A SIMPLE METHOD FOR THE ISOLATION AND ENUMERATION OF SPARSE POPULATIONS OF CYANOBACTERIA IN ESTUARINE WATERS1

A simple, sensitive assay method for the isolation and enumeration of sparse populations of cyanobacteria in an estuarine system is described. The method, based on the standard membrane-filter plate count technique, differentiates between viable and nonviable cells. It was found that an estuarine water-based agar medium was the most suitable medium for isolation of cyanobacteria. Because of the restricted nature of colony development, isolation of individual species is easily accomplished.     

Method for isolating cyanobacteria-lysing streptomycetes from soil

A new technique is described for the isolation and enumeration of cyanobacteria-lysing Streptomyces spp from soil or water. Two cyanobacteria, Anabaena cylindrica (ACTT 27899) and Tolypothrix tenuis (ATCC 27914) were used as the test organisms. Ten-day-old cyanobacterial cultures were vacuum-filtered to form a lawn on a 7 cm Whatman No. 1 filter paper which was then supported on Allen's agar. When the lawn was inoculated with dilutions of a heavy clay prairie soil and incubated at 27 PT 1dEC under constant illumination, white or grey colonies of streptomycetes grew. These colonies were surrounded by zones where a yellowing and lysis of the algal cells occurred. Streptomyces achromogenes was identified as a major lytic species within a population of 5 times 10₃ plaque-producing streptomycetes/g (dry weight) soil.     

Plate-Dilution Frequency Technique for Assay of Microbial Ecology

The plate-dilution frequency technique described facilitates simultaneous enumeration of a wide range of physiologically different microorganisms in complex systems with a precision comparable to dilution tube (most probable number) methods. Replicate microsamples are inoculated from each member of a dilution series onto areas delineated on plates of prepoured solid media; the plates are incubated, and the occurrence of growth or specific biochemical transformation is recorded for each inoculated area. Microbial enumeration is accomplished by reference to appropriate tables. Details of the experimental procedures are described, and tables are presented from which microbial numbers with 95% confidence limits can be obtained and compared for significant difference directly for 10-fold and 4-fold dilution series. Results of experiments in which microbial populations were estimated simultaneously by the plate-dilution frequency and conventional plate count methods are compared. The potential of the technique for broad-spectrum microbial assay is also discussed.     

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.     

Identification and quantification of suspended algae and bacteria populations using flow cytometry: applications for algae biofuel and biochemical growth systems

Species diversity in algae biofuel and biochemical culturing systems can affect yield; in many large-scale algae growth systems, it is not practical to maintain a monoculture. To better understand and monitor these complex systems, techniques are required which can quickly and effectively quantify the species distribution and overall growth of a mixed microbial community in suspension. A flow cytometric method has been developed which can be used to differentiate populations of three Chlorophyta species, one diatom species, cyanobacteria, and heterotrophic bacteria according to their fluorescence and morphology. The nucleic acid stain SYTO9 was used to discriminate species with similar natural autofluorescence and to identify heterotrophic bacteria. Absolute cell enumeration was performed with counting beads and validated with a hemocytometer. Species identification was validated by analyzing known mixtures of axenic cell cultures. The utility of the method was demonstrated by studying the effect of light intensity on species succession, growth, and biomass accumulation in small algae growth systems over 22 days. Flow cytometric analysis, augmented with SYTO9 stain and counting beads, can be utilized to monitor algae biofuel and biochemical growth systems involving multiple species. This method allows for monitoring of contamination, succession, and overall growth in both natural and intentionally created microbial communities.     

Evaluation of coliphage detection as a rapid indicator of water quality.

A rapid coliphage analysis technique for enumerating coliphages in natural waters has been evaluated by water quality laboratories located throughout the United States. Correlations were established between coliphages and coliforms in natural water systems. These correlations were highly significant. This relationship can thus be used to determine the number of fecal or total coliforms present in natural water samples based on an enumeration of coliphages. With this method, coliphages in natural water systems (containing greater than or equal to six coliphages per 100 ml) can be enumerated within 6 h.     

