Most Probable Number Tubes Research Articles

The most-probable-number enumeration of dichlobenil and 2,6-dichlorobenzamide (BAM) degrading microbes in Finnish aquifers

In groundwater subsurface deposits and a topsoil from five aquifers having 2,6-dichlorobenzamide (BAM) in water, we determined the most-probable-number (MPN) of 2,6-dichlorobenzonitrile (dichlobenil) and metabolite BAM degrading microorganisms. Dichlobenil and BAM were combined nitrogen sources in the MPN tubes, which were scored positive at concentrations <75% after 1 month incubation. Aerobic and anaerobic microbes degrading dichlobenil and BAM were common in samples in low numbers of 3.6-210 MPN g dw(-1). Additional degradation occurred in high MPN dilutions of some samples, the microbial numbers being 0.11-120 x 10(5) MPN g dw(-1). The strains were isolated from low and high dilutions of one deposit, and degradation in pure cultures was confirmed by HPLC. According to the 16S rDNA sequencing, strains were from genera Zoogloea, Pseudomonas, Xanthomonas, Rhodococcus, Nocardioides, Sphingomonas, and Ralstonia. Dichlobenil (45.5 +/- 18.3%) and BAM (37.6 +/- 14%) degradation was low in the MPN tubes. Despite of microbial BAM degradation activity in subsurface deposits, BAM was measured from groundwater.

Streptococcus suis in retail markets: How prevalent is it in raw pork?

Streptococcus suis present in raw pork meats sold in local retail markets was enumerated by Most Probable Number (MPN)-PCR method. This method combined the conventional MPN technique with a specifically designed PCR assay based on the amplification of a 294-bp S. suis species-specific 16S rRNA gene sequence. A total of 78 raw pork lean meat samples purchased at two different supermarkets (Site A and B) and a wet market (Site C) were tested. Results indicated that S. suis could be detected from the enriched MPN tubes of all, except one, sample homogenates. The concentration of S. suis ranged from < 3 to 4600 MPN/g of pork meat, with a total bacterial count (TBC) varying from 3.6 log to 7.4 log CFU/g. Statistical analyses indicated that pork meats purchased from the supermarket at Site B in summer contained significantly higher concentration of S. suis organisms than those from other retailers in any season. A significant correlation existed between log S. suis concentration and log TBC of the samples. This study revealed that raw pork meats available in local supermarkets or wet markets could contain S. suis at concentrations that were usually difficult to detect with traditional culture method. Field application of this method may contribute to a measurable evaluation, and thus the effective control, of human S. suis infection due to raw pork or pig carcass handling.

Vertical distribution of sulfate-reducing bacteria at the oxic-anoxic interface in sediments of the oligotrophic Lake Stechlin

Vertical distribution of sulfate-reducing bacteria (SRB) and sulfate reduction rates were investigated in littoral and profundal sediments of the oligotrophic Lake Stechlin. SRB counted by the most probable number (MPN) technique showed two peaks, one at the oxic-anoxic interface, and the second deeper in the reduced sediment layer. Sulfate reduction rates determined as potential rates in anoxic sediment slurries showed a similar vertical distribution with the highest values in the anoxic zones. The highest cell numbers observed were 1.3×105 cells cm−3 with lactate as substrate. SRB were also found in oxic sediment layers in numbers similar to those detected in anoxic zones. From the highest positive MPN tubes 34 pure cultures were isolated. Physiological characterization including nutritional behaviour, tests for desulfoviridin and spore formation turned out that nearly all isolates belonged to the genera Desulfovibrio, Desulfomicrobium, and Desulfotomaculum. SRB from the oxic sediment layers revealed a higher oxygen tolerance and capacity of oxygen respiration than isolates from the anoxic sediment layers. However, no sulfate reduction was observed in the presence of oxygen, since oxygen was preferentially reduced.

