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.     

A versatile most probable number system to quantify lacZY-marked pseudomonads present at low cell numbers in the rhizosphere

Quantitation of introduced genetically modified micro-organisms (GMMs) in the rhizosphere is a key issue when their release in the environment is planned. An improved most probable number (MPN) system, using a titration plate for incubation of rhizosphere extracts and two microcomputer programmes made recently available, MPNES (Woomer et al. 1990) and MPN 2.80 (Klee 1993) to generate the MPNs, is described. The MPN system was compared with colony counts to assess colonization of sugarbeet roots by an introduced lac ZY-modified rhizosphere pseudomonad. The MPNs displayed wider confidence intervals compared with drop-plate counts but allowed the quantitation limit to be lowered to 2.30 log₁₀ cfu per root system. The MPN system proved useful to quantify GMMs present at low cell numbers in the rhizosphere of sugarbeet.     

A Method of Profiling Microbial Communities Based on a Most-Probable-Number Assay That Uses BIOLOG Plates and Multiple Sole Carbon Sources

A new approach to the community-level BIOLOG assay was proposed. This assay, which we call the BIOLOG-MPN assay, is a most-probable-number (MPN) assay that uses BIOLOG plates and multiple sole carbon sources, and the profiles obtained by this assay consist of MPNs estimated for the substrates in the BIOLOG plates. In order to demonstrate the performance of the BIOLOG-MPN assay, it was applied to pure cultures, model bacterial communities that contain two strains in different ratios, and microbial community samples. MPN estimation using BIOLOG plates worked well for the substrates on which utilizers can grow at a sufficiently high rate for color development under the conditions of the assay procedure. Furthermore, the results obtained using model communities showed that the MPNs obtained reflected the mixing ratios of pure cultures in the model communities. The profiles obtained using model communities and community samples were differentiated properly by statistical analyses. The results suggest that the BIOLOG-MPN assay is a promising procedure for obtaining a quantitative picture of the community structure.     

A method of profiling microbial communities based on a most-probable-number assay that uses BIOLOG plates and multiple sole carbon sources.

A new approach to the community-level BIOLOG assay was proposed. This assay, which we call the BIOLOG-MPN assay, is a most-probable-number (MPN) assay that uses BIOLOG plates and multiple sole carbon sources, and the profiles obtained by this assay consist of MPNs estimated for the substrates in the BIOLOG plates. In order to demonstrate the performance of the BIOLOG-MPN assay, it was applied to pure cultures, model bacterial communities that contain two strains in different ratios, and microbial community samples. MPN estimation using BIOLOG plates worked well for the substrates on which utilizers can grow at a sufficiently high rate for color development under the conditions of the assay procedure. Furthermore, the results obtained using model communities showed that the MPNs obtained reflected the mixing ratios of pure cultures in the model communities. The profiles obtained using model communities and community samples were differentiated properly by statistical analyses. The results suggest that the BIOLOG-MPN assay is a promising procedure for obtaining a quantitative picture of the community structure.     

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.     

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.     

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.     

Use of a commercial gene probe assay kit for rapid MPN enumeration of Escherichia coli in drinking water

A commercial gene probe assay kit for presence/absence determination of Escherichia coli in food samples has been used in the standard UK six tube format most probable number (MPN) method for enumerating E. coli in drinking water samples. Presence/absence analysis with the gene probe kit (requiring 3 h) of all MPN tubes after a 21–24 h incubation (minerals modified glutamate; 37°C) enumerated confirmed E. coli in 24–27 h which offered an improvement of up to 48 h over the standard UK MPN method. MPNs determined by the gene probe method and the standard UK method agreed in nine of the 16 water samples which were analysed and for which E. coli concentrations were within the detection limits of the six tube MPN format. This was consistent with the gene probe method detecting one E. coli in a tube. For the other seven water samples, the gene probe method registered positive only 20 of the 30 tubes which the standard UK method determined to be positive. The sensitivity of the gene probe method for drinking water samples, although encouraging, needs improvement perhaps through kit quality control procedures.     

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.     

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.     

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.     