Modern methods for isolation,purification, and cultivation of soil cyanobacteria

Up-to-date methods for isolation of cyanobacteria from soil samples, removal of accompanying microflora, obtaining axenic strains, and conditions and media for subsequent cultivation are reviewed. Characterization of soil as a specific habitat for cyanobacteria is provided. Comparative analysis of pH and elemental composition of the liquid phase of most soil types with the media for cultivating cyanobacteria is carried out. The functional role of the major components required for the cultivation of cyanobacteria is described. The problems associated with isolation, purification, and cultivation of soil cyanobacteria, as well as the relevant solutions, are discussed.     

Comparison of Blue Nucleic Acid Dyes for Flow Cytometric Enumeration of Bacteria in Aquatic Systems

Seven blue nucleic acid dyes from Molecular Probes Inc. (SYTO-9, SYTO-11, SYTO-13, SYTO-16, SYTO-BC, SYBR-I and SYBR-II) were compared with the DAPI (4′,6-diamidino-2-phenylindole) method for flow cytometric enumeration of live and fixed bacteria in aquatic systems. It was shown that SYBR-II and SYTO-9 are the most appropriate dyes for bacterial enumeration in nonsaline waters and can be applied to both live and dead bacteria. The fluorescence signal/noise ratio was improved when SYTO-9 was used to stain living bacteria in nonsaline waters. Inversely, SYBR-II is more appropriate than SYTO dyes for bacterial enumeration of unfixed and fixed seawater samples.     

盐度对稀释平板法研究红树林区土壤微生物数量的影响

在使用稀释平板法分离潮间带红树林及其对照光滩土壤微生物以及计数时,多数情况下使用陈海水制作培养基和稀释水,很少考虑培养基和稀释水的盐度对最终计数结果的影响.使用稀释平板法研究了盐度对福建九龙江口红树林区与深圳福田红树林保护区土壤微生物平板计数的影响,结果表明培养基与稀释水盐度对微生物数量有明显的影响.统计分析显示细菌的海水稀释效果优于淡水,而放线菌与真菌则刚好相反(P<0.05,一个例外).海水不适合配制红树林区土壤微生物平板计数的培养基,从0～35,高盐度的平板培养基会降低微生物的数量,尤其是放线菌的数量,尽管培养基的盐度对真菌影响无规律,但细菌数量在低盐度时比在高盐度和不加氯化钠时要多.根据盐度效应,提出了稀释平板技术应用于潮间带的红树林及其相应光滩时的优化方法,认为细菌应该用海水作无菌稀释水,而放线菌和真菌则应用淡水作稀释水;包括光滩在内的红树林区土壤微生物分离与计数的培养基宜控制较低盐度范围.     

Flow cytometry sorting of freshwater phytoplankton

We used the flow sorting capacities of a benchtop FACSCalibur flow cytometer to analyze the phytoplankton community of four different aquatic ecosystems. We show that despite the high optical, mechanistic, and hydrodynamic stress for the cells while sorted, most of the targeted populations could be isolated and grew in mixed culture media subsequent to sorting. Forty-five phytoplankton taxa were isolated, including green algae (29 species), cyanobacteria (eight), diatoms (seven), and cryptomonads (one). The isolation success average was high since 80% of the total sorted populations grew successfully and 47% constituted monocultures. It is noteworthy, however, that some groups could not be isolated, as for example colonial cyanobacteria, chrysophytes, euglenophytes, desmids, or dinoflagellates, and some species such as Cryptomonas sp. were very sensitive to the sorting process. It is proposed that flow cytometric analysis of freshwater phytoplankton might be a relevant tool for water managers and could be applied in some specific cases, such as early monitoring of blooming taxa or basic bio-monitorings of key species. The higher isolation average obtained from the flow sorting can also be powerful for the physiological or molecular study of some taxa after their cultivation.     

Evaluation of culture media for recovery of Staphylococcus aureus from swimming pools.

