Simplified MPN method for enumeration of soil naphthalene degraders using gaseous substrate

We describe a simplified microplate most-probable-number (MPN) procedure to quantify the bacterial naphthalene degrader population in soil samples. In this method, the sole substrate naphthalene is dosed passively via gaseous phase to liquid medium and the detection of growth is based on the automated measurement of turbidity using an absorbance reader. The performance of the new method was evaluated by comparison with a recently introduced method in which the substrate is dissolved in inert silicone oil and added individually to each well, and the results are scored visually using a respiration indicator dye. Oil-contaminated industrial soil showed slightly but significantly higher MPN estimate with our method than with the reference method. This suggests that gaseous naphthalene was dissolved in an adequate concentration to support the growth of naphthalene degraders without being too toxic. The dosing of substrate via gaseous phase notably reduced the work load and risk of contamination. The result scoring by absorbance measurement was objective and more reliable than measurement with indicator dye, and it also enabled further analysis of cultures. Several bacterial genera were identified by cloning and sequencing of 16S rRNA genes from the MPN wells incubated in the presence of gaseous naphthalene. In addition, the applicability of the simplified MPN method was demonstrated by a significant positive correlation between the level of oil contamination and the number of naphthalene degraders detected in soil.     

Comparison of culture-based methods to enumerate Escherichia coli in tropical and temperate freshwaters

Aims: The specificity of a method for the enumeration of Escherichia coli (chromocult agar, CC) was tested using freshwater samples from a tropical area (Cuba Island) by isolating colonies and identifying them with API (Appareillage et procédé d’identification) strips. Enumerations of E. coli by the most probable number (MPN) microplate method were compared with counts on chromogenic and fluorogenic agar media [CC, rapid E. coli (REC), fluorocult] in tropical and temperate freshwater samples. Methods and Results: A high percentage of specificity (95·7%) for the CC agar enumeration of E. coli was observed. High regression coefficients (log‐log linear regressions) were found between E. coli counts on agar media and by the MPN method. In the tropical environment, counts with REC medium were significantly different from those obtained with the other methods. MPN counts were found to be significantly higher than those obtained using the plate counts methods in the temperate environment. Conclusions: Escherichia coli enumeration methods based on glucuronidase activity appear to be suitable for the evaluation of microbiological quality in the tropical environment featured in this study. Significance and Impact of the Study: The methods for the enumeration of E. coli tested in this study should help improve the evaluation of microbiological contamination of Cuban freshwaters.     

Glucose and plant exudate enhanced enumeration of bacteria capable of degrading polycyclic aromatic hydrocarbons

Enumerating environmental microbial isolates capable of polycyclic aromatic hydrocarbon (PAH) degradation can provide insight into the microbe-plant interactions that facilitate PAH removal. We examined a known PAH degrader ( Pseudomonas putida G7), a nondegrader ( Agrobacterium tumefaciens LBA4404), and several microorganisms isolated from the environment by using a PAH cocktail in an enumeration medium?with or without 0.025% (m/v) glucose and (or) root exudates. Compared with the standard most probable number (MPN), the addition of glucose and root exudates in a modified MPN method resulted in a 3- to 11-fold enhancement of PAH degraders being enumerated among microorganisms found in PAH-contaminated soils. High-performance liquid chromatography analysis verified that PAH levels were reduced using this modified enumeration method. Low levels of glucose, perhaps in concert with other materials in exudates, may promote microbial metabolism, thereby enhancing PAH degradation.     

Effect of a Pseudomonas rhamnolipid biosurfactant on cell hydrophobicity and biodegradation of octadecane.

In this study, the effect of a purified rhamnolipid biosurfactant on the hydrophobicity of octadecane-degrading cells was investigated to determine whether differences in rates of octadecane biodegradation resulting from the addition of rhamnolipid to four strains of Pseudomonas aeruginosa could be related to measured differences in hydrophobicity. Cell hydrophobicity was determined by a modified bacterial adherence to hydrocarbon (BATH) assay. Bacterial adherence to hydrocarbon quantitates the preference of cell surfaces for the aqueous phase or the aqueous-hexadecane interface in a two-phase system of water and hexadecane. On the basis of octadecane biodegradation in the absence of rhamnolipid, the four bacterial strains were divided into two groups: the fast degraders (ATCC 15442 and ATCC 27853), which had high cell hydrophobicities (74 and 55% adherence to hexadecane, respectively), and the slow degraders (ATCC 9027 and NRRL 3198), which had low cell hydrophobicities (27 and 40%, respectively). Although in all cases rhamnolipid increased the aqueous dispersion of octadecane at least 10(4)-fold, at low rhamnolipid concentrations (0.6 mM), biodegradation by all four strains was initially inhibited for at least 100 h relative to controls. At high rhamnolipid concentrations (6 mM), biodegradation by the fast degraders was slightly inhibited relative to controls, but the biodegradation by the slow degraders was enhanced relative to controls. Measurement of cell hydrophobicity showed that rhamnolipids increased the cell hydrophobicity of the slow degraders but had no effect on the cell hydrophobicity of the fast degraders. The rate at which the cells became hydrophobic was found to depend on the rhamnolipid concentration and was directly related to the rate of octadecane biodegradation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Impedance as an alternative to MPN enumeration of coliforms in pasteurized milks

