Use of fluorochromes for direct enumeration of total bacteria in environmental samples: past and present.

Understanding the role of bacteria in microbial food webs is intimately connected to the methods applied in the direct enumeration of bacteria. We have examined over 220 papers describing studies in which fluorochrome staining followed by epifluorescent microscopic direct counts was used to estimate total bacterial abundances. In this review, we summarize patterns in the use of 3,6-bis[dimethylamino]acridinium chloride (acridine orange) and 4',6-diamidino-2-phenylindole (DAPI), the two stains most frequently used in bacterial enumeration. The staining of samples with these fluorochromes, followed by filtration and direct counting of bacterial cells on filter surfaces, has become routine over the past 10 years. We examine trends in features of the standard direct count methods, such as sample preservation and preparation techniques, membrane filter types used, applied stain concentrations, duration of staining, and counting strategies, in relation to the types of samples being examined. The high variability in bacterial counts observed within similar sample types may be partially accounted for by differences in methods. Synthesizing review findings, we include a recommended method for the direct enumeration of bacteria in environmental samples.     

Enumeration of heterotrophs, fecal coliforms and Escherichia coli in water: comparison of 3M Petrifilm plates with standard plating procedures

A total of 177 naturally contaminated water samples were analyzed by membrane filtration according to the Standard Methods for the Examination of Water and Wastewater published by the American Public Health Association. Filters were incubated in parallel on mHPC-agar and 3M™ Petrifilm™ Aerobic Count Plates (Petrifilm™ AC plates) for heterotrophic counts. Fecal coliforms and Escherichia coli were enumerated on mFC-agar and 3M™ Petrifilm™ E. coli/Coliform Count Plates (Petrifilm™ EC plates). Typical colonies on each media type were confirmed following standard procedures. Heterotrophic counts were between 10³ and 10⁴ CFU/mL and the average log₁₀ counts obtained on Petrifilm™ AC plates were about two-fold lower than on mHPC-agar. Counts for fecal coliforms and E. coli were between 10² and 10³ CFU/mL. Average log₁₀ counts for confirmed fecal coliforms obtained on Petrifilm™ EC plates were slightly lower than on mFC agar with a correlation coefficient of 0.949. The average log₁₀ counts for confirmed E. coli on Petrifilm™ EC plates and on mFC agar were statistically not different (P=0.126) with a correlation coefficient of 0.879. Specificity of Petrifilm™ EC plates and mFC agar was evaluated by comparing typical colony counts with confirmed counts. On mFC agar, counts for typical colonies were by 2 log₁₀ CFU higher than the actual confirmed counts. In contrast, on Petrifilm™ EC plates typical colony counts were almost identical to confirmed colony counts for both fecal coliforms and E. coli. This comparison illustrates the high specificity of Petrifilm™ EC plates for enumeration of both fecal coliforms and E. coli in water.     

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.     

Application of laser scanning for the rapid and automated detection of bacteria in water samples

It is widely accepted that the heterotrophic plate count method may not support the growth of all viable bacteria which may be present within a water sample and that alternative procedures using 'viability markers' may yield additional information. In this study, ChemChrome B (CB), which is converted to a fluorescent product by esterase activity, was used to stain viable bacteria (captured by membrane filtration) from potable water samples. The labelled bacteria from each sample were subsequently enumerated using a novel laser scanning instrument (ChemScan). Analysis of 107 potable water samples using this procedure demonstrated the presence of a significantly greater number of bacteria than were detected by culture (z-test, P < 0.05). The mean number of bacteria isolated by culture on R2A agar incubated at 22 degrees C for 7 d was only 25.2% of the total number of viable bacteria detected using the CB/ChemScan viability assay. Further analysis of 81 water samples using a 5-cyano-2,3,4-tolyl-tetrazolium chloride (CTC) viability assay also demonstrated the presence of many viable bacteria which were not capable of growth under the culture conditions employed in this study. However, the results indicate that ChemChrome B has the ability to stain a significantly greater number of heterotrophs than CTC (z-test, P < 0.05). In contrast, six potable waters were identified in which the CTC viability assay resulted in counts greater than those obtained using CB. The ChemScan instrument was successfully used for rapid and accurate enumeration of labelled micro-organisms, allowing information on the total viable microbial load of a water sample to be determined within 1 h. Furthermore, the ChemScan system has the potential for use in detecting specific organisms labelled with fluorescently-labelled antibodies or nucleic acid probes.     