Confidence Intervals for Microbial Density Using Serial Dilutions with MPN Estimates

An alteration to Woodward's methods is recommended for deriving a 1 — α confidence interval for microbial density using serial dilutions with most-probable-number (MPN) estimates. Outcomes of the serial dilution test are ordered by their MPNs. A lower limit for the confidence interval corresponding to an outcome y is the density for which y and all higher ordered outcomes have total probability α/2. An upper limit is derived in the analogous way. An alteration increases the lowest lower limits and decreases the highest upper limits. For comparison, a method that is optimal in the sense of null hypothesis rejection is described. This method ranks outcomes dependent upon the microbial density in question, using proportional first derivatives of the probabilities. These and currently used methods are compared. The recommended method is shown to be more desirable in certain respects, although resulting in slightly wider confidence intervals than De Man's (1983) method.     

Most probable number methodology for quantifying dilute concentrations and fluxes of Escherichia coli O157:H7 in surface waters

Aims:  To better understand the transport and enumeration of dilute densities of Escherichia coli O157:H7 in agricultural watersheds, we developed a culture-based, five tube-multiple dilution most probable number (MPN) method.
Methods and Results:  The MPN method combined a filtration technique for large volumes of surface water with standard selective media, biochemical and immunological tests, and a TaqMan confirmation step. This method determined E. coli O157:H7 concentrations as low as 0·1 MPN per litre, with a 95% confidence level of 0·01–0·7 MPN per litre. Escherichia coli O157:H7 densities ranged from not detectable to 9 MPN per litre for pond inflow, from not detectable to 0·9 MPN per litre for pond outflow and from not detectable to 8·3 MPN per litre for within pond. The MPN methodology was extended to mass flux determinations. Fluxes of E. coli O157:H7 ranged from <27 to >10⁴ MPN per hour.
Conclusion:  This culture-based method can detect small numbers of viable/culturable E. coli O157:H7 in surface waters of watersheds containing animal agriculture and wildlife.
Significance and Impact of the Study:  This MPN method will improve our understanding of the transport and fate of E. coli O157:H7 in agricultural watersheds, and can be the basis of collections of environmental E. coli O157:H7.     

Second derivative UV absorbance analysis to monitor nitrate-reduction by bacteria in most probable number determinations

Heterotrophic and autotrophic nitrate-reducing bacteria (NRB) play important roles in many environments. These bacteria are often enumerated by most probable number (MPN) methods. Measuring NO(3)(-) depletion in the MPN cultures is the definitive way to determine the presence of NRB. Media used for MPN determinations of NRB in oil field waters usually contain high Cl(-) concentrations, matching those in the water samples. Many methods for measuring NO(3)(-) concentrations, such as ion chromatography (IC), cadmium reduction and ion electrode methods, are adversely affected by high concentrations of Cl(-) and organic compounds. A second derivative UV absorbance method proved to be a fast and reliable means for measuring NO(3)(-) depletion in MPN media used for enumerating autotrophic and heterotrophic NRB, without interferences from Cl(-) or the organic components in the latter medium. The MPN results for heterotrophic NRB determined by the second derivative UV absorbance agreed well with those determined by the production of nitrous oxide, and were often higher than those determined by measuring nitrate depletion by the diphenylamine spot test.     

Improved Microtechnique for the Leptospiral Microscopic Agglutination Test

A method for improving the original Galton microtechnique for detecting leptospiral antibodies has been developed. Simultaneous titrations were performed on 281 animal and human sera and 17 hyperimmune sera with the microscopic agglutination (MA) test and the improved microtechnique. Reproducibility of the improved microtechnique was determined independently on 65 animal sera by two laboratory sections. The results obtained by comparing positive test data from human and animal sera indicated that agreement between the original MA test and this new method exceeded 94%, whereas the original Galton microtechnique and the original MA test agreed in a maximum of 77% of the tests. This study indicates that the results obtained with the improved microtechnique are much more comparable to results obtained with the original MA test than are those obtained with the original Galton microtechnique.     

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.     

Nitrification and denitrification in soil: A comparison of enzyme assay,incubation and enumeration methods

Summary Three methods for assaying nitrification and denitrification were compared in agricultural field plots in the Southeastern USA. Nitrification was measured using the chlorate inhibition enzyme assay, by measuring (NO ₃ ⁻ −N) production in controlled incubations and by most probable number (MPN) determinations. Denitrification was measured by the phase I enzyme assay, by incubation of soil cores and by MPN determinations. The methods were compared in terms of their representation of seasonal patterns and treatment differences. The enzyme assays were most effective for showing treatment differences because they measure maximum potential enzyme biomass activity which is an integrated product of treatment effects. The incubation methods required minimal alteration ofin situ soil conditions but were confounded by other biological processes and by high spatial and temporal variabiltiy. MPN determinations were time consuming and were least effective for illustrating treatment differences and seasonal patterns.