Epifluorescence microscope methods for bacterial enumeration in a 4-chlorophenol degrading consortium

Epifluorescence microscope methods, namely BacLight, direct epifluorescence filter technique and Rhodamine 123, consistently underestimated plate bacterial counts in a 4-chlorophenol degrading consortium. Cells capable of passing through 0.2 microm filters, referred as 'ultramicrocells', were found. Although cell counts were higher when traditional methods were used, BacLight and direct epifluorescence filter technique were convenient techniques for the systematic monitoring of bacteria involved in biodegradation processes, as results were consistent and available within a short time.     

Use of nitrocellulose Synpor filters for counting soil bacteria by epifluorescence microscopy

A modified acridine orange staining method for estimating soil bacterial numbers by epifluorescence microscopy using Synpor filters (VCHZ Synthesia, Czechoslovakia) was elaborated. Comparing with the method of direct count of soil bacteria estimated in a Bürker chamber higher counts of bacteria and a lower variation of results were obtained. To verify the sensitivity of the method, microflora from various soil horizons was tested.     

Enumerating bacterial cells on bioadhesive coated slides

Quantifying bacterial abundance and biomass is fundamental to many microbiological studies. Directly counting via epifluorescence microscopy has become the method of choice, especially for environmental samples, and conventional techniques require filtration of cells onto black polycarbonate membrane filters. We investigated the utility of instead capturing stained bacterial suspensions on bioadhesive slides, performing tests using pure cultures of bacteria, mixtures of cultured bacteria, and environmental samples from five habitat types. When compared to the standard filtration and flow cytometric approaches, bioadhesive slides were found to be an accurate and precise platform for rapid enumeration of bacteria. Total bacterial counts made using the three methods were positively correlated for acridine orange and Live/Dead® (L/D) staining (0.81 ≤ r ≤ 0.95, all p ≤ 0.002). All platforms had similar precision, though counts obtained using bioadhesive slides were significantly higher than those made with polycarbonate filters and flow cytometry. The specific bioadhesive slides we used resulted in substantial cell mortality for certain pure cultures and river water samples, limiting their use for L/D determination. Cell enumeration using bioadhesive slides is particularly effective because it is highly precise at a wide range of cell concentrations, allows observation of cells that are not readily discernible on filters, reduces the number of steps and processing materials associated with sample analysis, and increases throughput.     

An Investigation of Errors in Direct Counts of Aquatic Bacteria by Epifluorescence Microscopy, with Reference to a New Method for Dyeing Membrane Filters

A number of methods for observing freshwater bacteria by epifluorescence (incident light fluorescence) microscopy are examined. The suitability of each method for quantitative studies using black membrane filters is assessed. In spite of inadequacies it was considered that the use of acridine-based fluorochromes provided the best available estimate of the bacterial population. The errors which may arise when these stains are used were examined and it was noted that small changes in methodology could cause significant differences in the results obtained. The largest errors were associated with changes in volume of sample filtered and the pore size of the membrane used. A procedure for sample treatment is suggested and a new method for dyeing membrane filters is given which allows the use of 0·22-μm pore size membranes of the cellulose ester and polycarbonate type.     

Comparative study of the abundance of various bacterial morphotypes in an eutrophic freshwater environment determined by AODC and TEM

Transmission electron microscopy (TEM) and epifluorescence microscopy were used to obtain comparative measurements of total bacterial counts, and to enumerate abundances of various bacterial morphotypes in an eutrophic freshwater habitat. Although particulate matter would have been expected to interfere with counting by obscuring large areas of the electron microscope grids, estimates of total bacterial abundance made by TEM were on average 1.2 times greater than those obtained using the acridine orange direct counting method (AODC). However, the precision of the AODC method was greater than that for TEM, with a coefficient of variation (C.V.) of 4.0% versus 8.8%, respectively. The total bacterial abundance ranged from 1.1 to 3.2 x 10(6) ml(-1). As was the case for total bacterial density, the numbers of rod- and vibrio-shaped cells were lower when counted in the epifluorescence microscope, indicating the presence of potential starvation forms or ultramicrobacteria. Greatest variations in counts made by TEM and AODC were found for filamentous and coccoid bacteria. Counts of filamentous bacteria made by AODC were only about half of those detected by TEM. In contrast, cocci were on average 1.5 times greater when counted by AODC compared to TEM estimates. Both counting differences were probably caused by the morphology and low density of filamentous and coccoid bacteria (1.7 and 1.4 x 10(5) ml(-1), respectively), which led to an uneven distribution on polycarbonate filters as well as on electron microscope grids. Besides, cocci might easily be mistaken for large viral particles when counted by AODC. Hence, the study supports the use of TEM over AODC for obtaining accurate estimates of total bacterial abundance and especially bacterial morphotypes in natural waters.     

