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

1999 ◽  
Vol 65 (10) ◽  
pp. 4419-4424 ◽  
Author(s):  
Masashi Gamo ◽  
Tadashi Shoji
Keyword(s):  
Bacterial Communities ◽  
Carbon Sources ◽  
High Rate ◽  
Most Probable Number ◽  
Probable Number ◽  
New Approach ◽  
Mixing Ratios ◽  
Color Development ◽  
Assay Procedure ◽  
Pure Cultures

ABSTRACT 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.

Enumeration of wax-degrading microorganisms in water repellent soils using a miniaturised Most-Probable-Number method

Soil Research ◽  
2005 ◽  
Vol 43 (2) ◽  
pp. 171 ◽  
Author(s):  
Margaret M. Roper ◽  
V. V. S. R. Gupta
Keyword(s):  
Agricultural Soils ◽  
Carbon Sources ◽  
Coconut Oil ◽  
Water Repellency ◽  
Most Probable Number ◽  
Water Repellent ◽  
Natural Habitats ◽  
Probable Number ◽  
Degrading Bacteria ◽  
Pure Cultures

The ability of a soil to reduce waxes that cause water repellency depends, in part, on the presence of populations of wax-degrading microorganisms. Therefore, estimating the size of populations of wax-degrading bacteria in water repellent soils is likely to be a useful indicator of the potential for bioremediation of water repellency. A Most Probable Number (MPN) method that selects specifically for the function of wax degradation in bacteria was developed and tested using both pure cultures and natural populations of wax-degrading bacteria. Two carbon sources (coconut oil and hexadecane) that contain fatty acids or hydrocarbons implicated as causative agents of water repellency were compared in terms of growth and emulsification by a range of known wax-degrading bacteria. Emulsification resulting from surfactant production, a direct measure of wax degradation, was the criterion for a positive test. All the bacteria tested emulsified coconut oil, which was chosen as the carbon source in subsequent testing of the MPN assay. Population estimates of pure cultures of wax-degrading bacteria (liquid suspensions and inoculated into sterile soils) made using the MPN and a plating technique showed good agreement between the 2 methods, indicating that the MPN method gives an accurate measure of size of the wax-degrading population. Extensive replication of MPN counts of known wax-degrading bacteria inoculated into sterile soils or populations in soils collected from a range of natural habitats showed little variation within samples, indicating that the new method was reproducible. Based on its accuracy and reproducibility the MPN method was deemed suitable for estimating populations in a range of soils. Monitoring wax-degrading populations under different agricultural managements and at different times of the year, i.e. seasonal dynamics, could provide clues for the development of new managements that minimise the risk of water repellency in agricultural soils.

Selective enumeration of aromatic and aliphatic hydrocarbon degrading bacteria by a most-probable-number procedure

1996 ◽  
Vol 42 (3) ◽  
pp. 252-258 ◽  
Author(s):  
Brian A. Wrenn ◽  
Albert D. Venosa
Keyword(s):  
Polycyclic Aromatic Hydrocarbon ◽  
Aromatic Hydrocarbon ◽  
Oxidation Products ◽  
Most Probable Number ◽  
Probable Number ◽  
Microtiter Plates ◽  
Degrading Bacteria ◽  
Polycyclic Aromatic ◽  
Pure Cultures ◽  
Plate Counts

A most-probable-number (MPN) procedure was developed to separately enumerate aliphatic and aromatic hydrocarbon degrading bacteria, because most of the currently available methods are unable to distinguish between these two groups. Separate 96-well microtiter plates are used to estimate the sizes of these two populations. The alkane-degrader MPN method uses hexadecane as the selective growth substrate and positive wells are detected by reduction of iodonitrotetrazolium violet, which is added after incubation for 2 weeks at 20 °C. Polycyclic aromatic hydrocarbon degraders are grown on a mixture of phenanthrene, anthracene, fluorene, and dibenzothiophene in a second plate. Positive wells turn yellow to greenish-brown from accumulation of the partial oxidation products of the aromatic substrates and they can be scored after a 3-week incubation period. These MPN procedures are accurate and selective. For pure cultures, heterotrophic plate counts on a nonselective medium and the appropriate MPN procedure provide similar estimates of the population density. Bacteria that cannot grow on the selective substrates do not produce false positive responses even when the inoculum density is very high. Thus, this method, which is simple enough for use in the field, provides reliable estimates for the density and composition of hydrocarbon-degrading microbial populations.Key words: most probable number, polycyclic aromatic hydrocarbon, alkane, hydrocarbon, bacteria.

