Comparison of two direct-count techniques for enumerating aquatic bacteria.

Planktonic bacteria from an estuary were concentrated on membrane filters and counted with both a scanning electron microscope and an epi-illuminated fluorescent microscope. Counts on 0.2 micron Nuclepore filters (polycarbonate) were significantly higher (P less than 0.001) than counts on 0.2-micron Sartorius filters (cellulose). In contrast, there was not a statistically significant difference between the two techniques when Nuclepore filters were used (0.5 less than P less than 0.9). The average cell volume from this study area was 0.047 micron3. The estimated number of bacteria ranged from 10(6) to 10(7) bacteria per ml, representing from 4 to 40 mg of C per m3.     

Collection of airborne micro-organisms on Nuclepore filters, estimation and analysis--CAMNEA method

The total number of airborne micro-organisms collected on Nuclepore filters was determined by acridine orange staining and epifluorescence microscopy. The viable fraction of the total numbers varied significantly when actinomycete and fungal spores from different environments were stored on the filter surface for 1 week, although the microflora composition was not altered. A high correlation between viable and total counts was noted in environments where the airborne flora was dominated by fungal spores, while a low correlation was found for airborne bacteria. Peak values of the total counts registered in some work environments varied between 10(7) and 10(11) micro-organisms/m3. Size analysis showed a dominating fraction of respirable micro-organisms (aerodynamical diameter less than 5 micron). The investigation shows that it is of the utmost importance to combine viable counts with total count enumeration in the study of exposure to micro-organisms in work-related situations.     

Rapid and simple method for double staining of bacteria with 4',6-diamidino-2-phenylindole and fluorescein isothiocyanate-labeled antibodies.

Bacteria were either heat fixed on microscope slides or filtered with 0.2 micron-pore-size Nuclepore filters. The samples were stained with 4',6-diamidino-2-phenylindole (DAPI) for total staining and with polyvalent rabbit antibodies and fluorescein isothiocyanate-conjugated swine anti-rabbit antibodies for specific staining. By switching between two different optical filter packages in the microscope, only one sample was needed for determining both total and specific counts of bacteria. False-positive counts and other artifacts that occur with antibody staining were easily distinguished when individual fluorescent particles were checked for DAPI fluorescence. The method for applying the general stain to membrane filters was performed quickly and simply by using a DAPI-soaked polypropylene filter that lay beneath the Nuclepore filter which collected the sample.     

Rapid and simple method for double staining of bacteria with 4',6-diamidino-2-phenylindole and fluorescein isothiocyanate-labeled antibodies

Bacteria were either heat fixed on microscope slides or filtered with 0.2 micron-pore-size Nuclepore filters. The samples were stained with 4',6-diamidino-2-phenylindole (DAPI) for total staining and with polyvalent rabbit antibodies and fluorescein isothiocyanate-conjugated swine anti-rabbit antibodies for specific staining. By switching between two different optical filter packages in the microscope, only one sample was needed for determining both total and specific counts of bacteria. False-positive counts and other artifacts that occur with antibody staining were easily distinguished when individual fluorescent particles were checked for DAPI fluorescence. The method for applying the general stain to membrane filters was performed quickly and simply by using a DAPI-soaked polypropylene filter that lay beneath the Nuclepore filter which collected the sample.     

Chlorine resistance patterns of bacteria from two drinking water distribution systems.

The relative chlorine sensitivities of bacteria isolated from chlorinated and unchlorinated drinking water distribution systems were compared by two independent methods. One method measured the toxic effect of free chlorine on bacteria, whereas the other measured the effect of combined chlorine. Bacteria from the chlorinated system were more resistant to both the combined and free forms of chlorine than those from the unchlorinated system, suggesting that there may be selection for more chlorine-tolerant microorganisms in chlorinated waters. Bacteria retained on the surfaces of 2.0-microns Nuclepore membrane filters were significantly more resistant to free chlorine compared to the total microbial population recovered on 0.2-micron membrane filters, presumably because aggregated cells or bacteria attached to suspended particulate matter exhibit more resistance than unassociated microorganisms. In accordance with this hypothesis, scanning electron microscopy of suspended particulate matter from the water samples revealed the presence of attached bacteria. The most resistant microorganisms were able to survive a 2-min exposure to 10 mg of free chlorine per liter. These included gram-positive spore-forming bacilli, actinomycetes, and some micrococci. The most sensitive bacteria were readily killed by chlorine concentrations of 1.0 mg liter-1 or less, and included most gram-positive micrococci, Corynebacterium/Arthrobacter, Klebsiella, Pseudomonas/Alcaligenes, Flavobacterium/Moraxella, and Acinetobacter.     

