Estimating microbial population counts by 'most probable number' using Microsoft Excel

A computer-assisted method for determining population counts using the 'most probable number' (MPN) was developed. The Microsoft Excel spreadsheet and its Solver tool were used to generate MPNs, error estimates and confidence limits. Our method was flexible, allowing the use of unbalanced replication schemes and varying replication numbers and inoculation volumes. Furthermore, it required no programming skills and generated fast results, which were comparable to those of standard MPN tables and MPN software.     

Molecular analysis of ammonia-oxidising bacteria in soil of successional grasslands of the Drentsche A (The Netherlands)

Changes in the community structure of chemolitho-autotrophic ammonia-oxidising bacteria of the beta-subgroup Proteobacteria were monitored during nutrient-impoverishment management of slightly acidic, peaty grassland soils, which decreased in pH with succession. Specific PCR, cloning and sequence analysis, denaturing gradient gel electrophoresis (DGGE) and probe hybridisation were used to analyse rDNA sequences directly recovered from successional soils. Four previously characterised ammonia oxidiser sequence clusters were recovered from each soil, three associated with the genus Nitrosospira and one with the genus Nitrosomonas. All samples were dominated by Nitrosospira-like sequences. Nitrosospira cluster 3 was the most commonly recovered ammonia oxidiser group in all fields, but a greater representation of Nitrosospira clusters 2 and 4 was observed in older fields. Most probable number (MPN) counts were conducted using neutral and slightly acid conditions. Neutral pH (7.5) MPNs suggested a decrease in ammonia oxidiser numbers in later successional fields, but this trend was not observed using slightly acid (pH 5.8) conditions. Analysis of terminal MPN dilutions revealed a distribution of sequence clusters similar to direct soil DNA extractions. However, an increased relative recovery of Nitrosospira cluster 2 was observed for acid pH MPNs compared to neutral pH MPNs from the most acidic soil tested, in agreement with current hypotheses on the relative acid tolerance of this group.     

Culturable populations of Sporomusa spp. and Desulfovibrio spp. in the anoxic bulk soil of flooded rice microcosms.

Most-probable-number (MPN) counts were made of homoacetogenic and other bacteria present in the anoxic flooded bulk soil of laboratory microcosms containing 90- to 95-day-old rice plants. MPN counts with substrates known to be useful for the selective enrichment or the cultivation of homoacetogenic bacteria (betaine, ethylene glycol, 2, 3-butanediol, and 3,4,5-trimethoxybenzoate) gave counts of 2.3 x 10(3) to 2.8 x 10(5) cells per g of dry soil. Homoacetogens isolated from the terminal positive steps of these dilution cultures belonged to the genus Sporomusa. Counts with succinate, ethanol, and lactate gave much higher MPNs of 5.9 x 10(5) to 3.4 x 10(7) cells per g of dry soil and led to the isolation of Desulfovibrio spp. Counting experiments on lactate and ethanol which included Methanospirillum hungatei in the medium gave MPNs of 2.3 x 10(6) to 7.5 x 10(8) cells per g of dry soil and led to the isolation of Sporomusa spp. The latter strains could grow with betaine, ethylene glycol, 2, 3-butanediol, and/or 3,4,5-trimethoxybenzoate, but apparently most cells of Sporomusa spp. did not initiate growth in counting experiments with those substrates. Spores apparently accounted for 2. 2% or less of the culturable bacteria. It appears that culturable Desulfovibrio spp. and Sporomusa spp. were present in approximately equal numbers in the bulk soil. Multiple, phylogenetically-distinct, phenotypically-different, strains of each genus were found in the same soil system.     

The occurrence of chemolitho-autotrophic nitrifiers in water-saturated grassland soils

Relatively high most probable number (MPN) counts of chemolithotrophic nitrite oxidizers were present in water-saturated soils compared with MPNs and activity of ammonia oxidizers. These high numbers of nitrite oxidizers were confirmed by fluorescent antibody counts and potential activity measurements. Application of different nitrite concentrations in the MPN procedure discriminated within the community of nitrite oxidizers and revealed a large number of nitrite-sensitive nitrite oxidizers and a subcommunity of nitrite-insensitive nitrite oxidizers. The size of this subcommunity was small but corresponded with the low numbers of ammonium oxidizers. Numbers of nitrite-sensitive nitrite oxidizers outnumbered the ammonia oxidizing bacteria by 2–4 orders of magnitude in these soils. The possibility is discussed that the fraction of the nitrite-insensitive cells was active as aerobic nitrite oxidizers, whereas the nitrite-sensitive cells represented an inactive group of nitrite oxidizers growing as heterotrophs or as anaerobes reducing nitrite. In this situation, both MPN enumerations at a low nitrite concentration and activity measurements could give false information about the size of the in situ nitrite-oxidizing community.     