Colony-Forming Unit Enumeration by a Plate-MPN Method

1983 ◽  
Vol 46 (10) ◽  
pp. 836-841 ◽  
Author(s):  
S-T. TAN ◽  
R. B. MAXCY ◽  
W. W. STROUP
Keyword(s):  
Standard Technique ◽  
Most Probable Number ◽  
Plate Method ◽  
Probable Number ◽  
Microbial Counts ◽  
Colony Forming Unit ◽  
Pure Cultures ◽  
Petri Plate ◽  
Mpn Test ◽  
Surface Plate

Concepts of the standard surface plate method and the most probable number method (MPN) were combined to provide a new enumeration technique (plate-MPN). Three discrete 0.01-ml samples of an appropriate decimal dilution were inoculated onto each quadrant of a pre-dried petri plate. The discrete spots from the inoculum were then observed for growth after incubation. Results were interpreted analogous to a 3-tube MPN test using presently available tables. Application of the test to pure cultures and mixed flora provided no evidence to indicate the plate-MPN technique to be any less accurate than the standard technique for microbial counts. The plate-MPN technique was less precise than the standard technique. However, the plate-MPN technique has many advantages over traditional methods.

Occurrence and Characterization of Aeromonas Species in Pasteurized Milk and White Cheese in Rio De Janeiro, Brazil

1993 ◽  
Vol 56 (1) ◽  
pp. 62-65 ◽  
Author(s):  
ANGELA CÔRREA FREITAS ◽  
MARLY PAIVA NUNES ◽  
ARLETE MOREIRA MILHOMEM ◽  
ILVAN DELGADO RICCIARDI
Keyword(s):  
Rio De Janeiro ◽  
Foodborne Pathogen ◽  
Food Samples ◽  
High Rate ◽  
Most Probable Number ◽  
Aeromonas Species ◽  
Probable Number ◽  
Pasteurized Milk ◽  
Milk Samples ◽  
White Cheese

A total of 35 samples (1000 ml each) of pasteurized milk and 25 samples (100 g each) of white cheese purchased at supermarkets in Rio de Janeiro were analyzed for the presence of Aeromonas. Strains of Aeromonas were isolated from 28.5% of pasteurized milk and 32% of white cheese samples. Standard Plate counts in the pasteurized milk samples ranged from 7.2 × 10* to 2.5 × 105 CFU/ml. Total and fecal coliform counts in white cheese samples ranged from 1.9 × 10* to 2.4 × 105 most probable number per g and 3.2 × 102 to 1.2 × 105 most probable number per g, respectively. It was possible to identify Aeromonas caviae (58.9%), Aeromonas hydrophila (12.8%), and Aeromonas schubertii (2.5%) among the cultures isolated from pasteurized milk samples. Twenty-five percent of the strains could only be classified as Aeromonas spp. In white cheese samples, unclassified strains were the most frequent isolates (61.5%) followed by A. hydrophila (26.9%), A. caviae (7.6%) and Aeromonas sobria (3.8%). Only strains of A. hydrophila and A. sobria showed high rate of positive results when tested for the production of hemolysin, cytotoxin, and staphylolytic activity. Heat-stable enterotoxin and autoagglutination test did not correlate as virulence factors. The presence of Aeromonas species in refrigerated food samples suggests that this microorganism could be a potential foodborne pathogen, and dairy products may represent an important vehicle of its transmission.

scholarly journals Composition and Function of Sulfate-Reducing Prokaryotes in Eutrophic and Pristine Areas of the Florida Everglades

2002 ◽  
Vol 68 (12) ◽  
pp. 6129-6137 ◽  
Author(s):  
Hector Castro ◽  
K. R. Reddy ◽  
Andrew Ogram
Keyword(s):  
Carbon Sources ◽  
Original System ◽  
Agricultural Runoff ◽  
Florida Everglades ◽  
Most Probable Number ◽  
Probable Number ◽  
Sulfate Reducing ◽  
Limited Degree ◽  
Sulfate Reduction Rates ◽  
And Function