Specific uptake rates of amino acids by attached and free-living bacteria in a mesotrophic lake

Seasonal and spatial patterns of specific uptake rates of amino acids by bacteria in Lake Constance were studied. The total bacterial population was divided into small (0.2- to 1.0-micron) and large (1.0- to 3.0-micron) free-living bacteria and attached bacteria by fractionated filtration. Data for attached bacteria, received by retention on 3.0-micron-pore Nuclepore filters, were corrected for free-living bacteria in this fraction. Specific uptake rates based on autoradiography were also recorded. Specific uptake rates for attached bacteria ranged from 9.41 X 10(-11) to 6.11 X 10(-8) ng of C h-1 cell-1 and were therefore significantly greater than those for free-living bacteria during most time periods. However, they were not significantly different from those for cells proven to be active by autoradiography. Specific uptake rates for small free-living bacteria ranged between 7.68 X 10(-11) and 4.60 X 10(-9) ng of C h-1 cell-1. They were nearly in the same range of those for large free-living bacteria (5.10 X 10(-11) to 1.07 X 10(-8) ng of C h-1 cell-1), although both fractions exhibited pronounced differences in their seasonal and vertical distributions.     

Specific uptake rates of amino acids by attached and free-living bacteria in a mesotrophic lake.

Seasonal and spatial patterns of specific uptake rates of amino acids by bacteria in Lake Constance were studied. The total bacterial population was divided into small (0.2- to 1.0-micron) and large (1.0- to 3.0-micron) free-living bacteria and attached bacteria by fractionated filtration. Data for attached bacteria, received by retention on 3.0-micron-pore Nuclepore filters, were corrected for free-living bacteria in this fraction. Specific uptake rates based on autoradiography were also recorded. Specific uptake rates for attached bacteria ranged from 9.41 X 10(-11) to 6.11 X 10(-8) ng of C h-1 cell-1 and were therefore significantly greater than those for free-living bacteria during most time periods. However, they were not significantly different from those for cells proven to be active by autoradiography. Specific uptake rates for small free-living bacteria ranged between 7.68 X 10(-11) and 4.60 X 10(-9) ng of C h-1 cell-1. They were nearly in the same range of those for large free-living bacteria (5.10 X 10(-11) to 1.07 X 10(-8) ng of C h-1 cell-1), although both fractions exhibited pronounced differences in their seasonal and vertical distributions.     

Adherence to uroepithelial cells of Providencia stuartii isolated from the catheterized urinary tract

The long-term catheterized urinary tract appears to offer a niche for Providencia stuartii, otherwise an unusual clinical isolate. P. stuartii, the most frequent and persistent isolate from the urine of 51 long-term catheterized patients, was recovered from 761 of 1230 (62%) weekly urine specimens. To test the hypothesis that prevalence of this species may be due to adherence properties of the organism, 20 selected strains from 14 patients at two nursing homes, representing six distinct serotypes and harbouring combinations of nine different plasmid species, were tested for adherence to uroepithelial cells (UEC). Optimal conditions were determined for differentiating strains on the basis of in vitro adherence to UEC. These strains, grown in nutrient broth, were incubated with UEC isolated from the urine of a healthy adult female (10(8) bacteria per 10(5) cells). Washed UEC, retained on 8 micron pore diameter filters, were transferred to slides, fixed and stained; bacteria were counted on each of 40 cells. Fourteen of the 20 strains were defined as adherent to UEC by comparison of mean adherent bacteria and percentage of uroepithelial cells with more than 10 bacteria. Adherence was compared to that of a P-fimbriated strain of Escherichia coli. It was not inhibited by 50 mM-mannose. We conclude that the majority of P. stuartii isolates are adherent to UEC in vitro and suggest that this may play a role in the persistence of this organism in the catheterized urinary tract.     

Electropositively charged filters for the recovery of yeasts and bacteria from beverages

The ability of electropositively charged filters to recover yeasts and lactic acid bacteria from a variety of beverages was evaluated. Filtration through 'Zeta plus', grade O5S, filters recovered nearly all of the yeast contaminants from table wines, sherry and port. Recovery of yeasts from cream liqueurs and egg-based beverages was also good but it was not possible to filter drinks containing orange juice, even through filters with nominal pore sizes of 2 to 10 micron. Lactic acid bacteria proved more difficult to recover than yeasts, even though smaller pore-sized filters (1 to 4 micron) were employed. However, a sufficiently high percentage of bacteria were recovered to justify use of these filters for quality assurance. The advantage of concentrating contaminants by using charged filters, and the influence of product composition on the efficiency of microbial adsorption are discussed. The growth of wine-spoiling yeasts and lactic acid bacteria were not inhibited by water- or ethanol-soluble extracts of the filter material.     

