Computer-Assisted Laser Scanning and Video Microscopy for Analysis of Cryptosporidium parvum Oocysts in Soil, Sediment, and Feces

A computer-assisted laser scanning microscope equipped for confocal laser scanning and color video microscopy was used to examine Cryptosporidium parvum oocysts in two agricultural soils, a barnyard sediment, and calf fecal samples. An agar smear technique was developed for enumerating oocysts in soil and barnyard sediment samples. Enhanced counting efficiency and sensitivity (detection limit, 5.2 x 10(sup2) oocysts(middot)g [dry weight](sup-1)) were achieved by using a semiautomatic counting procedure and confocal laser scanning microscopy to enumerate immunostained oocysts and fragments of oocysts in the barnyard sediment. An agarose-acridine orange mounting procedure was developed for high-resolution confocal optical sectioning of oocysts in soil. Stereo images of serial optical sections revealed the three-dimensional spatial relationships between immunostained oocysts and the acridine orange-stained soil matrix material. In these hydrated, pyrophosphate-dispersed soil preparations, oocysts were not found to be attached to soil particles. A fluorogenic dye permeability assay for oocyst viability (A. T. Campbell, L. J. Robertson, and H. V. Smith, Appl. Environ. Microbiol. 58:3488-3493, 1992) was modified by adding an immunostaining step after application of the fluorogenic dyes propidium iodide and 4(prm1),6-diamidino-2-phenylindole. Comparison of conventional color epifluorescence and differential interference contrast images on one video monitor with comparable black-and-white laser-scanned confocal images on a second monitor allowed for efficient location and interpretation of fluorescently stained oocysts in the soil matrix. This multi-imaging procedure facilitated the interpretation of the viability assay results by overcoming the uncertainties caused by matrix interference and background fluorescence.     

Distribution and activity of microorganisms in subsurface sediments of a pristine study site in Oklahoma

Distribution and activity of microorganisms in surface soil and subsurface sediments were studied in depth profiles of six different microbial biomass and activity indicators (total direct counts, number of cells capable of electron transport system activity, viable cell plate counts, most Probable numbers of protozoa, and 4-hydroxybenzoate-degrading microorganisms, and ATP content). The profiles showed the same general trends on two different dates (January and June 1985). Seasonal variations were noted, but they were not extreme. Biomass and activity values declined sharply with depth in the unsaturated zone, reaching minima in a clay confining layer in the interface zone between 3 and 4 m. Contiguous 10-cm samples from the interface zone showed significant textural and microbiological variability. Higher and more stable biomass and activity values were detected in the saturated zone, the highest being a very permeable gravelly loamy sand layer at approximately 7.5 m. In this layer, viable counts were nearly equal to total counts and they approached the viable counts in surface soil. Surface-type protozoa and cyanobacteria also were detected in this layer, suggesting that it was connected hydrologically to a nearby river. Lowest values were detected in an underlying bedrock clay layer at 8 m, which, despite its impermeability and low viable counts, did contain measurable total counts, 4-hydroxybenzoate-degrading microorganisms, and ATP. Correlations were noted between sediment texture and microbial activity (i.e., sandy texture=high activity, clayey texture=low activity), but other hydrogeological and geochemical factors probably also influenced microbial distribution and activity in the profile.     

ULTRASTRUCTURE OF THE THERMOPHILIC BLUE-GREEN ALGA,SYNECHOCOCCUS LIVIDUS COPELAND1

The ultrastructure of Synechococcus lividus Copeland, a thermophilic blue-green alga, was studied in thin sections. The cell envelope reveals striking similarities with that of some gram-negative bacteria. In contrast to bacteria and to many other species of blue-green algae, ribosomes are predominantly found in the central nuclear region and appear to be associated with the DNA fibrils. Thylakoids (photo-synthetic lamellae) are arranged as concentric shells, around the nuclear equivalent, lying nearly parallel to one another and to the plasma membrane. Both plasma and thylakoidal membranes, as described by other authors for different Cyanophyceae, are of the unit membrane dimension and morphology. Various types of intracellular inclusions are found: (1) Lipid inclusions, located in the cytoplasm are similar to the osmiophilic globules of higher plant chloroplasts. (2) Polyphosphate inclusions (or volutin) resembling those of other species are generally found at the cell poles but within the nuclear region. (3) Polyhedral inclusions also located in the nuclear region are clearly recognized to be different from the polyphosphate bodies, but their function remains unknown.     