Enumeration of Total Coliforms and E. coli in Foods by the SimPlate® Coliform and E. coli Color Indicator Method and Conventional Culture Methods: Collaborative Study

The relative effectiveness of the SimPlate Coliform and E. coli Color Indicator (CEc-CI) method was compared to the AOAC 3-tube Most Probable Number (MPN) methods for enumerating and confirming coliforms and Escherichia coli in foods (966.23 and 966.24). In this study, test portions were prepared and analyzed according to the conditions stated in both the AOAC methods and SimPlate directions for use. Six food types were artificially contaminated with coliform bacteria and E. coli: frozen burritos, frozen broccoli, fluid pasteurized milk, whole almond nut meats, cheese, and powdered cake mix. Method comparisons were conducted. Overall, the SimPlate method demonstrated <0.3 log difference for total coliform and E. coli counts compared to the AOAC reference methods for the majority of food types and levels analyzed. In all cases, the repeatability and reproducibility of the SimPlate CEc-CI method were not different from those of the reference methods and in certain cases, were statistically better than those of the AOAC 3-tube MPN methods. These results indicate that the SimPlate CEc-CI method and the reference culture methods are comparable for enumeration of both total coliforms and E. coli in foods.

Applying a most probable number method for enumerating planktonic, dissimilatory, ammonium-producing, nitrate-reducing bacteria in oil field waters

A most probable number (MPN) method was used to enumerate dissimilatory ammonium-producing, nitrate-reducing bacteria (DAP-NRB) in oil field waters and to determine whether they were stimulated by nitrate addition used to control hydrogen sulfide production. An ammonium production medium with 5 carbon and energy sources (acetate, glucose, glycerol, pyruvate, and succinate) and nitrate was used in a 3-tube MPN procedure to enumerate DAP-NRB. These bacteria were detected in 12 of 18 oil field water samples, but they were seldom detected in wellhead samples. Three oil field water samples were amended with nitrate in serum bottles and the numbers of different NRB were determined over a 38-day incubation time. This amendment stimulated increases in the numbers of heterotrophic NRB and autotrophic nitrate-reducing, sulfide-oxidizing bacteria, but DAP-NRB remained a minor portion of these communities. Overall, DAP-NRB were present in many of the oil field waters that were examined but their numbers were low. It appears that DAP-NRB would play a minor role in the consumption of nitrate injected into oil field waters for the control of hydrogen sulfide production.

Comparison of a Chromogenic Medium with Thiosulfate-Citrate-Bile Salts-Sucrose Agar for DetectingVibrio parahaemolyticus

: The widely used most probable number (MPN) method for detecting Vibrio parahaemolyticus cannot differentiate growth of V. parahaemolyticus from Vibrio vulnificus or Vibrio mimicus on the thiosulfate-citrate-bile salts-sucrose agar (TCBS). Presumptive positive colonies grown onTCBS need to be confirmed with lengthy biochemical tests. This study compared a chromogenic medium, Bio-Chrome Vibrio medium (BCVM), with TCBS for detecting V. parahaemolyticus in seawater, sediment, and oysters using a 3-tube MPN method. Among the 296 samples tested, 136 and 92 samples produced presumptive positive results on TCBS and BCVM, respectively. Biochemical tests and a multiplex polymerase chain reaction (PCR) assay confirmed 74 of 83 samples that were presumptive positive on both TCBS and BVCM as V. parahaemolyticus. Although false-positive results were reported when either medium was used, there were 62 reported for TCBS whereas only 15 were reported for BCVM. The specificities of TCBS and BCVM for V. parahaemolyticus detection were determined to be 77% and 94%, respectively. The accuracies of detecting V. parahaemolyticus were 54% for TCBS and 84% for BCVM. The Bio-Chrome Vibrio medium can be used in the MPN method to reduce the number of biochemical tests needed for V. parahaemolyticus confirmation.