ABSTRACT As a result of agricultural activities in regions adjacent to the northern boundary of the Florida Everglades, a nutrient gradient developed that resulted in physicochemical and ecological changes from the original system. Sulfate input from agricultural runoff and groundwater is present in soils of the Northern Everglades, and sulfate-reducing prokaryotes (SRP) may play an important role in biogeochemical processes such as carbon cycling. The goal of this project was to utilize culture-based and non-culture-based approaches to study differences between the composition of assemblages of SRP in eutrophic and pristine areas of the Everglades. Sulfate reduction rates and most-probable-number enumerations revealed SRP populations and activities to be greater in eutrophic zones than in more pristine soils. In eutrophic regions, methanogenesis rates were higher, the addition of acetate stimulated methanogenesis, and SRP able to utilize acetate competed to a limited degree with acetoclastic methanogens. A surprising amount of diversity within clone libraries of PCR-amplified dissimilatory sulfite reductase (DSR) genes was observed, and the majority of DSR sequences were associated with gram-positive spore-forming Desulfotomaculum and uncultured microorganisms. Sequences associated with Desulfotomaculum fall into two categories: in the eutrophic regions, 94.7% of the sequences related to Desulfotomaculum were associated with those able to completely oxidize substrates, and in samples from pristine regions, all Desulfotomaculum-like sequences were related to incomplete oxidizers. This metabolic selection may be linked to the types of substrates that Desulfotomaculum spp. utilize; it may be that complete oxidizers are more versatile and likelier to proliferate in nutrient-rich zones of the Everglades. Desulfotomaculum incomplete oxidizers may outcompete complete oxidizers for substrates such as hydrogen in pristine zones where diverse carbon sources are less available.

scholarly journals Distribution and Physiological Characteristics of Hyperthermophiles in the Kubiki Oil Reservoir in Niigata, Japan

2000 ◽  
Vol 66 (1) ◽  
pp. 73-79 ◽  
Author(s):  
Yoh Takahata ◽  
Miyuki Nishijima ◽  
Toshihiro Hoaki ◽  
Tadashi Maruyama
Keyword(s):  
Cell Density ◽  
Carbon Sources ◽  
Most Probable Number ◽  
Oil Reservoir ◽  
Formation Water ◽  
Probable Number ◽  
Initial Cell ◽  
Initial Cell Density ◽  
Inorganic Substances ◽  
Subterranean Environment

ABSTRACT The distribution of culturable hyperthermophiles was studied in relation to environmental conditions in the Kubiki oil reservoir in Japan, where the temperature was between 50 and 58°C. Dominant hyperthermophilic cocci and rods were isolated and shown to belong to the genera Thermococcus and Thermotoga, respectively, by 16S rDNA analyses. Using the most-probable-number method, we found that hyperthermophilic cocci were widely distributed in several unconnected fault blocks in the Kubiki oil reservoir. In 1996 to 1997, their populations in the production waters from oil wells were 9.2 × 103 to 4.6 × 104cells/ml, or 10 to 42% of total cocci. On the other hand, hyperthermophilic rods were found in only one fault block of the reservoir with populations less than 10 cells/ml. DominantThermococcus and Thermotoga spp. grew at reservoir temperatures and utilized amino acids and sugars, respectively, as sole carbon sources. While organic carbon was plentiful in the environment, these hyperthermophiles were unable to grow in the formation water due to lack of essential nutrients. Concentrations of some organic and inorganic substances differed among fault blocks, indicating that the movement of formation water between fault blocks was restricted. This finding suggests that the supply of nutrients via fluid current is limited in this subterranean environment and that the organisms are starved in the oil reservoir. Under starved conditions at 50°C, culturable cells of Thermococcus sp. remained around the initial cell density for about 200 days, while those of Thermotoga sp. decreased exponentially to 0.01% of the initial cell density after incubation for the same period. The difference in survivability between these two hyperthermophiles seems to reflect their populations in the fault blocks. These results indicate that hyperthermophilic cocci and rods adapt to the subterranean environment of the Kubiki oil reservoir by developing an ability to survive under starved conditions.

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

2001 ◽  
Vol 67 (7) ◽  
pp. 3168-3173 ◽  
Author(s):  
Linping Kuai ◽  
Arjun A. Nair ◽  
Martin F. Polz
Keyword(s):  
Dry Weight ◽  
Environmental Water ◽  
Microtiter Plate ◽  
Most Probable Number ◽  
Precipitation Reaction ◽  
Simple Method ◽  
Probable Number ◽  
Cell Counts ◽  
Pure Cultures ◽  
Reducing Bacteria

ABSTRACT 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.