A simple filtration device for separating nonadherent microorganisms from mammalian cells in in vitro studies on microbial adherence

In adherence studies, the removal of nonadherent microorganisms is essential for the valid enumeration of microorganisms that adhere to host cells. Although filtration devices are available commercially for the removal of nonadherent microorganisms, these are expensive and not reusable. In this article, we describe a simple, inexpensive, and reusable filtration device composed of two chambers of nylon, a nylon membrane of desired pore size, a rubber washer, and supporting stainless steel mesh. The device was effective in in vitro adherence assays for removing nonadherent endospores of Rhinosporidium seeberi from human buccal epithelial cells, providing valid counts of adherent microorganisms.     

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.     

Comparison of gauze swabs and membrane filters for isolation of Campylobacter spp. from surface water

The epidemiology of Campylobacter jejuni indicates that waterborne transmission is important; the organism has been isolated from seawater, fresh water, and estuarine sites. Membrane filtration, with and without use of an enrichment broth, has been the most common method for isolating C. jejuni from water. We evaluated two methods for isolating C. jejuni from water: membrane filtration and gauze filtration. The membrane filters evaluated included 0.22- and 0.45-micron-pore Millipore filters (Millipore Corp., Bedford, Mass.), 0.2- and 0.4-micron-pore Nuclepore filters (Nucleopore Corp., Pleasanton, Calif.), and a 0.45-micron-pore Zetapor filters (AMF Cuno, Meridian, Conn.). The gauze filters included both Moore and Spira swabs. Of the membrane filters evaluated, the 0.45-micron-pore Millipore and Zetapor filters were the most sensitive for recovery of C. jejuni from seeded waters. The 0.45-micron-pore Millipore filter placed in Oosterom broth was better for recovery of C. jejuni from seeded stationary surface waters than either the Spira or Moore swab. However, the 0.45-micron-pore Millipore filter placed on a plate or in enrichment broth was equivalent to the Spira gauze swab when used to examine water from Atlanta area streams. C. jejuni organisms were isolated from 9 of 24 surface water samples representing 5 of 12 streams.     

Comparison of the hydrophobic-grid membrane filter procedure and standard methods for coliform analysis of water

The hydrophobic-grid membrane filter (HGMF) has been proposed as an alternate method to the standard membrane filter (MF) procedure for the detection and enumeration of coliforms from water. Eight samples of nonchlorinated wastewater effluents were analyzed by the HGMF, standard MF, and tube fermentation most-probable-number methods for fecal coliforms, and eight samples each of polluted surface and dosed drinking waters were analyzed by the same methods for total coliforms. The drinking waters were dosed with coliforms and other heterotrophs concentrated from nonchlorinated domestic wastewater and treated with chlorine to reduce the numbers of organisms and simulate stress caused by chlorination. Statistical analyses determined that recoveries of fecal coliforms were significantly higher by the filtration methods for the nonchlorinated domestic wastewaters but not for the other waters. The results also indicated that recoveries of fecal and total coliforms did not differ significantly when either MFs or HGMFs were used. Total coliform results obtained with HGMFs having greater than 100 positive grid cells were significantly more precise than estimates obtained by the standard MF method only for polluted surface waters.     

Comparison of the hydrophobic-grid membrane filter procedure and standard methods for coliform analysis of water.

The hydrophobic-grid membrane filter (HGMF) has been proposed as an alternate method to the standard membrane filter (MF) procedure for the detection and enumeration of coliforms from water. Eight samples of nonchlorinated wastewater effluents were analyzed by the HGMF, standard MF, and tube fermentation most-probable-number methods for fecal coliforms, and eight samples each of polluted surface and dosed drinking waters were analyzed by the same methods for total coliforms. The drinking waters were dosed with coliforms and other heterotrophs concentrated from nonchlorinated domestic wastewater and treated with chlorine to reduce the numbers of organisms and simulate stress caused by chlorination. Statistical analyses determined that recoveries of fecal coliforms were significantly higher by the filtration methods for the nonchlorinated domestic wastewaters but not for the other waters. The results also indicated that recoveries of fecal and total coliforms did not differ significantly when either MFs or HGMFs were used. Total coliform results obtained with HGMFs having greater than 100 positive grid cells were significantly more precise than estimates obtained by the standard MF method only for polluted surface waters.     