Wound fiberglass depth filters as a less expensive approach for the concentration of viruses from water

Wound fiberglass depth cartridge filters (25.4 cm) with a nominal porosity of 1 micron were used to concentrate viruses from large volumes of surface water. They were found to be an excellent, less expensive alternative to the 0.2-micron pleated cartridge filters normally used for the concentration of enteric viruses from water. More than 99% of experimentally seeded poliovirus was adsorbed to these filters when the pH of the water was adjusted to pH 3.5 and aluminium chloride was added to a final concentration of 0.001 M, as recommended for electronegative filters. In comparative recovery of indigenous viruses from river water, similar results were obtained with two 1-micron or a 3-microns + 0.2-micron filter combination. The cost of the two 1-micron filters is about Can. $26, while it is about Can. $58 for the other combination.     

Use of nuclepore filters for counting bacteria by fluorescence microscopy.

Polycarbonate Nuclepore filters are better than cellulose filters for the direct counting of bacteria because they have uniform pore size and a flat surface that retains all of the bacteria on top of the filter. Although cellulose filters also retain all of the bacteria, many are trapped inside the filter where they cannot be counted. Before use, the Nuclepore filters must be dyed with irgalan black to eliminate autofluorescence. Direct counts of bacteria in lake and ocean waters are twice as high with Nuclepore filters as with cellulose filters.     

Neutrophil retention in model capillaries: deformability, geometry, and hydrodynamic forces

The initial retention of neutrophils within the pulmonary microvascular bed occurs in both physiological and pathological states, yet the factors responsible for this retention are poorly understood. Because the diameter of the neutrophil is approximately 7.03 micron and the mean pulmonary capillary diameter is 5.5 micron, we postulated that geometric constraints imposed by the microvascular bed, the deformability of the neutrophil, and the hydrodynamic characteristics of blood were important determinants of neutrophil retention. We used a filtration system wherein 111In-labeled human neutrophils (111In-N) suspended in a serum-containing buffer were passed through Nuclepore filters of known pore size. Compared with 99mTc-labeled erythrocytes (99mTc-RBC), the passage of 111In-N was delayed and a higher percentage was retained within the filter. Because the neutrophil and RBC are approximately equal in diameter, the deformability of the neutrophil must be less than that of RBC. As the flow rate increased, retention in the filters decreased logarithmically from 72 +/- 5% (flow rate 0.5 ml/min) to 15 +/- 4% (10.0 ml/min). As the number of RBC in the buffer increased, neutrophil retention in 5-micron filters decreased in a linear fashion from 65 +/- 6% at hematocrit of 0 to 33 +/- 2% at hematocrit of 10. The perfusion pressure and shear stress were of critical importance, and there was a logarithmic relationship between retention and perfusion pressure or shear stress (tau), whether the increase in pressure or tau was generated by increasing flow or by increasing the hematocrit of the perfusate. As the pore size of the filter increased, the retention of neutrophils decreased in a logarithmic fashion: from 75 +/- 5% in the 3-micron filter to 4 +/- 1.3% in the 12-micron filter.(ABSTRACT TRUNCATED AT 250 WORDS)     

Use of nuclepore filters for counting bacteria by fluorescence microscopy.

Polycarbonate Nuclepore filters are better than cellulose filters for the direct counting of bacteria because they have uniform pore size and a flat surface that retains all of the bacteria on top of the filter. Although cellulose filters also retain all of the bacteria, many are trapped inside the filter where they cannot be counted. Before use, the Nuclepore filters must be dyed with irgalan black to eliminate autofluorescence. Direct counts of bacteria in lake and ocean waters are twice as high with Nuclepore filters as with cellulose filters.     

