the successful detection of 27 different viral strains representing 12 common genotypes of NVs. A total of 718 bottled and natural mineral water samples were investigated, including 640 samples of finished, spring, and line products (mostly 1 to 1.5 liters), collected from 36 different water brands of various types and from diverse geographic origins over a 2-year period. In addition, 78 samples of larger volume (10 and 400 to 500 liters) and environmental swabs were investigated. From the 1,436 analyses that were performed for the detection of NVs belonging to genogroups I and II, 34 samples (2.44%) were presumptively positive by seminested RT-PCR. However, confirmation by DNA sequence analysis revealed that all presumptive positive results were either due to nonspecific amplification or to cross-contamination. In conclusion, these results do not provide any evidence for the presence of NV genome sequences in bottled waters. Noroviruses (NVs [formerly known as Norwalk-like viruses])play a major role in nonbacterial outbreaks of acute gastroenteritis (28) and are regarded as the main foodborne viruses. Most cases are caused by person-to-person spread and by consumption of shellfish or other contaminated food items. There have been relatively few outbreaks that have been attributed to contact with or drinking of contaminated water (11,17,(19)(20)(21)(22) despite the large degree of exposure of humans to water. Furthermore, in all of these cases, the origin of NVs or the route of contamination could clearly be identified. To the best of our knowledge, there is no documented epidemiological evidence that bottled water has ever served as a vehicle for spreading NVs. Nevertheless, it was recently reported that NV genome sequences could be detected in natural mineral waters sold in Switzerland and other countries (7,8). Although it was clearly emphasized in those studies that detection of NV-specific sequences by itself does not imply that infectious virus particles are also present, the findings caused serious concern about the safety of bottled waters.These results prompted us to screen for the presence of NV genomes in a number of brands of bottled water. For this purpose, we have used a previously described seminested reverse transcription (RT)-PCR procedure (15) that was also applied in the Swiss survey of bottled waters (7,8). The different steps of the procedure were evaluated for their efficiency, and the sensitivity of the detection method was further optimized. Here we present results of a study performed on 718 samples, including finished, spring, and line products from 36 different brands of bottled and natural mineral...

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Estimating microbial population counts by ‘most probable number’ using Microsoft Excel®

Cited by 101 publications

References 6 publications

Methylocella Species Are Facultatively Methanotrophic

Methylocella Species Are Facultatively Methanotrophic

Adsorption ofMycobacterium aviumsubsp.paratuberculosisto Soil Particles

Reverse Transcription-PCR Analysis of Bottled and Natural Mineral Waters for the Presence of Noroviruses

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