Several selective media were evaluated for the primary isolation and enumeration of Staphylococcus aureus from halogenated indoor swimming pool waters. Standard plate counts of the viable population and total coliform densities were also determined to ascertain their value as indicator systems. All studies were done with membrane filters. The most selective, accurate, and reliable medium was Vogel-Johnson (VJ) medium supplemented with 0.5% pyruvate. This medium recovered two times more typical colonies than VJ medium alone, and subsequent identification of these well-defined black colonies proved that approximately 80% were S. aureus. The S. aureus recoveries correlated well with halogen levels and bather density use also. In contrast, VJ medium alone was 60% selective for S. aureus, and VJ medium supplemented with catalase did not increase either the percent recovery or the selectivity over that of VJ medium alone. Standard plate counts did not correlate with halogen levels, bather density, or total viable colonies. Coliforms were rarely recovered from indoor pool waters and were not considered to be useful indicators of water quality.     

Evaluation of culture media for recovery of Staphylococcus aureus from swimming pools

Several selective media were evaluated for the primary isolation and enumeration of Staphylococcus aureus from halogenated indoor swimming pool waters. Standard plate counts of the viable population and total coliform densities were also determined to ascertain their value as indicator systems. All studies were done with membrane filters. The most selective, accurate, and reliable medium was Vogel-Johnson (VJ) medium supplemented with 0.5% pyruvate. This medium recovered two times more typical colonies than VJ medium alone, and subsequent identification of these well-defined black colonies proved that approximately 80% were S. aureus. The S. aureus recoveries correlated well with halogen levels and bather density use also. In contrast, VJ medium alone was 60% selective for S. aureus, and VJ medium supplemented with catalase did not increase either the percent recovery or the selectivity over that of VJ medium alone. Standard plate counts did not correlate with halogen levels, bather density, or total viable colonies. Coliforms were rarely recovered from indoor pool waters and were not considered to be useful indicators of water quality.     

Identification and Enumeration of Marine Chroococcoid Cyanobacteria by Immunofluorescence

We used an indirect immunofluorescence technique to permit the identification and enumeration of individual or closely related strains of chroococcoid cyanobacteria of the general Synechococcus and Synechocystis in natural seawater samples. Antisera directed against each of five strains (two phycoerythrin-containing Synechococcus strains, two phycocyanin-containing Synechococcus strains, and one Synechocystis strain) were produced and tested for cross-reactions with cultures of a variety of cyanobacteria and representatives of other algae and bacteria. Each antiserum was relatively specific. The observed cross-reactions occurred between strains that were isolated from similar oceanic environments. We were able, therefore, to apply this technique to field samples. Preliminary results for April to December 1982 in Great South Bay, New York, show that Synechocystis populations are present only during spring and summer, phycocyanin-containing Synechococcus strains are only a minor component in the spring and summer, and phycoerythrin-containing Synechococcus populations become significant in summer and remain so until late fall or winter.     

An assessment of three selective media for bifidobacteria in faeces

Three previously described media enumerating Bifidobacterium spp. in faeces were compared with respect to their selectivity and quantitative recovery. The results of this study indicate that of the three media studied, Beerens'agar is the most suitable medium for isolation and enumeration of Bifidobacterium spp. from the gut microflora.     

Automation of species-specific cyanobacteria phycocyanin fluorescence compensation using machine learning classification

High-frequency cyanobacteria monitoring often uses in-situ fluorescence of phycocyanin (f-PC). However, f-PC must be calibrated for the dominant cyanobacteria species, and it cannot distinguish cyanobacteria taxa, which relies on conventional time-consuming cyanobacteria identification methods. This study proposes a framework to automate f-PC species-specific compensation through three components: (1) prediction of the dominant cyanobacteria species using data-driven models and routine environmental monitoring data; (2) determination of species-specific f-PC per biomass in controlled laboratory experiments; and (3) automation of f-PC species compensation. The framework was validated by applying it to Myponga drinking water reservoir in South Australia. Three machine learning techniques using only high-frequency water temperature data were compared to predict the dominant cyanobacteria species. The framework application to Myponga drinking water reservoir improved the agreement of f-PC with conventional cyanobacteria biovolume measurements, and provided rapid, low-cost identification of the dominant cyanobacteria species, which can support proactive species-targeted cyanobacteria management.     