Assessing degradation capability of aerobic indigenous microflora in PAH-contaminated brackish sediments

A study was conducted to determine polycyclic aromatic hydrocarbons (PAHs) distribution and microbial population changes in brackish sediments from an Italian lagoon included in the Ramsar List of Wetlands of International Importance. The presence and level of PAH-degrading bacteria were estimated by the most probable number (MPN) enumeration technique, whereas degradation capability towards target compounds was checked against loss of spiked PAHs (Phenanthrene, Anthracene and Fluoranthene) in MPN tubes after incubation in optimal conditions. Chemical analyses and microbiological counts suggested a potential for PAHs biodegradation by natural occurring populations of sediment microorganisms, thus indicating an "optimal range" in sediment PAHs concentrations, outside of which the natural selection of the indigenous microflora did not occur. The MPN procedure here described, provided an effective and reliable way to simultaneously determine microbial population densities and subsequent confirmation of the biodegradation capability of sediment indigenous microflora when exposed to laboratory and environmental concentrations of PAHs.

Enumeration of Campylobacter in New Zealand recreational and drinking waters.

To use a published polymerase chain reaction (PCR) method for the detection and identification of thermotolerant Campylobacter species (Camp. jejuni, Camp. coli and Camp. lari) in tandem with a Most Probable Number (MPN) technique to enumerate these species in water samples. An initial study of 42 river water samples compared the use of conventional culture and PCR methods for the detection of Campylobacter in MPN enrichment tubes. It was found that all samples positive by culture were also positive by PCR. Thirty-seven percent more MPN tubes were positive by PCR compared with culture. The MPN/PCR technique was subsequently applied to 96 additional samples collected from rivers, drinking, roof and shallow ground water. Campylobacter was especially prevalent in river water (60% positive) and shallow ground water (75% positive) samples. Drinking water (29.2% positive) and roof water (37.5% positive) also contained Campylobacter, but the numbers detected were very low (maximum 0.3 and 0.56 MPN 100 ml-1, respectively). River waters contained Campylobacter at higher levels than any other water type and in a high percentage of the samples. Although Campylobacter was present in treated drinking water, the levels detected were low. These results suggest that water may act as a significant transmission route for human campylobacteriosis.

Communities of methanogenic bacteria in paddy field soils with long-term application of organic matter

Methanogenic communities were investigated in paddy field soil that had received long-term (over 30 y) applications of organic matter. Methanogenic bacteria were enumerated by the most probable number (MPN) method and by the analysis of ether-linked lipids. Three fields, one with chemical fertilizer (CF), one with application of rice straw plus chemical fertilizer (RS), and one with application of rice straw compost plus chemical fertilizer (RSC), were examined. The numbers of H 2-CO 2, methanol, and acetate utilizers estimated by the MPN method was 10 3–10 4, 10 4–10 5 and 10 4–10 5 g −1 dry soil in the CF and RS fields, respectively, and 10 5–10 6, 10 5–10 6 and 10 5–10 6 g −1 dry soil in the RSC field, respectively. Long-term application of rice straw compost significantly increased methanogenic numbers. Methanogenic cell numbers estimated by lipid analysis was 10 7–10 8 g −1 dry soil in the CF, RS and RSC fields. Rod-, coccus-, and sarcina-shaped cells were observed in the MPN tubes. Of the core lipids, hydroxyarchaeol was more abundant than caldarchaeol, which suggests that methanogenic bacteria of the family Methanosarcinaceae are predominant in these paddy field soils.

Comparison of Antibody-Direct Epifluorescent Filter Technique with the Most Probable Number Procedure for Rapid Enumeration of Listeria in Fresh Vegetables