Raman spectroscopy and chemical imaging for quantification of filtered waterborne bacteria

Rapid and reliable assessment of pathogenic microbial contamination in water is critically important. In the present work we evaluated the suitability of Raman Spectroscopy and Chemical Imaging as enumeration techniques for waterborne pathogens. The prominent C-H stretching band observed between 2800-3000 cm(-1) of the spectrum is used for quantification purposes. This band provides the highest intensity of the bacterial-spectrum bands facilitating the detection of low number of microorganisms. The intensity of the Raman response correlates with number of cells present in drops of sample water on aluminum-coated slides. However, concentration of pathogens in drinking and recreational water is low, requiring a concentration step, i.e., filtering. Subsequent evaluation of filtering approaches for water sampling for Raman detection showed significant background signal from alumina and silver membranes that reduces method sensitivity. Samples concentrated by filtration show good correlation between Raman spectroscopy and other quantification methods including turbidity (R(2)=0.92), plate counts (R(2)=0.87) and dry weight (R(2)=0.97). Background interferences did not allow for evaluation of this relationship at low cell concentrations.     

Comparison of gauze swabs and membrane filters for isolation of Campylobacter spp. from surface water.

The epidemiology of Campylobacter jejuni indicates that waterborne transmission is important; the organism has been isolated from seawater, fresh water, and estuarine sites. Membrane filtration, with and without use of an enrichment broth, has been the most common method for isolating C. jejuni from water. We evaluated two methods for isolating C. jejuni from water: membrane filtration and gauze filtration. The membrane filters evaluated included 0.22- and 0.45-micron-pore Millipore filters (Millipore Corp., Bedford, Mass.), 0.2- and 0.4-micron-pore Nuclepore filters (Nucleopore Corp., Pleasanton, Calif.), and a 0.45-micron-pore Zetapor filters (AMF Cuno, Meridian, Conn.). The gauze filters included both Moore and Spira swabs. Of the membrane filters evaluated, the 0.45-micron-pore Millipore and Zetapor filters were the most sensitive for recovery of C. jejuni from seeded waters. The 0.45-micron-pore Millipore filter placed in Oosterom broth was better for recovery of C. jejuni from seeded stationary surface waters than either the Spira or Moore swab. However, the 0.45-micron-pore Millipore filter placed on a plate or in enrichment broth was equivalent to the Spira gauze swab when used to examine water from Atlanta area streams. C. jejuni organisms were isolated from 9 of 24 surface water samples representing 5 of 12 streams.     

Comparison of m-Endo LES, MacConkey, and Teepol media for membrane filtration counting of total coliform bacteria in water.

Total coliform counts obtained by means of standard membrane filtration techniques, using MacConkey agar, m-Endo LES agar, Teepol agar, and pads saturated with Teepol broth as growth media, were compared. Various combinations of these media were used in tests on 490 samples of river water and city wastewater after different stages of conventional purification and reclamation processes including lime treatment, and filtration, active carbon treatment, ozonation, and chlorination. Endo agar yielded the highest average counts for all these samples. Teepol agar generally had higher counts then Teepol broth, whereas MacConkey agar had the lowest average counts. Identification of 871 positive isolates showed that Aeromonas hydrophila was the species most commonly detected. Species of Escherichia, Citrobacter, Klebsiella, and Enterobacter represented 55% of isolates which conformed to the definition of total coliforms on Endo agar, 54% on Teepol agar, and 45% on MacConkey agar. Selection for species on the media differed considerably. Evaluation of these data and literature on alternative tests, including most probable number methods, indicated that the technique of choice for routine analysis of total coliform bacteria in drinking water is membrane filtration using m-Endo LES agar as growth medium without enrichment procedures or a cytochrome oxidase restriction.     