Constitutive MAP kinase activation in hematopoietic stem cells induces a myeloproliferative disorder

Myelodysplastic syndromes/myeloproliferative neoplasms (MDS/MPNs) are a group of myeloid neoplasms in which abnormal activation of the Ras signaling pathway is commonly observed. The PI3K/Akt pathway is a known target of Ras; however, activation of the PI3K/Akt pathway has been shown to lead to neoplastic transformation of not only myeloid but also lymphoid cells, suggesting that pathways other than the PI3K/Akt pathway should play a central role in pathogenesis of Ras-mediated MDS/MPN. The MEK/ERK pathway is another downstream target of Ras, which is involved in regulation of cell survival and proliferation. However, the role of the MEK/ERK pathway in the pathogenesis of MDS/MPN remains unclear. Here, we show that introduction of a constitutively activated form of MEK into hematopoietic stem cells (HSCs) causes hematopoietic neoplasms that are limited to MDS/MPNs, despite the multipotent differentiation potential of HSCs. Active MEK-mediated MDS/MPNs are lethal, but are not considered a frank leukemia because it cannot be transplanted into naïve animals. However, transplantation of MDS/MPNs co-expressing active MEK and an anti-apoptotic molecule, Bcl-2, results in T-cell acute lymphocytic leukemia (T-ALL), suggesting that longevity of cells may impact transplantability and alter disease phenotype. Our results clearly demonstrate the proto-oncogenic property of the MEK/ERK pathway in hematopoietic cells, which manifest in MDS/MPN development.     

Reconsideration of the derivation of Most Probable Numbers,their standard deviations,confidence bounds and rarity values

Aims: The purpose of this work was to derive a simple Excel spreadsheet and a set of standard tables of most probable number (MPN) values that can be applied by users of International Standard Methods to obtain the same output values for MPN, SD of the MPN, 95% confidence limits and test validity. With respect to the latter, it is considered that the Blodgett concept of ‘rarity’ is more valuable than the frequently used approach of improbability (vide de Man). Methods and Results: The paper describes the statistical procedures used in the work and the reasons for introducing a new set of conceptual and practical approaches to the determination of MPNs and their parameters. Examples of MPNs derived using these procedures are provided. The Excel spreadsheet can be downloaded from http://www.wiwiss.fu‐berlin.de/institute/iso/mitarbeiter/wilrich/index.html . Conclusions: The application of the revised approach to the determination of MPN parameters permits those who wish to use tabulated values, and those who require access to a simple spreadsheet to determine values for nonstandard test protocols, to obtain the same output values for any specific set of multiple test results. The concept of ‘rarity’ is a more easily understood parameter to describe test result combinations that are not statistically valid. Provision of the SD of the log MPN value permits derivation of uncertainty parameters that have not previously been possible. Significance and Impact of the Study: A consistent approach for the derivation of MPNs and their parameters is essential for coherence between International Standard Methods. It is intended that future microbiology standard methods will be based on the procedures described in this paper.     

Repeated introduction of genetically modified Pseudomonas putida WCS358r without intensified effects on the indigenous microflora of field-grown wheat