On the microparticles which pass through glass fiber filter type GF/F in coastal and open waters

The chlorophyll a content of nicroparticles which passed throughglass fiber filters Whatman type GF/F but were retained on 0.2µm Nuclepore membranes was analyzed on a weekly basisover the course of 1 year in Kaneohe Bay, Hawaii. Depth profileswere also obtained at four oceanic stations off the islandsof Maui and Molokai, Hawaii. Experimental evidence indicatedthat these microparticles were photosynthetically active. Theproportion of microparticulate chlorophyll a could be up to35% of picoplankton chlorophyll a (2.0–0.2 µm sizerange) retained on a single pass through a 0.2 p.m Nucleporefilter. The filtrate from both GFIF and 0.2µm Nucleporefilters was found to contain chlorophyll a which could be retainedon a subsequent pass through either 0.2 µm Nuclepore orGF/F filters. Only serial filtration can ensure that essentiallyall picoplankton have been filtered from the water when eitherof these types of filters is used.     

Negative correlation between protozoal and bacterial levels in rumen samples and its relation to the determination of dietary effects on the rumen microbial population.

The bacterial protein content and protozoal protein content of unfractionated samples from the liquid-small particle phase of the rumen were determined on the basis of direct microscopic measurement of bacteria numbers and protozoa numbers and cell volumes. Standard values of 8.7 X 10(-11) mg of protein per bacterial cell and 5.9 X 10(-11) mg/micron 3 of protozoa cell volume, obtained from analysis of isolated cells, were used to convert the microscopic measurements to an estimate of the protein content of the rumen sample. When the correlation between bacterial and protozoal protein levels was examined within groups of animals, a highly significant negative correlation between these two parameters was found (P less than 0.001). The variation among animals for total (bacterial plus protozoal) microbial protein was smaller than the variation among animals for bacterial or protozoal protein alone. There was also a highly significant positive correlation (P less than 0.001) between protozoal protein level and total microbial protein level. The variation found among animals in total microbial protein level could be reduced by using a regression equation determined for bacterial versus protozoal protein to correct for the different population dynamics of the two groups.     

Dome formation in the human colon carcinoma cell line Caco-2 in culture. Influence of ouabain and permeable supports

We studied formation of domes in cell monolayers of the human colon carcinoma cell line Caco-2 which has been shown to exhibit signs of enterocytic differentiation and transport properties. After a 24 hr incubation with 4 X 10(-8) M ouabain, the number of domes seen on Caco-2 cell monolayers grown on plastic dishes was not significantly altered. After a 90 min preincubation with ouabain, 86rubidium uptake by Caco-2 cells was inhibited by ouabain, indicating that the cells have an ouabain-sensitive Na+, K+-ATPase, while dome formation was unaffected by ouabain. Domes were observed in Caco-2 cell monolayers grown on Nuclepore filters when the pore size was 0.015 micron but not when it was 0.030 micron. Our results suggest that dome formation in the Caco-2 cell line could be independent of Na+, K+-ATPase activity and might be due to accumulation of molecules having an effective hydrodynamic radius comprised between 0.015 and 0.030 micron.     

Detection of viable, but non-culturable Pseudomonas fluorescens DF57 in soil using a microcolony epifluorescence technique

Abstract Kanamycin-resistant Pseudomonas fluorescens DF57-3 cells (Tn5 modified) inoculated in soil microcosms rapidly lost their culturability, as defined by visible colony formation on Kings B agar supplemented with kanamycin. Thus, after 40 days only 0.02–0.35% of the initial inoculum was culturable. A microcolony epifluorescence technique was developed to determine the viable, but non-culturable subpopulation. A suspension of bacteria from the soil was prepared in salt solution after a sonication procedure and a sample was filtered onto a 0.2 μm Nuclepore filter. The filter was then placed for 3–4 days on the surface of Kings B agar before staining with acridine orange for epifluorescence microscopy. By staining and washing the filters carefully, disruption of microcolonies could be avoided. A majority of the microcolonies resulted from 2–3 cell divisions during the first 2 days of the incubation period, after which the cell divisions stopped. These microcolonies were taken to represent a population of viable, but non-culturable cells and comprised about 20% of the initial inoculum. A similar recovery was obtained when the filters were incubated on the surface of citrate minimal medium or soil extract medium. A few microcolonies showed continued growth on the filters, however, and their number corresponded well with that of visible macrocolonies. Observation by microscopy of a few (2–3) cell divisions (microcolony epifluorescence technique) is proposed for determination of subpopulations of viable, but non-culturable bacteria in soil.     

