Browsing by Author "Lisle, John T."

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Comparison of fluorescence microscopy and solid-phase cytometry methods for counting bacteria in water
(2004-09) Lisle, John T.; Hamilton, Martin A.; Willse, Alan Ray; McFeters, Gordon A.
Total direct counts of bacterial abundance are central in assessing the biomass and bacteriological quality of water in ecological and industrial applications. Several factors have been identified that contribute to the variability in bacterial abundance counts when using fluorescent microscopy, the most significant of which is retaining an adequate number of cells per filter to ensure an acceptable level of statistical confidence in the resulting data. Previous studies that have assessed the components of total-direct-count methods that contribute to this variance have attempted to maintain a bacterial cell abundance value per filter of approximately 106 cells filter–1. In this study we have established the lower limit for the number of bacterial cells per filter at which the statistical reliability of the abundance estimate is no longer acceptable. Our results indicate that when the numbers of bacterial cells per filter were progressively reduced below 105, the microscopic methods increasingly overestimated the true bacterial abundance (range, 15.0 to 99.3%). The solid-phase cytometer only slightly overestimated the true bacterial abundances and was more consistent over the same range of bacterial abundances per filter (range, 8.9 to 12.5%). The solid-phase cytometer method for conducting total direct counts of bacteria was less biased and performed significantly better than any of the microscope methods. It was also found that microscopic count data from counting 5 fields on three separate filters were statistically equivalent to data from counting 20 fields on a single filter.
Fluorescent probes applied to the physiological characterization of bacterial biofilms
(1999) Lisle, John T.; Stewart, Philip S.; McFeters, Gordon A.
Microbial formation of labile organic carbon in Antarctic glacial environments
(2017-04) Smith, Heidi J.; Foster, Rachel A.; McKnight, Diane M.; Lisle, John T.; Littmann, Sten; Kuypers, Marcel M. M.; Foreman, Christine M.
Roughly six petagrams of organic carbon are stored within ice worldwide. This organic carbon is thought to be of old age and highly bioavailable. Along with storage of ancient and new atmospherically deposited organic carbon, microorganisms may contribute substantially to the glacial organic carbon pool. Models of glacial microbial carbon cycling vary from net respiration to net carbon fixation. Supraglacial streams have not been considered in models although they are amongst the largest ecosystems on most glaciers and are inhabited by diverse microbial communities. Here we investigate the biogeochemical sequence of organic carbon production and uptake in an Antarctic supraglacial stream in the McMurdo Dry Valleys using nanometre-scale secondary ion mass spectrometry, fluorescence spectroscopy, stable isotope analysis and incubation experiments. We find that heterotrophic production relies on highly labile organic carbon freshly derived from photosynthetic bacteria rather than legacy organic carbon. Exudates from primary production were utilized by heterotrophs within 24 h, and supported bacterial growth demands. The tight coupling of microbially released organic carbon and rapid uptake by heterotrophs suggests a dynamic local carbon cycle. Moreover, as temperatures increase there is the potential for positive feedback between glacial melt and microbial transformations of organic carbon.
Microbial growth under humic-free conditions in a supraglacial stream system on the Cotton Glacier, Antarctica
(2013-07) Foreman, Christine M.; Cory, R. M.; Morris, Cindy E.; SanClements, M. D.; Smith, Heidi J.; Lisle, John T.; Miller, P. L.; Chin, Yu-Ping; McKnight, Diane M.
During the austral summers of 2004 and 2009, we sampled a supraglacial stream on the Cotton Glacier, Antarctica. The stream dissolved organic matter (DOM) was low (44â€“48 Âµ M C) and lacked detectable humic fluorescence signatures. Analysis of the excitation emissions matrices (EEMs) indicated that amino-acid fluorophores dominated, consistent with DOM of microbial origin, with little humic-like fluorescence. In most aquatic ecosystems, humic DOM attenuates harmful UV radiation and its absence may represent an additional stressor influencing the microbial community. Nonetheless, the stream contained an active microbial assemblage with bacterial cell abundances from 2.94 x 104 to 4.97 x 105 cells ml-1, and bacterial production ranging from 58.8 to 293.2 ng C l-1 d-1. Chlorophyll-a concentrations ranged from 0.3 to 0.53 Âµ g 1-1 indicating that algal phototrophs were the probable source of the DOM. Microbial isolates produced a rainbow of pigment colors, suggesting adaptation to stress, and were similar to those from other cryogenic systems (Proteobacteria and Bacteroidetes lineages). Supraglacial streams provide an example of contemporary microbial processes on the glacier surface and a natural laboratory for studying microbial adaptation to the absence of humics.
Physicochemical and biological dynamics in a coastal Antarctic lake as it transitions from frozen to open water
(2013-03) Dieser, Markus; Foreman, Christine M.; Jaros, C.; Lisle, John T.; Greenwood, Mark C.; Laybourn-Parry, Johanna; Miller, P. L.; Chin, Yu-Ping; McKnight, Diane M.