Methanotrophic Activity,Abundance, and Diversity in Forested Swamp Pools: Spatiotemporal Dynamics and Influences on Methane Fluxes

Methane oxidation (methanotrophy) in the water column and sediments of forested swamp pools likely control seasonal and annual emission of CH₄ from these systems, but the methanotrophic microbial communities, their activities, locations, and overall impact, is poorly understood. Several techniques including ¹⁴CH₄ oxidation assays, culture-based most probable number (MPN) estimates of methane-oxidizing bacteria (MOB) and protozoan abundance, MOB strain isolation and characterization, and PCR techniques were used to investigate methanotrophy at a forested swamp near Ithaca, New York. The greatest methanotrophic activity and largest numbers of MOB occurred predominantly at the low oxygen sediment/water interface in the water column. Seasonally, methanotrophic activity was very dynamic, ranging from 0.1 to 61.9 μ moles CH₄ d^?1 g^?1 dry sediment, and correlated most strongly with dissolved inorganic carbon (r = 0.896). Incorporation of methanotrophic variables (abundance and activity) into existing CH₄ flux regression models improved model fit, particularly during mid summer when CH₄ fluxes were most dynamic. Annually integrated methane flux and methanotrophic activity measurements indicate that differences in methanotrophic activity at the sediment/water interface likely accounted for differences in the annual CH₄ emission from the field site. Direct isolations of MOB resulted in the repeated isolation of organisms most closely related to Methylomonas methanica S1. A single acidophilic, type II MOB related to Methylocella palustris K was also isolated. Using a PCR-based MPN method and 16S rRNA genome copy number from isolates and control strains, type I and type II MOB were enumerated and revealed type I dominance of the sediment-associated MOB community.     

Determination of Spatial Distributions of Zinc and Active Biomass in Microbial Biofilms by Two-Photon Laser Scanning Microscopy

The spatial distributions of zinc, a representative transition metal, and active biomass in bacterial biofilms were determined using two-photon laser scanning microscopy (2P-LSM). Application of 2P-LSM permits analysis of thicker biofilms than are amenable to observation with confocal laser scanning microscopy and also provides selective excitation of a smaller focal volume with greater depth localization. Thin Escherichia coli PHL628 biofilms were grown in a minimal mineral salts medium using pyruvate as the carbon and energy source under batch conditions, and thick biofilms were grown in Luria-Bertani medium using a continuous-flow drip system. The biofilms were visualized by 2P-LSM and shown to have heterogeneous structures with dispersed dense cell clusters, rough surfaces, and void spaces. Contrary to homogeneous biofilm model predictions that active biomass would be located predominantly in the outer regions of the biofilm and inactive or dead biomass (biomass debris) in the inner regions, significant active biomass fractions were observed at all depths in biofilms (up to 350 μm) using live/dead fluorescent stains. The active fractions were dependent on biofilm thickness and are attributed to the heterogeneous characteristics of biofilm structures. A zinc-binding fluorochrome (8-hydroxy-5-dimethylsulfoamidoquinoline) was synthesized and used to visualize the spatial location of added Zn within biofilms. Zn was distributed evenly in a thin (12 μm) biofilm but was located only at the surface of thick biofilms, penetrating less than 20 μm after 1 h of exposure. The relatively slow movement of Zn into deeper biofilm layers provides direct evidence in support of the concept that thick biofilms may confer resistance to toxic metal species by binding metals at the biofilm-bulk liquid interface, thereby retarding metal diffusion into the biofilm (G. M. Teitzel and M. R. Park, Appl. Environ. Microbiol. 69:2313-2320, 2003).     

Microbial diversity in hot synthetic compost as revealed by PCR-amplified rRNA sequences from cultivated isolates and extracted DNA

High-temperature (>/=60 degrees C) synthetic food waste compost was examined by cultivation-dependent and -independent methods to determine predominant microbial populations. Fluorescent direct counts totaled 6.4 (+/-2.5)x10(10) cells gdw(-1) in a freeze-dried 74 degrees C compost sample, while plate counts for thermophilic heterotrophic aerobes averaged 2.6 (+/-1.0)x10(8) CFU gdw(-1). A pre-lysis cell fractionation method was developed to obtain community DNA and a suite of 16S and 18S rDNA-targeted PCR primers was used to examine the presence of Bacteria, Archaea and fungi. Bacterial 16S rDNA, including a domain-specific 1500-bp fragment and a 300-bp fragment specific for Actinobacteria, was amplified by PCR from all compost samples tested. Archaeal rDNA was not amplified in any sample. Fungal 18S rDNA was only amplified from a separate dairy manure compost that reached a peak temperature of 50 degrees C. Amplified rDNA restriction analysis (ARDRA) was used to screen isolated thermophilic bacteria and a clone library of full-length rDNA fragments. ARDRA screening revealed 14 unique patterns among 63 isolates, with one pattern accounting for 31 of the isolates. In the clone library, 52 unique patterns were detected among 70 clones, indicating high diversity of uncultivated bacteria in hot compost. Phylogenetic analysis revealed that the two most abundant isolates belonged in the genera Aneurinibacillus and Brevibacillus, which are not commonly associated with hot compost. With the exception of one Lactobacillus-type sequence, the clone library contained only sequences that clustered within the genus Bacillus. None of the isolates or cloned sequences could be assigned to the group of obligate thermophilic Bacillus spp. represented by B. stearothermophilus, commonly believed to dominate high-temperature compost. Amplified partial fragments from Actinobacteria, spanning the V3 variable region (Neefs et al. (1990) Nucleic Acids Res. 18, 2237-2242), included sequences related to the genera Saccharomonospora, Gordonia, Rhodococcus and Corynebacterium, although none of these organisms were detected among the isolates or full-length cloned rDNA sequences. All of the thermophilic isolates and sequenced rDNA fragments examined in this study were from Gram-positive organisms.     