Relationships between Biovolume and Biomass of Naturally Derived Marine Bacterioplankton

Microscopic estimation of bacterial biomass requires determination of both biovolume and biovolume-to-biomass conversion. Both steps have uncertainty when applied to the very small bacteria typically found in natural seawater. In the present study, natural bacterioplankton assemblages were freshly collected, passed through 0.6-μm-pore-size Nuclepore filters to remove larger particulate materials, and diluted for growth in 0.22-μm-pore-size Millipore filter-sterilized unenriched seawater. This provided cells comparable in size and morphology to those in natural seawater, but the cultures were free of the interfering particulate detritus naturally present. Cells were collected on glass-fiber GF/F filters, and biovolumes were corrected for cells passing these filters; C and N were measured with a CHN analyzer. Our criteria for size measurement by epifluorescence photomicrography were confirmed with fluorescent microspheres of known diameters. Surprisingly, in six cultures with average per-cell biovolumes ranging from 0.036 to 0.073 μm³, the average per-cell carbon biomass was relatively constant at 20 ± 0.08 fg of C (mean ± standard error of the mean). The biovolume-to-biomass conversion factor averaged 0.38 ± 0.05 g of C cm⁻³, which is about three times higher than the value previously estimated from Escherichia coli, and decreased with increasing cell volume. The C:N ratio was 3.7 ± 0.2. We conclude that natural marine bacterial biomass and production may be higher than was previously thought and that variations in bacterial size may not reflect variations in biomass per cell.     

Effects of an abrupt diet change from hay to concentrate on microbial numbers and physical environment in the cecum of the pony

Microbial numbers, pH, fluid volume, and turnover rate in the pony cecum were measured during an abrupt change from an all-forage to an all-concentrate diet, both fed at maintenance energy levels. Concentrate feeding resulted in increased (P less than 0.01) numbers of total viable anaerobic bacteria. The numbers of organisms growing on selective starch medium increased (P less than 0.01) when concentrate was fed, while numbers on xylan and pectin media decreased (P less than 0.025). Seven days after the diet change to concentrate, the number of bacteria growing on lactate medium increased (P less than 0.01), followed by a gradual decline. Cellulolytic bacteria occurred in low numbers, ranging from 1.1 x 10(4) to 4.4 x 10(4) per g of cecal contents. Feeding all concentrate decreased both the number of genera (P less than 0.01) and total protozoan numbers (P less than 0.01) in the cecum. Minimum cecal pH values of 6.4 and 5.8 were obtained when forage and concentrate, respectively, were fed, with the minimum pH occurring 6 h postfeeding. Dry-matter percentage of cecal contents followed a diurnal pattern which was the inverse of the pH curve. During forage feeding, the cecum contained an average of 2.2 liters (1.6 to 3.4 liters), which turned over 3.9 times per day. When concentrate was fed, cecal volume averaged 3.9 liters (0.6 to 8.6 liters), with a mean liquid turnover of 4.2 times per day. Microbial numbers and pH changes in the pony cecum associated with an abrupt change in diet from hay to concentrate resembled those which occur in the rumen under similar feeding conditions.     

Effects of an abrupt diet change from hay to concentrate on microbial numbers and physical environment in the cecum of the pony.

Microbial numbers, pH, fluid volume, and turnover rate in the pony cecum were measured during an abrupt change from an all-forage to an all-concentrate diet, both fed at maintenance energy levels. Concentrate feeding resulted in increased (P less than 0.01) numbers of total viable anaerobic bacteria. The numbers of organisms growing on selective starch medium increased (P less than 0.01) when concentrate was fed, while numbers on xylan and pectin media decreased (P less than 0.025). Seven days after the diet change to concentrate, the number of bacteria growing on lactate medium increased (P less than 0.01), followed by a gradual decline. Cellulolytic bacteria occurred in low numbers, ranging from 1.1 x 10(4) to 4.4 x 10(4) per g of cecal contents. Feeding all concentrate decreased both the number of genera (P less than 0.01) and total protozoan numbers (P less than 0.01) in the cecum. Minimum cecal pH values of 6.4 and 5.8 were obtained when forage and concentrate, respectively, were fed, with the minimum pH occurring 6 h postfeeding. Dry-matter percentage of cecal contents followed a diurnal pattern which was the inverse of the pH curve. During forage feeding, the cecum contained an average of 2.2 liters (1.6 to 3.4 liters), which turned over 3.9 times per day. When concentrate was fed, cecal volume averaged 3.9 liters (0.6 to 8.6 liters), with a mean liquid turnover of 4.2 times per day. Microbial numbers and pH changes in the pony cecum associated with an abrupt change in diet from hay to concentrate resembled those which occur in the rumen under similar feeding conditions.