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A novel method for rapid determination of total microbial cell numbers was investigated. The method involves the application of most-probable-number estimation statistics to direct microscopic counting of microbial cells by using a particle sizing graticule. Its accuracy and reliability were tested with computer simulations of bacterial cell distri...
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... assess the effectiveness of our technique on randomly dispersed bacteria, we used it to enumerate cells in Breed smears (1) of contaminated milk samples. The MPN value was calculated simply by counting the total number of fertile quadrats in five fields and applying equation 3 (s = 15, n = 5, a = 2) and the appropriate K values in Table 2. Specimens were stained with Newman stain and observed under transmitted light with an Olympus binocular microscope and a 10Ox oil immersion lens. ...
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... Before the discovery of the 16S rRNA gene as a potential marker for microbial identification, microbiologists were limited by the requirement to isolate microorganisms in pure cultures, in order to subsequently analyze them using various physiological and biochemical traits. Quantification of cells in environmental samples was performed via viable plate counts or "most-probable-number" techniques 95 . These methods are clearly not appropriate for diversity estimation in environmental samples because they tend to select for certain organisms. ...
... Each membrane was mounted in immersion oil and examined with a Nikon Optiphot microscope inco~orating an epi~uore~n~ attachment (100 W Hg lamp; " G " filter set providing green excitation and red emission; non-fluorescing objettives ) at a magnification of 500 or 1250 x . Cell numbers were estimated using a " Most-Probable-Number " technique (Roser et al., 1987). Average cell size was estimated as follows. ...
A range of measures of microbial biomass were compared for their effectiveness and internal consistency in characterizing ornithogenic soils. Excellent correspondences (r = 0.86–0.94, slope = 0.84–1.12) were found between four measures of total biomass (ATP, fluorescein diacetate hydrolysis rate, substrate induced respiration rate and algal + bacterial biomass) when they were compared using regression analysis. Bacterial numbers, estimated from agar plate counts, were found to be highly correlated with microscopy counts and total biomass measurements, though the best-fit relationship was curvilinear rather than linear.Measurement of the procaryotic and eucaryotic components of the soil microbiota by selective antibiotic inhibition was found to be unreliable. Other problems were noted when assay units were converted into microbial C using multiplication factors published in the literature. The substrate-induced-respiration method overestimated the bacterial biomass of occupied penguin colony soil by at least 4-fold when the standard conversion factor (1 mg C = 25μ1CO2 h−1) was used. Total biomass was also overestimated when fluorescein-diacetate-hydrolysis rates were converted to microbial C. While all four approaches could provide relative measures of the biomass of the Antarctic microbiota, it was concluded that they should not be employed in isolation but always in parallel when such soils are initially analysed.
Distribution of airborne microorganisms was determined with two different types of air samplers, the Anderson cascade sampler and the Aerobioscope sampler, in the vicinity of Taejon. The size distribution of particles carrying bacteria and fungi was concurrently measured. The concentration of detected viable airborne particles was greatly varied. It was observed that the number of microbial particles increased in April and October. The most size of particles carrying bacteria was larger than 4.7 μm in mean aerodiameter, which made up 69.8% of the total particle fraction. About 63.2% of fungi-carrying particles were smaller than 4.7 μm in aero-diameter. The distribution of particles on Yellow Sand Phenomena-days was also analyzed. The number of fine particles having mass median aero-diameter from 1.0 to 10 μm increased on Yellow Sand Phenomena days to about 6 times that on normal days and the number of colony forming unit (CFU/m3) of airborne bacteria also increased by 4.3 times in April. The results from the Anderson sampler showed that the concentration of bacteria increased greatly on the fraction of fine particles ranging from 0.6 μm to 4.7 μm in diameter. Unlike the increase in bacterial flora on Yellow Sand Phenomena days, the fungal concentration slightly decreased and showed a normal size distribution pattern. This study suggests that a long-range transmission of bacteria results from bacteria adsorbing onto the fine particles during the Yellow Sand Phenomena.
A marked decrease in the vase life of cut Rosa cultivar ‘Sonia’ was observed when water-washed viable or heat-inactivated cells of either Bacillus subtilis, Enterobacter agglomerans, Pseudomonas fluorescens or P. putida were added to the vase water. The concentrations of viable or heat-killed bacterial cells influenced the vase life, but different bacterial strains exerted similar effects. High initial numbers of bacterial cells added to the vase water reduced the water conductivity, shortened the vase life and delayed the onset of flower-bud development. Similar numbers of viable cells added to the vase water (<107-⩾05cells ml−1) decreased the vase life of the roses more than did heat-inactivated cells. Water-conductivity measurement was a rapid, reproducible method of assessing the effects of bacteria after only 24 h of vase life. Vascular plugging caused by bacterial cells was not the sole cause of decreased water conductivity in the stem segments of the roses.