Comparing recovering efficiency of immunomagnetic separation and centrifugation of mycobacteria in metalworking fluids

The accurate detection and enumeration of Mycobacterium immunogenum in metalworking fluids (MWFs) is imperative from an occupational health and industrial fluids management perspective. We report here a comparison of immunomagnetic separation (IMS) coupled to flow-cytometric enumeration, with traditional centrifugation techniques for mycobacteria in a semisynthetic MWF. This immunolabeling involves the coating of laboratory-synthesized nanometer-scale magnetic particles with protein A, to conjugate a primary antibody (Ab), specific to Mycobacterium spp. By using magnetic separation and flow-cytometric quantification, this approach enabled much higher recovery efficiency and fluorescent light intensities in comparison to the widely applied centrifugation technique. This IMS technique increased the cell recovery efficiency by one order of magnitude, and improved the fluorescence intensity of the secondary Ab conjugate by 2-fold, as compared with traditional techniques. By employing nanometer-scale magnetic particles, IMS was found to be compatible with flow cytometry (FCM), thereby increasing cell detection and enumeration speed by up to two orders of magnitude over microscopic techniques. Moreover, the use of primary Ab conjugated magnetic nanoparticles showed better correlation between epifluorescent microscopy counts and FCM analysis than that achieved using traditional centrifugation techniques. The results strongly support the applicability of the flow-cytometric IMS for microbial detection in complex matrices.     

Membrane filtration of dairy products for microbiological analysis

Incubation with protease or Tween 80 or both dramatically improved the membrane filterability of liquid milks, powdered skim milk, and frozen dairy products without reducing the viability of five common species of bacteria. The technique can permit isolation and enumeration of microorganisms from test samples of these foods as large as 5 g. Flow direction through the filter was an important factor in filterability of dairy products.     

Membrane filtration of dairy products for microbiological analysis.

Incubation with protease or Tween 80 or both dramatically improved the membrane filterability of liquid milks, powdered skim milk, and frozen dairy products without reducing the viability of five common species of bacteria. The technique can permit isolation and enumeration of microorganisms from test samples of these foods as large as 5 g. Flow direction through the filter was an important factor in filterability of dairy products.     

Virus and prokaryote enumeration from planktonic aquatic environments by epifluorescence microscopy with SYBR Green I

Viruses are the most abundant biological entities in aquatic environments, typically exceeding the abundance of bacteria by an order of magnitude. The reliable enumeration of virus-like particles in marine microbiological investigations is a key measurement parameter. Although the size of typical marine viruses (20-200 nm) is too small to permit the resolution of details by light microscopy, such viruses can be visualized by epifluorescence microscopy if stained brightly. This can be achieved using the sensitive DNA dye SYBR Green I (Molecular Probes-Invitrogen). The method relies on simple vacuum filtration to capture viruses on a 0.02-microm aluminum oxide filter, and subsequent staining and mounting to prepare slides. Virus-like particles are brightly stained and easily observed for enumeration, and prokaryotic cells can easily be counted on the same slides. The protocol provides an inexpensive, rapid (30 min) and reliable technique for obtaining counts of viruses and prokaryotes simultaneously.     

Comparing recovering efficiency of immunomagnetic separation and centrifugation of mycobacteria in metalworking fluids

The accurate detection and enumeration of Mycobacterium immunogenum in metalworking fluids (MWFs) is imperative from an occupational health and industrial fluids management perspective. We report here a comparison of immunomagnetic separation (IMS) coupled to flow-cytometric enumeration, with traditional centrifugation techniques for mycobacteria in a semisynthetic MWF. This immunolabeling involves the coating of laboratory-synthesized nanometer-scale magnetic particles with protein A, to conjugate a primary antibody (Ab), specific to Mycobacterium spp. By using magnetic separation and flow-cytometric quantification, this approach enabled much higher recovery efficiency and fluorescent light intensities in comparison to the widely applied centrifugation technique. This IMS technique increased the cell recovery efficiency by one order of magnitude, and improved the fluorescence intensity of the secondary Ab conjugate by 2-fold, as compared with traditional techniques. By employing nanometer-scale magnetic particles, IMS was found to be compatible with flow cytometry (FCM), thereby increasing cell detection and enumeration speed by up to two orders of magnitude over microscopic techniques. Moreover, the use of primary Ab conjugated magnetic nanoparticles showed better correlation between epifluorescent microscopy counts and FCM analysis than that achieved using traditional centrifugation techniques. The results strongly support the applicability of the flow-cytometric IMS for microbial detection in complex matrices.