Separation of solids and disinfection for agronomical use of the effluent from a UASB reactor

2013 ◽  
Vol 69 (1) ◽  
pp. 25-30
Author(s):  
G. C. Sundefeld Junior ◽  
R. P. Piveli ◽  
S. A. Cutolo ◽  
S. S. Ferreira Filho ◽  
J. G. Santos
Keyword(s):  
Suspended Solids ◽  
High Rate ◽  
Upflow Anaerobic Sludge Blanket ◽  
World Health ◽  
Uasb Reactor ◽  
Most Probable Number ◽  
Anaerobic Sludge ◽  
Uv Disinfection ◽  
Surface Load ◽  
Probable Number

The present work addresses the preparation of the effluent from a full-scale upflow anaerobic sludge blanket (UASB) reactor for drip irrigation of orange crops. The pilot plant included a lamella plate clarifier followed by a geo-textile blanket filter and a UV disinfection reactor. The clarifier operated with a surface load of 115 m3m−2d−1, whereas the filter operated with 10 m3m−2d−1. The UV reactor was an open-channel type and the effective dose was approximately 2.8 W h m−3. The effluent of the UASB reactor received 0.5 mg L−1 cationic polyelectrolyte before entering the high-rate clarifier. Suspended solids’ concentrations and Escherichia coli and helminth egg's densities were monitored throughout the treatment system for 12 months. Results showed that the total suspended solids concentration in the filter effluent was lower than 7 mg L−1 and helminth density was below 1.0 egg L−1. The UV disinfection demonstrated the ability to produce a final effluent with E. coli density lower than 103MPN/100 mL (MPN: most probable number) during the entire process. Thus, the World Health Organization standards for unrestricted crop use were met. Agronomic interest parameters were controlled and it was possible to identify the important contribution of treated sewage in terms of the main nutrients.

scholarly journals Enumeration and Inoculation of Nitrifying Bacteria in Soilless Potting Media

1997 ◽  
Vol 122 (5) ◽  
pp. 709-714 ◽  
Author(s):  
Harvey J. Lang ◽  
George C. Elliott
Keyword(s):  
Counting Efficiency ◽  
Nitrifying Bacteria ◽  
Most Probable Number ◽  
Probable Number ◽  
Potting Medium ◽  
Pure Cultures ◽  
Potting Media ◽  
Nitrobacter Agilis ◽  
Inoculum Volume ◽  
Rate Limiting

Autotrophic nitrifying organisms were enumerated in soilless potting media using the most probable number (MPN) technique. Populations of NH4+ and NO2- oxidizing organisms varied widely between two soilless media—Metro-Mix 220 and 350. Estimates for NH4+ oxidizing organisms ranged from 0.7 to 7.8 × 105 organisms/cm3, while NO2- oxidizers ranged from 1.3 to 9.5 × 105 organisms/cm3. Population numbers were similar to those typically reported in soils. There was a significant effect of medium type, NH4+ N : NO3- N fertilizer ratio, and planting on MPN counts of both groups of organisms, with significant interaction between several of the factors. Estimates of NH4+ oxidizers were not linearly correlated with NH4+ oxidizing activity, implying low counting efficiency, heterotrophic nitrification, or rate-limiting substrate NH4+ level. In a separate study, a soilless potting medium was inoculated with pure cultures of either Nitrosomonas europaea or Nitrobacter agilis. Rates of NH4+ and NO2- oxidation increased, respectively, as inoculum volume increased. Inoculation with nitrifying bacteria may help in the overall management of N in the rhizosphere and be feasible alternatives for the prevention of either NH4+ or NO2- phytotoxicity with fertilizers containing urea or NH4+.

Determination of the number of root nodule bacteria in the presence of other organisms

1962 ◽  
Vol 2 (4) ◽  
pp. 5 ◽  
Author(s):  
RA Date ◽  
JM Vincent
Keyword(s):  
Root Nodule ◽  
Plate Count ◽  
Most Probable Number ◽  
Dilution Method ◽  
Nodule Bacteria ◽  
Probable Number ◽  
Root Nodule Bacteria ◽  
Carrier Materials ◽  
Pure Cultures

Use of the plant-dilution method for the determination of the number of root-nodule bacteria in nonsterile carrier materials has been described for both small and large-seeded legume hosts. Calculation of the most probable number of rhizobia is described using values based on the appropriate tables of Fisher and Yates, and approximate factors of error for 95 per cent fiducial limits are stated. It was found that the plant-dilution method was likely to underestimate the number of rhizobia by a factor not greater than 2, when comparisons were made with pure cultures. However for non-sterile peat the tendency was for the plate count to give the lower estimate. This latter effect was no doubt due to the presence of other organisms and therefore the plant-dilution method is to be preferred in such cases.

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