To investigate the impact of genetically modified, antibiotic-producing rhizobacteria on the indigenous microbial community, Pseudomonas putida WCS358r and two transgenic derivatives were introduced as a seed coating into the rhizosphere of wheat in two consecutive years (1999 and 2000) in the same field plots. The two genetically modified microorganisms (GMMs), WCS358r::phz and WCS358r::phl, constitutively produced phenazine-1-carboxylic acid (PCA) and 2,4-diacetylphloroglucinol (DAPG), respectively. The level of introduced bacteria in all treatments decreased from 10(7) CFU per g of roots soon after sowing to less than 10(2) CFU per g after harvest 132 days after sowing. The phz and phl genes remained stable in the chromosome of WCS358r. The amount of PCA produced in the wheat rhizosphere by WCS358r::phz was about 40 ng/g of roots after the first application in 1999. The DAPG-producing GMMs caused a transient shift in the indigenous bacterial and fungal microflora in 1999, as determined by amplified ribosomal DNA restriction analysis. However, after the second application of the GMMs in 2000, no shifts in the bacterial or fungal microflora were detected. To evaluate the importance of the effects induced by the GMMs, these effects were compared with those induced by crop rotation by planting wheat in 1999 followed by potatoes in 2000. No effect of rotation on the microbial community structure was detected. In 2000 all bacteria had a positive effect on plant growth, supposedly due to suppression of deleterious microorganisms. Our research suggests that the natural variability of microbial communities can surpass the effects of GMMs.     

Identification of oncostatin M as a JAK2 V617F-dependent amplifier of cytokine production and bone marrow remodeling in myeloproliferative neoplasms

The JAK2 mutation V617F is detectable in a majority of patients with Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs). Enforced expression of JAK2 V617F in mice induces myeloproliferation and bone marrow (BM) fibrosis, suggesting a causal role for the JAK2 mutant in the pathogenesis of MPNs. However, little is known about mechanisms and effector molecules contributing to JAK2 V617F-induced myeloproliferation and fibrosis. We show that JAK2 V617F promotes expression of oncostatin M (OSM) in neoplastic myeloid cells. Correspondingly, OSM mRNA levels were increased in the BM of patients with MPNs (median 287% of ABL, range 22-1450%) compared to control patients (median 59% of ABL, range 12-264%; P < 0.0001). OSM secreted by JAK2 V617F+ cells stimulated growth of fibroblasts and microvascular endothelial cells and induced the production of angiogenic and profibrogenic cytokines (HGF, VEGF, and SDF-1) in BM fibroblasts. All effects of MPN cell-derived OSM were blocked by a neutralizing anti-OSM antibody, whereas the production of OSM in MPN cells was suppressed by a pharmacologic JAK2 inhibitor or RNAi-mediated knockdown of JAK2. In summary, JAK2 V617F-mediated up-regulation of OSM may contribute to fibrosis, neoangiogenesis, and the cytokine storm observed in MPNs, suggesting that OSM might serve as a novel therapeutic target molecule in these neoplasms.     

Microtechnique for Most-Probable-Number Analysis

A microtechnique based on the most-probable-number (MPN) method has been developed for the enumeration of the ammonium-oxidizing population in soil samples. An MPN table for a research design ([8 by 12] i.e., 12 dilutions, 8 replicates per dilution) is presented. A correlation of 0.68 was found between MPNs determined by the microtechnique and the standard tube technique. Higher MPNs were obtained with the microtechnique with increased accuracy in endpoint determinations being a possible cause. Considerable savings of time, space, equipment, and reagents are observed using this method. The microtechnique described may be adapted to other microbial populations using various types of media and endpoint determinations.     

In vitro megakaryocyte differentiation and proplatelet formation in Ph-negative classical myeloproliferative neoplasms: distinct patterns in the different clinical phenotypes

Background

Ph-negative myeloproliferative neoplasms (MPNs) are clonal disorders that include primary myelofibrosis (PMF), polycythemia vera (PV) and essential thrombocythemia (ET). Although the pathogenesis of MPNs is still incompletely understood, an involvement of the megakaryocyte lineage is a distinctive feature.

Methodology/Principal Findings

We analyzed the in vitro megakaryocyte differentiation and proplatelet formation in 30 PMF, 8 ET, 8 PV patients, and 17 healthy controls (CTRL). Megakaryocytes were differentiated from peripheral blood CD34⁺ or CD45⁺ cells in the presence of thrombopoietin. Megakaryocyte output was higher in MPN patients than in CTRL with no correlation with the JAK2 V617F mutation. PMF-derived megakaryocytes displayed nuclei with a bulbous appearance, were smaller than ET- or PV-derived megakaryocytes and formed proplatelets that presented several structural alterations. In contrast, ET- and PV-derived megakaryocytes produced more proplatelets with a striking increase in bifurcations and tips compared to both control and PMF. Proplatelets formation was correlated with platelet counts in patient peripheral blood. Patients with pre-fibrotic PMF had a pattern of megakaryocyte proliferation and proplatelet formation that was similar to that of fibrotic PMF and different from that of ET.