Pony Lake, at Cape Royds, Antarctica, is a shallow, eutrophic, coastal lake that freezes solid in the winter. Changes in Pony Lake's physicochemical parameters and microbial community were studied during the transition from ice to open water. Due to rising water temperatures, the progressive melt of the ice column and the gradual mixing of basal brines into the remaining water column, Pony Lake evolved physically and chemically over the course of the summer, thereby affecting the microbial community composition. Temperature, pH, conductivity, nutrients and major ion concentrations reached their maximum in January. Pony Lake was colonized by bacteria, viruses, phytoflagellates, ciliates, and a small number of rotifers. Primary and bacterial production were highest in mid-December (2.66 mg C 1-1 d-1 and 30.5 Âµg C 1-1 d-1, respectively). A 16S rRNA gene analysis of the bacterioplankton revealed 34 unique sequences dominated by members of the ÃŸ- and y-proteobacteria lineages. Cluster analyses on denaturing gradient gel electrophoresis (DGGE) banding patterns and community structure indicated a shift in the dominant members of the microbial community during the transition from winter ice, to early, and late summer lakewater. Our data demonstrate that temporal changes in physicochemical parameters during the summer months determine community dynamics and mediate changes in microbial species composition.
Rapid direct methods for enumeration of specific, active bacteria in water and biofilms
(1998-12) McFeters, Gordon A.; Pyle, Barry H.; Lisle, John T.; Broadaway, Susan C.
Conventional methods for detecting indicator and pathogenic bacteria in water may underestimate the actual population due to sublethal environmental injury, inability of the target bacteria to take up nutrients and other physiological factors which reduce bacterial culturability. Rapid and direct methods are needed to more accurately detect and enumerate active bacteria. Such a methodological advance would provide greater sensitivity in assessing the microbiological safety of water and food. The principle goal of this presentation is to describe novel approaches we have formulated for the rapid and simultaneous detection of bacteria plus the determination of their physiological activity in water and other environmental samples. The present version of our method involves the concentration of organisms by membrane filtration or immunomagnetic separation and combines an intracellular fluorochrome (CTC) for assessment of respiratory activity plus fluorescent-labelled antibody detection of specific bacteria. This approach has also been successfully used to demonstrate spatial and temporal heterogeneities of physiological activities in biofilms when coupled with cryosectioning. Candidate physiological stains include those capable of determining respiratory activity, membrane potential, membrane integrity, growth rate and cellular enzymatic activities. Results obtained thus far indicate that immunomagnetic separation can provide a high degree of sensitivity in the recovery of seeded target bacteria (Escherichia coli O157:H7) in water and hamburger. The captured and stained target bacteria are then enumerated by either conventional fluorescence microscopy or ChemScank, a new instrument that is very sensitive and rapid. The ChemScanR laser scanning instrument (Chemunex, Paris, France) provides the detection of individual fluorescently labelled bacterial cells using three emission channels in less than 5 min. A high degree of correlation has been demonstrated between results obtained with the ChemScan and traditional plate counts of mixed natural bacterial populations in water. The continuing evolution of these methods will be valuable in the rapid and accurate analysis of environmental samples.
When a habitat freezes solid: Microorganisms over-winter within the ice column of a coastal Antarctic lake
(2011-03) Foreman, Christine M.; Dieser, Markus; Greenwood, Mark C.; Cory, R. M.; Laybourn-Parry, Johanna; Lisle, John T.; Jaros, C.; Miller, P. L.; Chin, Yu-Ping; McKnight, Diane M.
A major impediment to understanding the biology of microorganisms inhabiting Antarctic environments is the logistical constraint of conducting field work primarily during the summer season. However, organisms that persist throughout the year encounter severe environmental changes between seasons. In an attempt to bridge this gap, we collected ice core samples from Pony Lake in early November 2004 when the lake was frozen solid to its base, providing an archive for the biological and chemical processes that occurred during winter freezeup. The ice contained bacteria and virus-like particles, while flagellated algae and ciliates over-wintered in the form of inactive cysts and spores. Both bacteria and algae were metabolically active in the ice core melt water. Bacterial production ranged from 1.8 to 37.9 μg C L−1 day−1. Upon encountering favorable growth conditions in the melt water, primary production ranged from 51 to 931 μg C L−1 day−1. Because of the strong H2S odor and the presence of closely related anaerobic organisms assigned to Pony Lake bacterial 16S rRNA gene clones, we hypothesize that the microbial assemblage was strongly affected by oxygen gradients, which ultimately restricted the majority of phylotypes to distinct strata within the ice column. This study provides evidence that the microbial community over-winters in the ice column of Pony Lake and returns to a highly active metabolic state when spring melt is initiated.