Utility of Green Fluorescent Nucleic Acid Dyes and Aluminum Oxide Membrane Filters for Rapid Epifluorescence Enumeration of Soil and Sediment Bacteria

High background fluorescence and unspecific staining hampered the epifluorescence enumeration of bacteria in 45% of the tested soil and sediment samples with 4′,6-diamidino-2-phenylindole (DAPI) and polycarbonate membrane filters. These problems of the determination of total cell counts can be circumvented by using green fluorescent high-affinity nucleic acid dyes and aluminum oxide membrane filters. Due to the bright staining of cells, we recommend SYBR Green II as dye.     

Determination of bacterial number and biomass in the marine environment.

Three techniques for the measurement of bacterial numbers and biomass in the marine environment are described. Two are direct methods for counting bacteria. The first employs an epifluorescence microscope to view bacteria that have been concentrated on membrane filters and stained with acridine orange. The second uses a transmission electron microscope for observing replicas of bacteria that are concentrated on membrane filters. The other technique uses Limulus amebocyte lysate, an aqueous extract from the amebocytes of the horseshoe crab, Limulus polyphemus, to quantitate lipopolysaccharide (LPS) in seawater samples. The biomass of gram-negative (LPS containing) bacteria was shown to be related to the LPS content of the samples. A factor of 6.35 was determined for converting LPS to bacterial carbon.     

Determination of bacterial number and biomass in the marine environment.

Three techniques for the measurement of bacterial numbers and biomass in the marine environment are described. Two are direct methods for counting bacteria. The first employs an epifluorescence microscope to view bacteria that have been concentrated on membrane filters and stained with acridine orange. The second uses a transmission electron microscope for observing replicas of bacteria that are concentrated on membrane filters. The other technique uses Limulus amebocyte lysate, an aqueous extract from the amebocytes of the horseshoe crab, Limulus polyphemus, to quantitate lipopolysaccharide (LPS) in seawater samples. The biomass of gram-negative (LPS containing) bacteria was shown to be related to the LPS content of the samples. A factor of 6.35 was determined for converting LPS to bacterial carbon.     

Use of Inorganic Membrane Filters (Anopore) for Epifluorescence and Scanning Electron Microscopy of Nanoplankton and Picoplankton

Inorganic membrane filters (Anopore) were examined qualitatively by epifluorescence and scanning electron microscopy to determine their suitability for the study of nanoplankton and picoplankton. Compared with filters currently used, the Anopore filters allowed for increased resolution of the specimen with epifluorescence microscopy because of filter flatness and increased illumination caused by the large number of pores cm^-2. The inorganic filters had a lower filtration rate than polycarbonate filters. For scanning electron microscopy, the metal oxide (Anopore) filters were efficient support for the plankton, with little charging of cells or background.     

THE FAILURE OF PHENOL TREATED ESCHERICHIA COLI TO GROW ON MEMBRANE FILTERS

SUMMARY: Counts of Escherichia coli were done on nutrient agar (control), on membrane filters on nutrient agar and on membrane filters on filter paper pads. With untreated bacteria counts were similar under all conditions, though membrane filters on nutrient agar tended to give slightly low counts. Phenol treated bacteria gave much lower counts when membrane filters were used: the mean counts for 3 strains of the test organism with filters on nutrient agar varied from 35–65% of the control, while counts with filters on filter paper pads were somewhat lower, varying from 30–47% of the control. The low counts on membrane filters on filter paper pads were not due to adsorption of phenol by the filters or to a low concentration of nutrients in the growth medium.     