Factors which Influence the Enumeration of Bdellovibrio bacteriovorus in Sewage and River Water

S ummary . A double layer plating technique was used for counting Bdellovibrio bacteriovorus in river water and sewage samples. The optimum conditions for enumeration were provided by dilute media with 0.6 % of agar as the top layer, incubated for 6 days at 30° with Achromobacter sp. as the host. Pretreatment of samples affected counts: centrifugation and membrane filtration reduced the bdellovibrio count, homogenization increased it. Protozoa in sewage samples caused large plaques that greatly hindered counting and attempts to eliminate these plaques were unsuccessful. The highest counts in sewage samples averaged 900 cells/ml.     

Evaluation of PetrifilmTM methods for enumeration of aerobic flora and coliforms in a wide range of foods

C. DE W. BLACKBURN, C.L. BAYLIS AND S.B. PETITT. 1996. Petrifilm^TM is a ready-to-use alternative to traditional microbial enumeration methods. The Petrifilm^TM Aerobic Count Plate (ACP) and Coliform Count Plate (CCP) were compared with standard methods for the enumeration of the aerobic mesophilic flora and coliform bacteria in 91 foods covering a wide range of different food commodities. There was good correlation between the Petrifilm^TM ACP and the standard aerobic colony count method ( r = 0.989) and between the Petrifilm^TM CCP and the standard Violet Red Bile Agar plating method ( r = 0.872). In both cases, the Petrifilm^TM methods had a better repeatability than the standard methods. The Petrifilm^TM ACP and CCP were shown to be practical and accurate alternatives to standard enumeration methods in a wide range of foods, with benefits of saving time, labour and incubator space.     

Comparison of m-Endo LES, MacConkey, and Teepol media for membrane filtration counting of total coliform bacteria in water.

Total coliform counts obtained by means of standard membrane filtration techniques, using MacConkey agar, m-Endo LES agar, Teepol agar, and pads saturated with Teepol broth as growth media, were compared. Various combinations of these media were used in tests on 490 samples of river water and city wastewater after different stages of conventional purification and reclamation processes including lime treatment, and filtration, active carbon treatment, ozonation, and chlorination. Endo agar yielded the highest average counts for all these samples. Teepol agar generally had higher counts then Teepol broth, whereas MacConkey agar had the lowest average counts. Identification of 871 positive isolates showed that Aeromonas hydrophila was the species most commonly detected. Species of Escherichia, Citrobacter, Klebsiella, and Enterobacter represented 55% of isolates which conformed to the definition of total coliforms on Endo agar, 54% on Teepol agar, and 45% on MacConkey agar. Selection for species on the media differed considerably. Evaluation of these data and literature on alternative tests, including most probable number methods, indicated that the technique of choice for routine analysis of total coliform bacteria in drinking water is membrane filtration using m-Endo LES agar as growth medium without enrichment procedures or a cytochrome oxidase restriction.     

Microbiological Comparisons within and across Contiguous Lacustrine, Paleosol, and Fluvial Subsurface Sediments

Twenty-six subsurface samples were collected from a borehole at depths of 173.3 to 196.8 m in the saturated zone at the Hanford Site in south-central Washington State. The sampling was performed throughout strata that included fine-grained lacustrine (lake) sediments, a paleosol (buried soil) sequence, and coarse-grained fluvial (river) sediments. A subcoring method and tracers were used to minimize and quantify contamination to obtain samples that were representative of subsurface strata. Sediment samples were tested for total organic carbon, inorganic carbon, total microorganisms by direct microscopic counts, culturable aerobic heterotrophs by plate counts, culturable anaerobes by most-probable-number enumeration, basal respiration rates, and mineralization of (sup14)C-labeled glucose and acetate. Total direct microscopic counts of microorganisms were low, ranging from below detection to 1.9 x 10(sup5) cells g (dry weight)(sup-1). Culturable aerobes and anaerobes were below minimum levels of detection in most samples. Direct microscopic counts, basal respiration rates, and (sup14)C-glucose mineralization were all positively correlated with total organic carbon and were highest in the lacustrine sediments. In contrast to previous subsurface studies, these saturated-zone samples did not have higher microbial abundance and activities than unsaturated sediments sampled from the same borehole, the fine-textured lacustrine sediment had higher microbial numbers and activities than the coarse-textured fluvial sands, and the paleosol samples did not have higher biomass and activities relative to the other sediments. The results of this study expand the subsurface microbiology database to include information from an environment very different from those previously studied.     