Conclusions/Significance

In conclusion, MPNs are associated with high megakaryocyte proliferative potential. Profound differences in megakaryocyte morphology and proplatelet formation distinguish PMF, both fibrotic and prefibrotic, from ET and PV.     

Rapid and simple method for the most-probable-number estimation of arsenic-reducing bacteria.

A rapid and simple most-probable-number (MPN) procedure for the enumeration of dissimilatory arsenic-reducing bacteria (DARB) is presented. The method is based on the specific detection of arsenite, the end product of anaerobic arsenate respiration, by a precipitation reaction with sulfide. After 4 weeks of incubation, the medium for the MPN method is acidified to pH 6 and sulfide is added to a final concentration of about 1 mM. The brightly yellow arsenic trisulfide precipitates immediately and can easily be scored at arsenite concentrations as low as 0.05 mM. Abiotic reduction of arsenate upon sulfide addition, which could yield false positives, apparently produces a soluble As-S intermediate, which does not precipitate until about 1 h after sulfide addition. Using the new MPN method, population estimates of pure cultures of DARB were similar to direct cell counts. MPNs of environmental water and sediment samples yielded DARB numbers between 10(1) and 10(5) cells per ml or gram (dry weight), respectively. Poisoned and sterilized controls showed that potential abiotic reductants in environmental samples did not interfere with the MPN estimates. A major advantage is that the assay can be easily scaled to a microtiter plate format, enabling analysis of large numbers of samples by use of multichannel pipettors. Overall, the MPN method provides a rapid and simple means for estimating population sizes of DARB, a diverse group of organisms for which no comprehensive molecular markers have been developed yet.     

Culturable Populations of Sporomusa spp. and Desulfovibrio spp. in the Anoxic Bulk Soil of Flooded Rice Microcosms

Most-probable-number (MPN) counts were made of homoacetogenic and other bacteria present in the anoxic flooded bulk soil of laboratory microcosms containing 90- to 95-day-old rice plants. MPN counts with substrates known to be useful for the selective enrichment or the cultivation of homoacetogenic bacteria (betaine, ethylene glycol, 2,3-butanediol, and 3,4,5-trimethoxybenzoate) gave counts of 2.3 × 10³ to 2.8 × 10⁵ cells per g of dry soil. Homoacetogens isolated from the terminal positive steps of these dilution cultures belonged to the genus Sporomusa. Counts with succinate, ethanol, and lactate gave much higher MPNs of 5.9 × 10⁵ to 3.4 × 10⁷ cells per g of dry soil and led to the isolation of Desulfovibrio spp. Counting experiments on lactate and ethanol which included Methanospirillum hungatei in the medium gave MPNs of 2.3 × 10⁶ to 7.5 × 10⁸ cells per g of dry soil and led to the isolation of Sporomusa spp. The latter strains could grow with betaine, ethylene glycol, 2,3-butanediol, and/or 3,4,5-trimethoxybenzoate, but apparently most cells of Sporomusa spp. did not initiate growth in counting experiments with those substrates. Spores apparently accounted for 2.2% or less of the culturable bacteria. It appears that culturable Desulfovibrio spp. and Sporomusa spp. were present in approximately equal numbers in the bulk soil. Multiple, phylogenetically-distinct, phenotypically-different, strains of each genus were found in the same soil system.     

Repeated Introduction of Genetically Modified Pseudomonas putida WCS358r without Intensified Effects on the Indigenous Microflora of Field-Grown Wheat

