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.     

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.     

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.     

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.     

Interference of tooth differentiation with interposed filters

The effect of interposed Nuclepore filters on the epithelio-mesenchymal interaction in embryonic mouse tooth was studied. Filters with pore sizes of 0.6 and 0.2 μm allowed differentiation of odontoblasts and ameloblasts in the bell-stage tooth germ. This differentiation progressed more rapidly when the 0.6-μm pore size filter was used. Nuclepore filters with 0.1-μm pores prevented differentiation. Electron microscopic examination revealed penetration of cell processes into the filter pores. Cytoplasmic material could be seen in the 0.6-μm pore-size filter within 3 days of cultivation, whereas, in the 0.2-μm filter pores, penetration was slight. After 6 days of cultivation, cytoplasmic material was found at all levels of the 0.2-μm pore-size filter, but not in the channels of the 0.1-μm pore-size filters, preventing differentiation. It is concluded that the 0.1-μm pore-size filter blocks tooth development at the level of mesenchymal cell differentiation into odontoblasts. It is suggested that this differentiation requires a close association between the interacting mesenchymal and epithelial cells.     

Picoplankton size distributions in marine and fresh waters: problems with filter fractionation studies

Abstract The retention of algal picoplankton by Nuclepore polycarbonate filters of 0.2, 1.0, 2.0 and 3.0 μm pore size was tested in 2 marine and 3 freshwater sites. When 1 μm Nuclepore filters were used, the percentage of the total cyanobacterial cells passing the filter varied between sites and with increasing depth within sites. As much as 99% of the Synechococcus -like cells was retained by a 1 μm filter. This could lead to an underestimation of the picoplanktonic contribution or, more seriously, an apparent distribution pattern that is an artifact of the choice of filter pore size. Filter retention was also dependent on vaccum pressure during filtration. This study emphasizes the need for direct observation of picoplankton numbers in filter fractionation studies.     

Adsorption of dissolved organic matter to the inorganic filter substrate and its implications for C uptake measurements

Inorganic carbon uptake rates for glass fiber-filtered samples are higher than those for membrane-filtered samples because of adsorption of dissolved organic matter to the filter substrate. Experimentally derived values for adsorption onto filters were as follows (relative units): GF/F filter, 1, quartz filter, 1.1, GF/C filter, 0.6; GN-6 Gelman filter, 0.1; Nuclepore and Poretics filter, 0.0; Anodisc filter, 0.4 to 1.9.     

Adsorption of Dissolved Organic Matter to the Inorganic Filter Substrate and Its Implications for 14C Uptake Measurements

Inorganic carbon uptake rates for glass fiber-filtered samples are higher than those for membrane-filtered samples because of adsorption of dissolved organic matter to the filter substrate. Experimentally derived values for adsorption onto filters were as follows (relative units): GF/F filter, 1, quartz filter, 1.1, GF/C filter, 0.6; GN-6 Gelman filter, 0.1; Nuclepore and Poretics filter, 0.0; Anodisc filter, 0.4 to 1.9.     

Comparison of different glass fibre and silver metal filters for the determination of particulate organic carbon

The most retentive glass fibre filters were able to retain almost all bacteria from the water of an oligotrophic lake. Having satisfactory speed and capacity, of filtration this type of filter is quite near to the ideal which should be able to include all algae and bacteria in the category of particulate organic carbon. Similar retention could also be achieved by silver filters but, because of their high blank values, price, and lower filtration speed and capacity, they are not able to compete with glass fibre filters in practical work.     

Mechanisms of Cell Interaction during Primary Embryonic Induction Studied in Transfilter Experiments

The transmission mechanisms operative at different stages of neutralisation during primary embryonic induction of the newt Triturus vulgaris were studied in experiments employing Nuclepore filters placed between interactive tissue explants. The transmission time of the neuralising effect was determined with 0.2 μm Nuclepore filter. In another series of experiments the transformation of neuralised ectoderm by archenteron roof mesoderm into other parts of the CNS was studied. Although sufficiently long induction times were used no transformation into hindbrain structures could be induced across filters with pore sizes from 0.1 μm to 1.0 μm. However, electron microscopy demonstrated cytoplasmic penetration into 0.6 μm filters at 15 h of induction. The results speak against free long-range diffusion of inductive material at the stage of transformation of the neuralised ectoderm to more caudal parts of CSN and warrant a more detailed structural study of the transmission phenomenon in question.     