Some special problems in the determination of viable counts of groundwater microorganisms

Factors affecting viable cell counts in groundwater or sediments were studied with samples from the Segeberg Forest test area in northern Germany. There was very little variation in results with the season (April, August, November) or depth of sampling; generally there were 10³–10⁴ aerobic cells per ml or g sediment. Long incubation times resulted in higher cell counts; groundwater samples required 4–5 weeks, and sediment extracts had to be cultured for 7 weeks. Total cell counts in sediment were 10²–10⁴ cell/g higher than viable cell counts of aerobes. This was explained partly by the additional presence of anaerobes and partly by the observation that some morphotypes may not have grown under our conditions. Viable cell counts were not influenced by cell extraction from the sediment with either Na-pyrophosphate or groundwater extracts. However, iron-precipitating or manganese-oxidizing bacteria were better extracted with sterile groundwater. The microflora of wells was more numerous than that of the free aquifer; consequently it was better to pump off all well water before aquifer water was sampled. The diameter of the well was also important; thinner tubes had higher cell counts than those with wider diameter. For sampling, wells should be at least 1 year old, since young wells contain higher numbers of microorganisms due to underground disturbances from the drilling. Turbid water samples could be clarified by filtration, but this reduced the viable counts by 1–2 orders of magnitude. Two different media inoculated with a sample dilution resulted in the same cell counts, but their microbial diversity was different. Storage of groundwater samples before processing resulted in up to 17-fold increases in cell counts and loss of diversity in the first 24 hours. Cell numbers decreased slowly during longer storage.     

A note on the effect of dilution and temperature on the bactericidal activity of potassium sorbate

O ragui , J.I. M ara , D.D.1984. A note on a modified membrane-Bovis agar for the enumeration of Streptococcus bovis by membrane filtration. Journal of Applied Bacteriology 56 , 179–181.
A modified membrane-Bovis agar, containing a reduced quantity of sodium azide, for the isolation and enumeration of Streptococcus bovis is described and evaluated. Higher counts, with larger colonies, were obtained from water and sewage samples with this medium than with the original formulation.     

Model studies on a membrane filtration method for the enumeration of coagulase-positive staphylococci in swimming-pool water using rabbit plasma-bovine fibrinogen agar

The recovery of Staphylococcus aureus from swimming-pool water by membrane filtration was studied in model experiments. On the nonselective medium tryptone soya agar (TSA) there was no difference in counts of noninjured S. aureus with all membrane filters tested and with pour plates. Chlorine-injured S. aureus was enumerated most efficiently on TSA by Gelman Tuffryn HT-450 and Sartorius SM 13806 filters. Tuffryn filters were also most productive when used in combination with the selective medium rabbit plasma - bovine fibrinogen agar (RPFA). Other filters, particularly Gelman GN-6 and Millipore HAWP, when used on RPFA were shown to have a synergistic inhibitory effect on both noninjured and chlorine-injured S. aureus. This effect was not found on Baird-Parker agar. Using Tuffryn filters, counts on RPFA were equal to those on TSA for noninjured S. aureus and 0.1-2.0 log units less for chlorine-injured S. aureus. Despite this, the possibility for reading the in situ coagulase reaction for individual colonies on RPFA is considered such an advantage of this medium that its general use for enumeration of S. aureus in swimming pools is recommended. Further studies should be carried out to allow better resuscitation.     

Rapid separation and quantitation of mixed microorganisms by filtration and bioluminescence

A membrane filtration/bioluminescence system was developed for the differentiation and quantitation of mixed populations of microorganisms. Samples containing microorganisms were filtered through two membrane filters of descending pore size. The microorganisms retained on the filter contain ATP that can be extracted and measured on the filter via the firefly luciferase-luciferin bioluminescence assay. Results, obtained in less than 20 min, show a good correlation (r greater than or equal to 0.95) between the light produced and the number of organisms in the sample. Using these techniques, Escherichia coli can be separated from yeast or mold and measured in samples containing both microorganisms. When lysostaphin is used to selectively lyse Staphylococci on the filter, the specific quantification of these bacteria among other microorganisms can also be accomplished. The filtration/bioluminescence technique offers the potential of being a rapid and sensitive method to differentiate and detect microorganisms, by selective sizing or lysing, in a variety of samples.