R.H. MADDEN AND A. GILMOUR. 1995. Samples (900) of pasteurized whole, semi-skimmed and skimmed milk were subjected to conventional enumeration of coliforms by a nine-tube most probable number (MPN) technique, and impedance enumeration, in parallel. Regression analysis of the positive samples (98) showed that impedance enumeration was at least as accurate as the MPN method but results were obtained faster, with all testing being completed in 20 h, rather than 48 h. Consumable requirements, and staffing levels, were also much less with the impedance system. The impedance method could therefore beneficially replace the conventional method.     

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).     

Enumeration of petroleum-degrading marine and estuarine microorganisms by the most probable number method.

Several media designed for use in a most probable number (MPN) determination of petroleum-degrading microorganisms were compared. The best results, i.e., largest numbers, were obtained using a buffered (32 mM PO4=) liquid medium containing 1% hydrocarbon substrate. Of 104 presumptive oil degraders tested, 20 grew on oil agar medium but did not utilize oil or a mixture of pure paraffinic hydrocarbons (C10 to C16 n-alkanes) in liquid (MPN) medium. Visible turbidity in the liquid medium was correlated with hydrocarbon utilization. Counts of petroleum degraders obtained using liquid medium (MPN) were in most cases higher than those obtained on an oil-amended silica gel medium. Both procedures yield an estimation of oil degraders, and the oil-amended agar permits growth of organisms which do not degrade crude oil. All strains of oil-degrading microorganisms examined in this study were lipolytic, but the converse was not always true.     

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.     

Most-probable-number technique for the enumeration of aromatic degraders in natural environments

A most-probable-number (MPN) method is described for the enumeration of heterotrophic populations capable of utilizing chlorinated and nonchlorinated benzoates and phenols as sole carbon sources. A correlation coefficient of 0.91 was obtained between the numbers determined by the MPN technique and the standard plate count. The MPN method gave realistic cell counts when population densities were low, and the presence of oligocarbophiles did not give spurious results.     

PAH Degradation Capacity of Soil Microbial Communities—Does It Depend on PAH Exposure?

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous pollutants of the environment. But is their microbial degradation equally wide in distribution? We estimated the PAH degradation capacity of 13 soils ranging from pristine locations (total PAHs ≈ 0.1 mg kg^?1) to heavily polluted industrial sites (total PAHs ≈ 400 mg kg^?1). The size of the pyrene- and phenanthrene-degrading bacterial populations was determined by most probable number (MPN) enumeration. Densities of phenanthrene degraders reflected previous PAH exposure, whereas pyrene degraders were detected only in the most polluted soils. The potentials for phenanthrene and pyrene degradation were measured as the mineralization of ¹⁴C-labeled spikes. The time to 10% mineralization of added ¹⁴C phenanthrene and ¹⁴C pyrene was inversely correlated with the PAH content of the soils. Substantial ¹⁴C phenanthrene mineralization in all soils tested, including seven unpolluted soils, demonstrated that phenanthrene is not a suitable model compound for predicting PAH degradation in soils. ¹⁴C pyrene was mineralized by all Danish soil samples tested, regardless of whether they were from contaminated sites or not, suggesting that in industrialized areas the background level of pyrene is sufficient to maintain pyrene degradation traits in the gene pool of soil microorganisms. In contrast, two pristine forest soils from northern Norway and Ghana mineralized little ¹⁴C pyrene within the 140-day test period. Mineralization of phenanthrene and pyrene by all Danish soils suggests that soil microbial communities of inhabited areas possess a sufficiently high PAH degradation capacity to question the value of bioaugmentation with specific PAH degraders for bioremediation.     

Two-temperature membrane filter method for enumerating fecal coliform bacteria from chlorinated effluents.