Comparison of Autoclave and Ethylene Oxide-Sterilized Membrane Filters Used in Water Quality Studies

Autoclave and ethylene oxide-sterilized membrane filters manufactured by Gelman, Millipore, and Sartorius were field tested for their recovery of total coliforms, fecal coliforms, fecal streptococci, and heterotrophs. The data were analyzed by using split-plot analysis of variance and significance tests. Membranes were also tested for pH and toxicity using Escherichia coli. The mean data summaries indicated that Gelman membrane filters generally produced the highest counts during the field studies. Statistical analyses of the March data showed that there were significant differences between membrane filters at 1% level; however, statistical analyses of June data revealed no significant differences except in total coliform recoveries. Toxicity tests at 35 C indicated that Gelman and Millipore autoclaved membrane filters were able to recover 92% of the test organisms. Toxicity tests performed at 44.5 C revealed that no membranes were able to recover more than 40% of the test organisms. Since differences were found in the ability of the three brands of membrane filters to recover bacteria from natural and controlled sources, membrane filters from different manufacturers cannot be readily interchanged. There is a need for a standardized procedure for testing bacterial recovery by membrane filters.     

Membrane filter staining method: bacterial plate counts in 24 H.

We describe a technique to stain bacterial colonies on membrane filters. The procedure yielded reliable and reproducible bacterial plate counts in 24 h. The procedure can be applied to treated and untreated water samples requiring prompt analysis.     

Membrane filter staining method: bacterial plate counts in 24 H

We describe a technique to stain bacterial colonies on membrane filters. The procedure yielded reliable and reproducible bacterial plate counts in 24 h. The procedure can be applied to treated and untreated water samples requiring prompt analysis.     

Relationship between bacterial counts and endotoxin concentrations in the air of wastewater treatment plants.

The relationship between bacterial counts and endotoxin concentrations in air samples was studied. Selective EMB medium favored the growth of a larger portion of airborne gram-negative bacteria than LES Endo or MacConkey medium and was a good predictor of the endotoxin levels determined with a chromogenic Limulus assay of the air of wastewater treatment plants. The bacterial counts determined with nonselective media correlated poorly with airborne endotoxin levels; however, R2A medium yielded higher viable bacterial counts than TYG medium. Direct counting by epifluorescence microscopy yielded the highest bacterial counts, but no correlation was obtained between total bacterial counts and endotoxin concentrations.     

Relationship between bacterial counts and endotoxin concentrations in the air of wastewater treatment plants.

The relationship between bacterial counts and endotoxin concentrations in air samples was studied. Selective EMB medium favored the growth of a larger portion of airborne gram-negative bacteria than LES Endo or MacConkey medium and was a good predictor of the endotoxin levels determined with a chromogenic Limulus assay of the air of wastewater treatment plants. The bacterial counts determined with nonselective media correlated poorly with airborne endotoxin levels; however, R2A medium yielded higher viable bacterial counts than TYG medium. Direct counting by epifluorescence microscopy yielded the highest bacterial counts, but no correlation was obtained between total bacterial counts and endotoxin concentrations.     

Fluorometric determination of the DNA concentration in municipal drinking water

DNA concentrations in municipal drinking water samples were measured by fluorometry, using Hoechst 33258 fluorochrome. The concentration, extraction, and detection methods used were adapted from existing techniques. The method is reproducible, fast, accurate, and simple. The amounts of DNA per cell for five different bacterial isolates obtained from drinking water samples were determined by measuring DNA concentration and total cell concentration (acridine orange epifluorescence direct cell counting) in stationary pure cultures. The relationship between DNA concentration and epifluorescence total direct cell concentration in 11 different drinking water samples was linear and positive; the amounts of DNA per cell in these samples did not differ significantly from the amounts in pure culture isolates. We found significant linear correlations between DNA concentration and colony-forming unit concentration, as well as between epifluorescence direct cell counts and colony-forming unit concentration. DNA concentration measurements of municipal drinking water samples appear to monitor changes in bacteriological quality at least as well as total heterotrophic plate counting and epifluorescence direct cell counting.     

Fluorometric determination of the DNA concentration in municipal drinking water.

DNA concentrations in municipal drinking water samples were measured by fluorometry, using Hoechst 33258 fluorochrome. The concentration, extraction, and detection methods used were adapted from existing techniques. The method is reproducible, fast, accurate, and simple. The amounts of DNA per cell for five different bacterial isolates obtained from drinking water samples were determined by measuring DNA concentration and total cell concentration (acridine orange epifluorescence direct cell counting) in stationary pure cultures. The relationship between DNA concentration and epifluorescence total direct cell concentration in 11 different drinking water samples was linear and positive; the amounts of DNA per cell in these samples did not differ significantly from the amounts in pure culture isolates. We found significant linear correlations between DNA concentration and colony-forming unit concentration, as well as between epifluorescence direct cell counts and colony-forming unit concentration. DNA concentration measurements of municipal drinking water samples appear to monitor changes in bacteriological quality at least as well as total heterotrophic plate counting and epifluorescence direct cell counting.     