Determination of spatial distributions of zinc and active biomass in microbial biofilms by two-photon laser scanning microscopy

The spatial distributions of zinc, a representative transition metal, and active biomass in bacterial biofilms were determined using two-photon laser scanning microscopy (2P-LSM). Application of 2P-LSM permits analysis of thicker biofilms than are amenable to observation with confocal laser scanning microscopy and also provides selective excitation of a smaller focal volume with greater depth localization. Thin Escherichia coli PHL628 biofilms were grown in a minimal mineral salts medium using pyruvate as the carbon and energy source under batch conditions, and thick biofilms were grown in Luria-Bertani medium using a continuous-flow drip system. The biofilms were visualized by 2P-LSM and shown to have heterogeneous structures with dispersed dense cell clusters, rough surfaces, and void spaces. Contrary to homogeneous biofilm model predictions that active biomass would be located predominantly in the outer regions of the biofilm and inactive or dead biomass (biomass debris) in the inner regions, significant active biomass fractions were observed at all depths in biofilms (up to 350 microm) using live/dead fluorescent stains. The active fractions were dependent on biofilm thickness and are attributed to the heterogeneous characteristics of biofilm structures. A zinc-binding fluorochrome (8-hydroxy-5-dimethylsulfoamidoquinoline) was synthesized and used to visualize the spatial location of added Zn within biofilms. Zn was distributed evenly in a thin (12 microm) biofilm but was located only at the surface of thick biofilms, penetrating less than 20 microm after 1 h of exposure. The relatively slow movement of Zn into deeper biofilm layers provides direct evidence in support of the concept that thick biofilms may confer resistance to toxic metal species by binding metals at the biofilm-bulk liquid interface, thereby retarding metal diffusion into the biofilm (G. M. Teitzel and M. R. Park, Appl. Environ. Microbiol. 69:2313-2320, 2003).     

Inactivation of Cryptosporidium parvum Oocysts by Ammonia

The survival of Cryptosporidium parvum oocysts in soil and water microhabitats may be affected by the environmental production and release of free ammonia. The objective of this study was to determine the effects of increasing free ammonia concentrations and times of exposure on oocyst viability. Wild-type oocysts were obtained from naturally infected calf feces by chemical (continuous-flow) centrifugation and sucrose gradients. Ammonia (NH₃) from a commercial solution was applied in concentrations ranging from 0.007 to 0.148 M. Exposure times ranged from 10 min to 24 h at a constant temperature of 24 ± 1°C. Viability of oocysts was determined with a dye permeability assay and an in vitro excystation assay (M. B. Jenkins, L. J. Anguish, D. D. Bowman, M. J. Walker, and W. C. Ghiorse, Appl. Environ. Microbiol. 63:3844–3850, 1997). Even the lowest concentration of ammonia decreased significantly the viability of oocysts after 24 h of exposure. Increasing concentrations of ammonia increased inactivation rates, which ranged from 0.014 to 0.066 h⁻¹. At the highest concentration of ammonia, a small fraction of viable oocysts still remained. Exposure to pH levels corresponding to those associated with the ammonia concentrations showed minimal effects of alkaline pH alone on oocyst viability. This study shows that environmentally relevant concentrations of free ammonia may significantly increase the inactivation of oocysts in ammonia-containing environments.     

Growth kinetics of Pseudomonas putida G7 on naphthalene and occurrence of naphthalene toxicity during nutrient deprivation

The objectives of this work were (1) to demonstrate how the chemostat approach could be modified to allow determination of kinetic parameters for a sparingly soluble, volatile substrate such as naphthalene and (2) to examine the influence of the interactions of various nutrients on possible growth-inhibitory effects of naphthalene. Pseudomonas putida G7 was used as a model naphthalene-degrading microorganism. Naphthalene was found to be toxic to P. putida G7 in the absence of a nitrogen source or oxygen. The death rate of cells grown on minimal medium plus naphthalene and then exposed to naphthalene under anoxic conditions was higher than that observed under oxic conditions in the absence of a nitrogen source. The presence of necessary nutrients for the biodegradation of PAH compounds is indicated to be important for the survival of microorganisms that are capable of PAH degradation. The amounts of ammonia and oxygen necessary for naphthalene biodegradation and for suppression of naphthalene toxicity were calculated from growth yield coefficients. A chemostat culture of P. putida G7 using naphthalene as a carbon and energy source was accomplished by using a feed augmented with a methanol solution of naphthalene so as to provide sufficient growth to allow accurate evaluation of kinetic parameters. When naphthalene was the growth-limiting substrate, the degradation of naphthalene followed Monod kinetics. Maximum specific growth rate (micrometer) and Monod constant (Ks) were 0.627 +/- 0.007 h-1 and 0.234 +/- 0.0185 mg/L, respectively. The evaluation of biodegradation parameters will allow a mathematical model to be applied to predict the long-term behavior of PAH compounds in soil when combined with PAH transport parameters.