Reports indicate that the standard membrane filter (MF) technique for recovery of fecal coliform bacteria from chlorinated sewage effluents is less effective than the multiple-tube (or most-probable-number [MPN]) procedure. A modified MF method was developed that requires a preincubation period of 5 h at 35 degrees C followed by 18+/-1 h at 44.5 degrees C. This procedure was evaluated by using both laboratory- and plant-chlorinated primary and secondary effluents. Results obtained by the modified MF method compared favorably with those of the MPN technique for the enumeration of fecal coliforms from chlorinated effluent. Agreement between these two methods was greatest with samples from secondary treatment plants. The average recovery of fecal coliforms by the standard MF procedure was only 14% that of the MPN method, whereas with the modified technique recovery was increased to 68% of the MPN counts. Enhanced recovery resulting from a simple modification in the incubation schedule makes the MF method a valuable adjunct for enumerating fecal coliforms from chlorinated effluents.     

Quantitative analysis of ammonia oxidising bacteria using competitive PCR

Culture-based methods for enumeration, such as most probable number (MPN) methodologies, have proved inefficient due to difficulties in the isolation and cultivation of ammonia oxidising bacteria in the laboratory. Biases are associated with the isolation of bacteria in selective media and organisms cultivated in the laboratory may not be truly representative of those in the environment. In this study, we developed a competitive PCR (cPCR)-based method based on the amplification of 16S rRNA genes specific for the beta-subgroup proteobacterial ammonia oxidising bacteria for enumeration of these organisms. Populations in both agricultural soils and estuarine sediments were quantified by traditional MPN and by cPCR. The numbers of ammonia oxidisers for both sample types were significantly underestimated by conventional MPN and were 1-3 orders of magnitude lower than those obtained by cPCR. Higher numbers of ammonia oxidisers found in fertilised plots in agricultural soils by the cPCR technique were not observed in MPN estimates. It was necessary to construct a separate standard curve for each sample type as differences in DNA extraction, quantity and purity had a significant bearing on the ease of PCR of both competitor and target DNA.     

Two-temperature membrane filter method for enumerating fecal coliform bacteria from chlorinated effluents.

Reports indicate that the standard membrane filter (MF) technique for recovery of fecal coliform bacteria from chlorinated sewage effluents is less effective than the multiple-tube (or most-probable-number [MPN]) procedure. A modified MF method was developed that requires a preincubation period of 5 h at 35 degrees C followed by 18+/-1 h at 44.5 degrees C. This procedure was evaluated by using both laboratory- and plant-chlorinated primary and secondary effluents. Results obtained by the modified MF method compared favorably with those of the MPN technique for the enumeration of fecal coliforms from chlorinated effluent. Agreement between these two methods was greatest with samples from secondary treatment plants. The average recovery of fecal coliforms by the standard MF procedure was only 14% that of the MPN method, whereas with the modified technique recovery was increased to 68% of the MPN counts. Enhanced recovery resulting from a simple modification in the incubation schedule makes the MF method a valuable adjunct for enumerating fecal coliforms from chlorinated effluents.     

EVALUATION OF TWO NONRADIOACTIVE GENE PROBES FOR THE ENUMERATION OF VIBRIO PARAHAEMOLYTICUS IN CRABMEAT

This study evaluated two nonradioactive DNA probe procedures for the detection and enumeration of Vibrio parahaemolyticus in crabmeat by comparing counts obtained by direct plating and by most probable number (MPN) procedures. The nonradioactive probes evaluated were an alkaline phosphatase-labeled thermolabile direct hemolysin (AP-tdh) and a digoxigenin-labeled thermostable direct hemolysin (DG-tdh) for detection and enumeration of total and pathogenic V. parahaemolyticus, respectively. Inoculated samples (50 g each) of steamed crabmeat were analyzed by nine analysts in seven laboratories. Samples were inoculated with various Vibrio strains and combinations of strains at different levels of inoculation (0 to 92,000 cells/g). The results indicated that the AP-tlh probe was reliable for identification and enumeration of total V. parahaemolyticus and the DG-tdh probe was specific for the pathogenic strains. Both probes required less effort and expense than the biochemical testing and hemolysin assays that have previously been used. The MPN and direct plating procedures using the nonradioactive probes were both effective for enumeration of total and pathogenic V. parahaemolyticus in the absence of competing microflora, but direct plating was preferable to MPN for enumeration of V. parahaemolyticus, especially pathogenic strains, in the presence of competitors.     