Resuscitation and quantification of stressed Escherichia coli K12 NCTC8797 in water samples

The aim of this study was to investigate the impact on numbers of using different media for the enumeration of Escherichia coli subjected to stress, and to evaluate the use of different resuscitation methods on bacterial numbers. E. coli was subjected to heat stress by exposure to 55 degrees C for 1h or to light-induced oxidative stress by exposure to artificial light for up to 8h in the presence of methylene blue. In both cases, the bacterial counts on selective media were below the limits of detection whereas on non-selective media colonies were still produced. After resuscitation in non-selective media, using a multi-well MPN resuscitation method or resuscitation on membrane filters, the bacterial counts on selective media matched those on non-selective media. Heat and light stress can affect the ability of E. coli to grow on selective media essential for the enumeration as indicator bacteria. A resuscitation method is essential for the recovery of these stressed bacteria in order to avoid underestimation of indicator bacteria numbers in water. There was no difference in resuscitation efficiency using the membrane filter and multi-well MPN methods. This study emphasises the need to use a resuscitation method if the numbers of indicator bacteria in water samples are not to be underestimated. False-negative results in the analysis of drinking water or natural bathing waters could have profound health effects.     

A note on 'plotless' methods for estimating bacterial cell densities

R oser , D., N edwell , D.B. & G ordon , A. 1984. A note on "plotless" methods for estimating bacterial cell densities. Journal of Applied Bacteriology 56 , 343–347.
'Plotless' techniques for determining population densities have been developed for, and applied to, higher plant populations. They can often be carried out more rapidly than techniques involving total counts of individuals in plots, or quadrants, but such plotless techniques have not been generally applied to the estimation of densities of bacterial cells. Direct microscopical counting of cell numbers in a field of view, an example of a plot-related method, has been traditionally used for micro-bial cell counts. In this study 'plot' and 'plotless' methods on a variety of bacterial samples are compared. Estimates of bacterial cell density were obtained by measuring the distance of cells from a fixed point in a field of view. The values, which were more rapidly obtained, were directly correlated with total cell counts. Although there was some apparent deviation from a perfect 1:1 relationship with total counts, as indicated by a correlation coefficient less than 1.0, there were no significant differences between the replicated counts of bacteria on samples of tissue from the surface of Hypholoma basidiocarps ( P < 0.05). This indicated that the methods of enumeration were comparable. The distance-related estimates could readily be obtained from fields of view with cell densities varying over several orders of magnitude. It was more rapidly applied, particularly at high density, and the method was applicable not only to random cell distributions but also to the non-random distributions encountered when microbial cells aggregated into micro-colonies. The method appears to be particularly well-suited for automated, digitized, direct counting procedures, as well as to estimating bacterial numbers on membrane filters and natural substrates.     

Comparison of Gelman and Millipore Membrane Filters for Enumerating Fecal Coliform Bacteria

Tests of two leading brands of membrane filters used for enumerating fecal coliform bacteria showed that Gelman GN-6 filters recovered statistically more colonies of bacteria than did Millipore HAWG 047SO filters from pure cultures incubated at either 35 C (the optimal growth temperature) or 44.5 C (the standard temperature for the fecal coliform test). Standard membrane filter procedures with M-FC broth base were used to enumerate the organisms. Densities of colonies incubated on Gelman filters at 44.5 C averaged 2.3 times greater than those on Millipore filters. Plate counts of the bacteria at both temperatures indicated that incubation at 44.5 C did not inhibit propagation of fecal coliform bacteria. For the pour plates, M-FC broth base plus 1.5% agar was used. This modified medium compared favorably to plate count agar for enumerating Escherichia coli. At 35 and 44.5 C, colony counts on Gelman filters agreed closely with plate counts prepared concurrently, but Millipore counts were consistently lower than plate counts, especially at 44.5 C. Comparative analyses of river water for fecal coliform bacteria by the membrane filter technique gave results comparable to those for the pure cultures.