To investigate the impact of genetically modified, antibiotic-producing rhizobacteria on the indigenous microbial community, Pseudomonas putida WCS358r and two transgenic derivatives were introduced as a seed coating into the rhizosphere of wheat in two consecutive years (1999 and 2000) in the same field plots. The two genetically modified microorganisms (GMMs), WCS358r::phz and WCS358r::phl, constitutively produced phenazine-1-carboxylic acid (PCA) and 2,4-diacetylphloroglucinol (DAPG), respectively. The level of introduced bacteria in all treatments decreased from 10⁷ CFU per g of roots soon after sowing to less than 10² CFU per g after harvest 132 days after sowing. The phz and phl genes remained stable in the chromosome of WCS358r. The amount of PCA produced in the wheat rhizosphere by WCS358r::phz was about 40 ng/g of roots after the first application in 1999. The DAPG-producing GMMs caused a transient shift in the indigenous bacterial and fungal microflora in 1999, as determined by amplified ribosomal DNA restriction analysis. However, after the second application of the GMMs in 2000, no shifts in the bacterial or fungal microflora were detected. To evaluate the importance of the effects induced by the GMMs, these effects were compared with those induced by crop rotation by planting wheat in 1999 followed by potatoes in 2000. No effect of rotation on the microbial community structure was detected. In 2000 all bacteria had a positive effect on plant growth, supposedly due to suppression of deleterious microorganisms. Our research suggests that the natural variability of microbial communities can surpass the effects of GMMs.     

Rapid and Simple Method for the Most-Probable-Number Estimation of Arsenic-Reducing Bacteria

A rapid and simple most-probable-number (MPN) procedure for the enumeration of dissimilatory arsenic-reducing bacteria (DARB) is presented. The method is based on the specific detection of arsenite, the end product of anaerobic arsenate respiration, by a precipitation reaction with sulfide. After 4 weeks of incubation, the medium for the MPN method is acidified to pH 6 and sulfide is added to a final concentration of about 1 mM. The brightly yellow arsenic trisulfide precipitates immediately and can easily be scored at arsenite concentrations as low as 0.05 mM. Abiotic reduction of arsenate upon sulfide addition, which could yield false positives, apparently produces a soluble As-S intermediate, which does not precipitate until about 1 h after sulfide addition. Using the new MPN method, population estimates of pure cultures of DARB were similar to direct cell counts. MPNs of environmental water and sediment samples yielded DARB numbers between 10¹ and 10⁵ cells per ml or gram (dry weight), respectively. Poisoned and sterilized controls showed that potential abiotic reductants in environmental samples did not interfere with the MPN estimates. A major advantage is that the assay can be easily scaled to a microtiter plate format, enabling analysis of large numbers of samples by use of multichannel pipettors. Overall, the MPN method provides a rapid and simple means for estimating population sizes of DARB, a diverse group of organisms for which no comprehensive molecular markers have been developed yet.     

Measurement of hydrocarbon-degrading microbial populations by a 96-well plate most-probable-number procedure

A 96-well microtiter plate most-probable-number (MPN) procedure was developed to enumerate hydrocarbondegrading microorganisms. The performance of this method, which uses number 2 fuel oil (F2) as the selective growth substrate and reduction of iodonitrotetrazolium violet (INT) to detect positive wells, was evaluated by comparison with an established 24-well microtiter plate MPN procedure (the Sheen Screen), which uses weathered North Slope crude oil as the selective substrate and detects positive wells by emulsification or dispersion of the oil. Both procedures gave similar estimates of the hydrocarbon-degrader population densities in several oil-degrading enrichment cultures and sand samples from a variety of coastal sites. Although several oils were effective substrates for the 96-well procedure, the combination of F2 with INT was best, because the color change associated with INT reduction was more easily detected in the small wells than was disruption of the crude oil slick. The method's accuracy was evaluated by comparing hydrocarbon-degrader MPNs with heterotrophic plate counts for several pure and mixed cultures. For some organisms, it seems likely that a single cell cannot initiate sufficient growth to produce a positive result. Thus, this and other hydrocarbon-degrader MPN procedures might underestimate the hydrocarbon-degrading population, even for culturable organisms.     