Calibration of the impedance method for rapid quantitative estimation of Escherichia coli in live marine bivalve molluscs

AIM: Calibration of impedance measurement was performed vs the Association Fran?oise de Normalisation (AFNOR) MPN method with a view to rapid enumeration of Escherichia coli in live marine bivalve molluscs. METHODS AND RESULTS: Linear regression models between log10 MPN and detection time (DT) were adjusted for several shellfish types, growth media, and impedance instruments (BacTrac and Malthus systems). Escherichia coli concentrations could be estimated from DT using a single regression line for BacTrac 4100 with M1 medium (R2 = 87.8%) and Malthus with M2 medium (R2 = 86.7%), and two regression lines for BacTrac 4110 with M2 medium (R2 = 86.4 and 88.2%). The uncertainty of the predicted bacterial concentration was around +/-0.43 log unit for duplicate sample analysis. The impedance signal was attributable to E. coli in 99% of cases. All cultures containing E. coli produced an impedance signal with BacTrac 4100 and BacTrac 4110, whereas 5.6% did not exhibit a signal with Malthus. CONCLUSIONS: Impedance measurement is a possible alternative to the MPN method for rapid quantitative estimation of E. coli in live bivalve shellfish. SIGNIFICANCE AND IMPACT OF THE STUDY: The impedance method reduces analysis handling time considerably and is much easier to use than the MPN method. Moreover, results can be obtained within 5-10 h, allowing rapid intervention to ensure public health protection in case of shellfish contamination.     

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.     

A statistical comparison of two culturing methods for enumerating sulfate-reducing bacteria

The addition of alkaline pyrogallol-soaked cotton wool plugs has been used by other workers to remove oxygen from the headspace gas in culture tubes for the growth of sulfate-reducing bacteria (SRB). This study compares the enumeration fo SRB using the most probable number (MPN) method in tubes with and without the pyrogallol plugs. In both cases, the liquid medium contained two iron finishing nails which help reduce the redox potential of the medium. For each of the 25 samples from oil fields, cooling water systems and natural environments, the time-consuming method using pyrogallol plugs in screw cap tubes yielded virtually the same MPN values as the same method without the plugs and using Kaput® closures on the tubes. However, the pyrogallol plug method, which requires approximately 10-fold more technician time, was superior for pure culture enumeration and in cases where SRB greatly outnumber heterotrophs.     

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.     

Comparison of nine brands of membrane filter and the most-probable-number methods for total coliform enumeration in sewage-contaminated drinking water.

Nine different brands of membrane filter were compared in the membrane filtration (MF) method, and those with the highest yields were compared against the most-probable-number (MPN) multiple-tube method for total coliform enumeration in simulated sewage-contaminated tap water. The water was chlorinated for 30 min to subject the organisms to stresses similar to those encountered during treatment and distribution of drinking water. Significant differences were observed among membranes in four of the six experiments, with two- to four-times-higher recoveries between the membranes at each extreme of recovery. When results from the membranes with the highest total coliform recovery rate were compared with the MPN results, the MF results were found significantly higher in one experiment and equivalent to the MPN results in the other five experiments. A comparison was made of the species enumerated by these methods; in general the two methods enumerated a similar spectrum of organisms, with some indication that the MF method was subject to greater interference by Aeromonas.     

Development of a vital fluorescent staining method for monitoring bacterial transport in subsurface environments

Previous bacterial transport studies have utilized fluorophores which have been shown to adversely affect the physiology of stained cells. This research was undertaken to identify alternative fluorescent stains that do not adversely affect the transport or viability of bacteria. Initial work was performed with a groundwater isolate, Comamonas sp. strain DA001. Potential compounds were first screened to determine staining efficiencies and adverse side effects. 5-(And 6-)-carboxyfluorescein diacetate, succinimidyl ester (CFDA/SE) efficiently stained DA001 without causing undesirable effects on cell adhesion or viability. Members of many other gram-negative and gram-positive bacterial genera were also effectively stained with CFDA/SE. More than 95% of CFDA/SE-stained Comamonas sp. strain DA001 cells incubated in artificial groundwater (under no-growth conditions) remained fluorescent for at least 28 days as determined by epifluorescent microscopy and flow cytometry. No differences in the survival and culturability of CFDA/SE-stained and unstained DA001 cells in groundwater or saturated sediment microcosms were detected. The bright, yellow-green cells were readily distinguished from autofluorescing sediment particles by epifluorescence microscopy. A high throughput method using microplate spectrofluorometry was developed, which had a detection limit of mid-10(5) CFDA-stained cells/ml; the detection limit for flow cytometry was on the order of 1,000 cells/ml. The results of laboratory-scale bacterial transport experiments performed with intact sediment cores and nondividing DA001 cells revealed good agreement between the aqueous cell concentrations determined by the microplate assay and those determined by other enumeration methods. This research indicates that CFDA/SE is very efficient for labeling cells for bacterial transport experiments and that it may be useful for other microbial ecology research as well.