Direct techniques in microbial ecology are by definition observational, and because microorganisms are very small microscopy is used. This type of approach can be used either qualitatively or quantitatively. As most research relies heavily on quantitative data, only this aspect of direct methods is considered in this chapter. This chapter concentrates on the methods for bacteria, because methods for other microorganisms are different. Epifluorescence microscopy is used for estimating total counts, sizes, biomass, growth rates, and numbers of active bacteria able to grow, take up nutrients, and respire. It can even be combined with immunological approaches to count specific bacterial species. This chapter focuses on the epifluorescence approaches. Quantitative microscopic observation always demands that the organisms are distributed randomly within the sample. Samples from different habitats require different treatment.
Planktonic larval dispersal and recruitment can be major determinants of the structure and dynamics of manne communit~es. However, these processes have been difficult to study because of their natural variability and the limitations of methods used to collect and analyze plankton samples. In part~cular, the use of microscopy to determine the composition of plankton samples is time-consuming and often llmited by a lack of reliable morphological characters for species identification. The need for methods of greater accuracy and efficiency has led to the development of molecular approaches to plankton analysis, including detecbon by DNA hybridization, amplification of DNA from plankton samples by the polymerase chain reaction (PCR) and taxonomic characterization by ribosomal DNA sequence analysis. Here we describe a PCR-based method that detects larval crabs in estuarine plankton samples. This technique is unusually expedent and relatively cost-effective. It is based on the detection of a middle repetitive sequence characteristic of the stone crab Menippe rnercenana, as well as the closely related species M. adina. Amphfication by PCR of a 585 base pair region of this sequence from plankton samples accurately indicates the presence of either species. Because of the high abun-dance of this sequence in the genome of Menippe, single larvae can be detected in typical plankton samples. Unlike methods based on 'universal' sequences (rRNA or regions of the mitochondrial genome), the amplification of a PCR product of the expected size is a reliable indication of the presence of the target species, and no further characterization is necessary. This technique is intended to facili-tate the large-scale process~ng of plankton samples that is necessary for accurate determination of the temporal and spatial distributions of individual species in plankton communities.
A modification of the most-probable-number (MPN) method for the determination of total viable microbial cells was investigated. This alternative method (AMPN) applies MPN statistics to microorganisms growing on solid instead of liquid medium which is used in the conventional method. To verify the validity of the proposed method, numbers obtained with the AMPN were compared to counts from drop-plate (DP) and spread-plate (SP) methods. Correlation and regression analysis showed a positive correlation between the AMPN and the DP and SP methods. Also, the Student t test applied for paired samples showed no difference between the mean values of replicates and the methods tested at the 5% level of significance. The advantages and disadvantages of the alternative method under defined conditions are discussed. Since the results obtained with the AMPN were equivalent to results from DP and SP methods we adopted this system for its reliability and cost-effectiveness.
The microbial biomass present in Windmill Islands soils, under varying degrees of Adélie penguin (Pygoscelis adelie) influence, was estimated using four different approaches. Average concentrations of 1200, 180, and 440μg microbial C g−1 (measured as ATP) were detected in active colonies, extinct colonies and soils possessing visible algal growth respectively. This compared with an average of 12μg C g−1 in control samples from sites remote from penguin colonies. Biomass estimates based on substrate-induced-respiration rates were 1–4 times higher than the equivalent ATP estimates.The soil microbiota of active penguin colonies was dominated by bacteria (92% of the total biomass) with tetrad-forming cocci comprising 35% of the total biovolume. Away from the immediate colony site, algal biomass, measured as chlorophyll a, accounted for 78–96% of the total biomass of carbon while bacterial biomass measured microscopically accounted for 4–22%. Yeasts accounted for < 1% of the total biomass. Too few fungal hyphae were detected to make useful direct counts and their biomass appeared to be less than for yeast-like forms. Increasing algal predominance corresponded with decreasing soil pH and a 10,000-fold increase in fungal biomass compared to bacterial microbiota, as measured by colony counts.