The antibody-direct epifluorescent filter (Ab-DEFT) technique was evaluated as a rapid alternative to the most probable number (MPN) method for enumeration of artificially inoculated Listeria monocytogenes in ready-to-eat packaged salads and other fresh vegetables. Ab-DEFT was performed by homogenization of food in mesh-lined Stomacher bags, followed by prefiltration of homogenate through a 5 microns pore nylon filter, and passage of filtrate through a 0.4 micron pore black polycarbonate filter to collect and concentrate Listeria cells. After cells were stained with a fluorochrome-labeled polyclonal antibody to Listeria, the filter surface was examined by epifluorescence microscopy, and fluorescent cells were counted. A 3-tube MPN procedure was performed by successive enrichments of homogenized foods in Listeria enrichment and Fraser broths, followed by selective plating. Ab-DEFT provided quantitative determinations of Listeria cells that correlated with plate counts and MPN estimates in a linear response over a range of cell concentrations from 10 to 10(7) colony forming units (CFU)/mL. Microbial backgrounds as high as 10(8) CFU/mL did not affect performance of Ab-DEFT. In contrast to the MPN method, which required 5 days to perform, quantitation by Ab-DEFT could be completed in less than 1 h. Despite cross-reactivities demonstrated by the polyclonal fluorescent antibody, the potential of Ab-DEFT as a rapid alternative to MPN for microbial cell enumeration was evident.

24-Hour Presumptive Enumeration of Escherichia coli O157:H7 in Foods by Using the ISO-GRID® Method with SD-39 Agar

Two 24-hour presumptive enumeration methods for Escherichia coli O157:H7 organisms based on the hydrophobic grid membrane filter (ISO-GRID) and using SD-39 agar, a new selective and differential culture medium, were developed and compared to a 3-tube MPN (most probable number) method using modified tryptone soy broth enrichment. The comparative study comprised 22 combinations of storage conditions and food products, including a variety of raw and cooked meats and several dairy products. The ISO-GRID direct filtration method produced counts which were equivalent to or significantly higher than the 3-tube MPN method for all food-storage combinations except for frozen pasteurized whole egg. The ISO-GRID resuscitation method produced counts equivalent to the 3-tube MPN method for the frozen egg.

Vertical distribution of sulfate-reducing bacteria at the oxic-anoxic interface in sediments of the oligotrophic Lake Stechlin

Vertical distribution of sulfate-reducing bacteria (SRB) and sulfate reduction rates were investigated in littoral and profundal sediments of the oligotrophic Lake Stechlin. SRB counted by the most probable number (MPN) technique showed two peaks, one at the oxic-anoxic interface, and the second deeper in the reduced sediment layer. Sulfate reduction rates determined as potential rates in anoxic sediment slurries showed a similar vertical distribution with the highest values in the anoxic zones. The highest cell numbers observed were 1.3×10 5 cells cm −3 with lactate as substrate. SRB were also found in oxic sediment layers in numbers similar to those detected in anoxic zones. From the highest positive MPN tubes 34 pure cultures were isolated. Physiological characterization including nutritional behaviour, tests for desulfoviridin and spore formation turned out that nearly all isolates belonged to the genera Desulfovibrio, Desulfomicrobium, and Desulfotomaculum. SRB from the oxic sediment layers revealed a higher oxygen tolerance and capacity of oxygen respiration than isolates from the anoxic sediment layers. However, no sulfate reduction was observed in the presence of oxygen, since oxygen was preferentially reduced.

Enumeration of acetogens by a colorimetric most-probable-number assay

Anaerobic O demethylation by acetogenic bacteria often is the first step in the mineralization of methoxylated aromatic compounds in anoxic environments. In this reaction, an ether bond is cleaved and the resulting methyl group is metabolized via the acetyl coenzyme A pathway (acetogenesis). Anaerobic O demethylation was used to assess acetogen populations. Environmental samples were diluted in anaerobic medium containing a methoxylated aromatic substrate (vanillate) and titanium(III), and acetogen titers were estimated by the most-probable-number (MPN) method. Complex formation between Ti(III) and vicinal hydroxyl groups of the aromatic products of anaerobic O demethylation results in the development of a yellow color in the medium, which can be detected by eye and monitored spectrophotometrically. High-performance liquid chromatography analysis of the yellow MPN tubes showed that they contained the product of anaerobic O demethylation of vanillate (protocatechuate). This assay was used to enumerate O-demethylating acetogen populations in environmental samples.