Methane oxidation and the competition for oxygen in the rice rhizosphere

A mechanistic approach is presented to describe oxidation of the greenhouse gas methane in the rice rhizosphere of flooded paddies by obligate methanotrophic bacteria. In flooded rice paddies these methanotrophs compete for available O(2) with other types of bacteria. Soil incubation studies and most-probable-number (MPN) counts of oxygen consumers show that microbial oxygen consumption rates were dominated by heterotrophic and methanotrophic respiration. MPN counts of methanotrophs showed large spatial and temporal variability. The most abundant methanotrophs (a Methylocystis sp.) and heterotrophs (a Pseudomonas sp. and a Rhodococcus sp.) were isolated and characterized. Growth dynamics of these bacteria under carbon and oxygen limitations are presented. Theoretical calculations based on measured growth dynamics show that methanotrophs were only able to outcompete heterotrophs at low oxygen concentrations (frequently < 5 microM). The oxygen concentration at which methanotrophs won the competition from heterotrophs did not depend on methane concentration, but it was highly affected by organic carbon concentrations in the paddy soil. Methane oxidation was severely inhibited at high acetate concentrations. This is in accordance with competition experiments between Pseudomonas spp. and Methylocystis spp. carried out at different oxygen and carbon concentrations. Likely, methane oxidation mainly occurs at microaerophilic and low-acetate conditions and thus not directly at the root surface. Acetate and oxygen concentrations in the rice rhizosphere are in the critical range for methane oxidation, and a high variability in methane oxidation rates is thus expected.     

A simple method to evaluate the concentration of pentachlorophenol degraders in contaminated soils

A new most probable number (MPN) method for the determination of pentachlorophenol (PCP) degraders in soil using the change in pH due to PCP degradation is compared with a well documented MPN method using radiolabeled PCP. The results of all MPN counts were similar within a 95% confidence limit. The results obtained in MPN per gram of dry soil using pH measurements were 1.8 (+3.1, -1.03) x10 (4) compared to 0.64 (+1.34, -0.42) x 10(4) when using production of [(14)C]CO(2).     

Mutational Disruption of the Biosynthesis Genes Coding for the Antifungal Metabolite 2,4-Diacetylphloroglucinol Does Not Influence the Ecological Fitness of Pseudomonas fluorescens F113 in the Rhizosphere of Sugarbeets

The ability of Pseudomonas fluorescens F113 to produce the antibiotic 2,4-diacetylphloroglucinol (DAPG) is a key factor in the biocontrol of the phytopathogenic fungus Pythium ultimum by this strain. In this study, a DAPG-producing strain (rifampin-resistant mutant F113Rif) was compared with a nearly isogenic DAPG-negative biosynthesis mutant (Tn5::lacZY derivative F113G22) in terms of the ability to colonize and persist in the rhizosphere of sugarbeets in soil microcosms during 10 plant growth-harvest cycles totaling 270 days. Both strains persisted similarly in the rhizosphere for 27 days, regardless of whether they had been inoculated singly onto seeds or coinoculated in a 1:1 ratio. In order to simulate harvest and resowing, the roots were removed from the soil and the pots were resown with uninoculated sugarbeet seeds for nine successive 27-day growth-harvest cycles. Strains F113Rif and F113G22 performed similarly with respect to colonizing the rhizosphere of sugarbeet, even after nine cycles without reinoculation. The introduced strains had a transient effect on the size of the total culturable aerobic bacterial population. The results indicate that under these experimental conditions, the inability to produce DAPG did not reduce the ecological fitness of strain F113 in the rhizosphere of sugarbeets.     

Microbial community dynamics and evaluation of bioremediation strategies in oil-impacted salt marsh sediment microcosms

Microbial community dynamics in wetlands microcosms emended with commercial products (surfactant, a biological agent, and nutrients) designed to enhance bioremediation was followed for 3 months. The effectiveness of enhanced degradation was assessed by determining residual concentrations of individual petroleum hydrocarbons by GC/MS. The size and composition of the sediment microbial community was assessed using a variety of indices, including bacterial plate counts, MPNs, and DNA hybridizations with domain- and group-specific oligonucleotide probes. The addition of inorganic nutrients was the most effective treatment for the enhancement of oil degradation, resulting in marked degradation of petroleum alkanes and a lesser extent of degradation of aromatic oil constituents. The enhanced degradation was associated with increases in the amount of extractable microbial DNA and Streptomyces in the sediment, although not with increased viable counts (plate counts, MPN). Bacteria introduced with one of the proprietary products were still detected in the microcosms after 3 months, but were not a major quantitative constituent of the community. The biological product enhanced oil degradation relative to the control, but to a lesser extent than the nutrient additions alone. In contrast, application of the surfactant to the oil-impacted sediment decreased oil degradation. Journal of Industrial Microbiology & Biotechnology (2001) 27, 72–79. Received 18 March 2001/ Accepted in revised form 09 June 2001