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 degrees 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.

Numbers, activities, and diversity of autotrophic ammonia-oxidizing bacteria in a freshwater, eutrophic lake sediment

The microbiological and chemical potential for ammonia oxidation in a freshwater, eutrophic lake sediment was examined in relation to environmental changes caused by seasonal, dimictic circulation. Poulations of both ammonia and nitrite oxidizers as estimated by most probable number (MPN) were sustained throughout extended anaerobic summer intervals, with nitrite oxidizers outnumbering ammonia oxidizers by a factor ranging from 3.0 to 8.1. Ammonia oxidation potential on a per cell basis was affected by seasonal changes and was seen to decrease as oxygen was removed from the sediments. Pure-culture isolations from a positive MPN tube inoculated with oxygenated sediment and representing a single point in a seasonal cycle produced ammonia-oxidizing strains belonging to the genus Nitrosospira. These strains did not react with known ammonia-oxidizer serotypes and, therefore, extend the serological diversity of this group of bacteria. An immunofluorescence analysis of MPN tubes from sediment collected during a period of lake stratification revealed progressive changes in the diversity of the ammonia-oxidizer population. The genera Nitrosomonas, Nitrosolobus, and Nitrosospira, including the novel serotype of Nitrosospira isolated from the sediment a year earlier, were found to coexist in well-oxygenated sediment. This diversity was seen to disappear, with Nistrosomonas surviving, as anaerobic conditions persisted. Key words: ammonia oxidizers, lake sediments, nitrifiers, nitrification.

Enumeration and characterization of nitrogen-fixing bacteria in an eelgrass (Zostera marina) bed

Marine nitrogen-fixing bacteria distributed in the eelgrass bed and seawater of Aburatsubo Inlet, Kanagawa, Japan were investigated using anaerobic and microaerobic enrichment culture methods. The present enrichment culture methods are simple and efficient for enumeration and isolation of nitrogen-fixing bacteria from marine environments. Mostprobable-number (MPN) values obtained for nitrogen-fixing bacteria ranged from 1.1×10(2) to 4.6×10(2)/ml for seawater, 4.0×10(4) to 4.3×10(5)/g wet wt for eelgrass-bed sediment, and 2.1 × 10(5) to 1.2 × 10(7)/g wet wt for eelgrass-root samples. More than 100 strains of halophilic, nitrogen-fixing bacteria belonging to the family Vibrionaceae were isolated from the MPN tubes. These isolates were roughly classified into seven groups on the basis of their physiological and biochemical characteristics. The majority of the isolates were assigned to the genusVibrio and one group to the genusPhotobacterium. However, there was also a group that could not be identified to the generic level. All isolates expressed nitrogen fixation activities under anaerobic conditions, and no organic growth factors were required for their activities.

Metabolism of monocrotophos and quinalphos by algae isolated from soil.

but selective with concentrations,enhancement in the growth of soll algae has been reported (Megharaj et al. 1986). The capabilities of five algal species, isolated from a vertisol, to degrade monoerotophos and quinalphos, the most effective insecticides in controlling cotton bollworms, were determined in the present study. MATERIALS AND METHODS To determine the role of soil algae in the degradation of monoorotophos (Dimethyl (E)-1-methyl-2- methyl carbamoylvinyl phosphate) and quinalphos (O,O-diethyl-Oquinoxalin -2- yl phosphorothioate),enPichment cultures were prepared by adding ! ml of I000 ppm aqueous solutions from the commercial formulation of each insecticide separately to 20-g portions of the soil samples, maintained at 50% of water-holding capacity. After five additions, at 15-day intervals, of each chemical, the soil samples were withdrawn for isolation of predominant algae following the most-probable number (MPN) method as described earlier (Muralikrishna and VenMateswarlu 1984). Axenic cultures were developed from the most common and consistently occurring forms in almost all the MPN tubes. Two green algae, Chlorella vulqaris Beljerinck and $~enedesm~ biJuaatus (Turpin) Kuetzing, and three species of blue-green algae (cyanobacteria)

ICMSF Methods Studies. XVI. Comparison of Salt Polymyxin Broth with Glucose Salt Teepol Broth for Enumerating Vibrio parahaemolyticus in Naturally Contaminated Samples

Two media, glucose salt teepol broth (GSTB) and salt polymyxin broth (SPB), were compared for their efficacy in enumerating Vibrio parahaemolyticus in naturally contaminated samples using the most probable number (MPN) procedure. Eleven laboratories in four countries participated, six of them using two analysts. One hundred ninety-six of 335 samples were found to contain V. parahaemolyticus. Neither enrichment medium was superior, and both media appeared necessary to prevent false-negative results. There was a high (52.5%) percentage of MPN patterns that were improbable with respect to Poisson distribution in those MPN tubes that yielded V. parahaemolyticus. Suspect colonies that developed on thiosulfate citrate bile salts sucrose (TCBS) plates streaked from GSTB were confirmed (1,438/1,929 = 75%) about as frequently as when streaked from SPB (2,155/3,038 = 71%).

Enumeration, Isolation, and Characterization of N 2 -Fixing Bacteria from Seawater

Marine pelagic N(2)-fixing bacteria have not, in general, been identified or quantified, since low or negligible rates of N(2) fixation have been recorded for seawater when blue-green algae (cyanobacteria) are absent. In the study reported here, marine N(2)-fixing bacteria were found in all samples of seawater collected and were analyzed by using a most-probable-number (MPN) method. Two different media were used which allowed growth of microaerophiles, as well as that of aerobes and facultative anaerobes. MPN values obtained for N(2)-fixing bacteria ranged from 0.4 to 1 x 10 per liter for water collected off the coast of Puerto Rico and from 2 to 5.5 x 10 per liter for Chesapeake Bay water. Over 100 strains of N(2)-fixing bacteria were isolated from the MPN tubes and classified, yielding four major groups of NaCl-requiring bacteria based on biochemical characteristics. Results of differential filtration studies indicate that N(2)-fixing bacteria may be associated with phytoplankton. In addition, when N(2)-fixing bacteria were inoculated into unfiltered seawater and incubated in situ, nitrogenase activity could be detected within 1 h. However, no nitrogenase activity was detected in uninoculated seawater or when bacteria were incubated in 0.2-mum-filtered (phytoplankton-free) seawater. The ability of these isolates to fix N(2) at ambient conditions in seawater and the large variety of N(2)-fixing bacteria isolated and identified lead to the conclusion that N(2) fixation in the ocean may occur to a greater degree than previously believed.

Examination of an acid forest soil for ammonia- and nitrite-oxidizing autotrophic bacteria

An acid forest soil (pH 3.9 – 4.4) from an undisturbed mixed oak stand in southern Indiana was examined for the occurrence of ammonia- and nitrite- oxidizing chemoautotrophs. Populations of both nitrifiers were detected in pH 7 most-probable-number (MPN) autotrophic media, and a Nitrosospira was isolated from highest dilution ammonia oxidizer MPN tubes. Populations of nitrite oxidizers were 10 to 1000 times higher than those of ammonia oxidizers. In pH 4.0 MPN media, ammonia oxidation was slight and unsustainable on 10% transfer to fresh medium, whereas nitrite oxidation was vigorous and sustainable. In pure culture the Nitrosospira isolate (Np IO1a) was completely inhibited by nitrapyrin at 5 μg mL−1, tolerant of 1.0 and 10.0 mM chlorate, and capable of growth only at pH 6.2 and above. Fluorescent antibodies raised against Np IO1a were used to confirm the predominance of Np IO1a in all MPN series examined. These results suggest that autotrophic ammonia oxidizers may be restricted to circumneutral microsites in this acid soil, whereas autotrophic nitrite oxidizers may not be limited to such sites.