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.     

The affinity of [3H]quinuclidinyl benzilate and some other radioligands for glass microfiber and cellulose filters in some common liquid scintillation solvents

The optimum conditions for measuring radioactivity in the filtration assay of muscarinic cholinoceptors with tritiated quinuclidinyl benzilate are to use Whatman GF/B filters and to add a simple toluene scintillant to them while they are still damp. Practically all the radioactive material is then slowly extracted into the scintillant and high counting efficiencies are obtained after 24 h. Dried filters, or dry filters in control experiments in the absence of receptor, adsorb much of the radioactivity with a 30% reduction in counting efficiency. Other scintillants were able to extract the radioactive material from dry filters, but were generally not preferable to toluene. The GF/B filters performed better than other glass microfiber and cellulose filters in terms of retention of receptor-bound ligand, rapid filtration rates, and low filter blanks. Toluene is unsuitable as a scintillant with GF/B filters for some other radioligands examined.     

Transfllter Studies on Neural Induction in the Newt

In order to study the transmission mechanism of neuralising signals during primary embryonic induction, the interacting components (competent newt gastrula ectoderm and dorsal lip tissues) were separated by filter membranes of varying pore size. Nuclepore filters with nominal pore size from 0.1 to 8 μm were employed and the neuralising effect was shown to traverse all of these membranes. Electron microscopic examination did not reveal any cytoplasmic processes in the pores and the authors conclude that the morphogenetic signals are carried by transmissable compounds rather than through direct cytoplasmic contacts.     

Transfilter studies on neural induction in the newt.

In order to study the transmission mechanism of neuralising signals during primary embryonic induction, the interacting components (competent newt gastrula ectoderm and dorsal lip tissues) were separated by filter membranes of varying pore size. Nuclepore filters with nominal pore size from 0.1 to 8 mum were employed and the neuralising effect was shown to traverse all of these membranes. Electron microscopic examination did not reveal any cytoplasmic processes in the pores and the authors conclude that the morphogenetic signals are carried by transmissable compounds rather than through direct cytoplasmic contacts.     

A Toluidine Blue-Membrane Filter Method for the Quantitative Staining of Bacteria

A method for measuring the uptake of toluidine blue by bacteria on membrane filters was developed. Bacteria were filtered out of solution onto a cellulose acetate filter and stained on the filter at 50 C with toluidine blue in citrate-phosphate buffer, pH 4.0. The filter was destained in ethanol, placed on a glass slide and subsequently made transparent in a 1,4-dioxan and cyclohexanone mixture. The absorbance of the stained bacteria on the slide was measured in a spectrophotometer at 590 nm. The uptake of dye by cells of Streptococcus cremoris and Escherichia coli could be explained using the Freundlich adsorption isotherm. Cell concentrations of both these organisms can be determined with this technique.     

A toluidine blue-membrane filter method for the quantitative staining of bacteria

A method for measuring the uptake of toluidine blue by bacteria on membrane filters was developed. Bacteria were filtered out of solution onto a cellulose acetate filter and stained on the filter at 50 C with toluidine blue in citrate-phosphate buffer, pH 4.0. The filter was destained in ethanol, placed on a glass slide and subsequently made transparent in a 1,4-dioxan and cyclohexanone mixture. The absorbance of the stained bacteria on the slide was measured in a spectrophotometer at 590 nm. The uptake of dye by cells of Streptococcus cremoris and Escherichia coli could be explained using the Freundlich adsorption isotherm. Cell concentrations of both these organisms can be determined with this technique.     

Comparison of a new inorganic membrane filter (Anopore) with a track-etched polycarbonate membrane filter (Nuclepore) for direct counting of bacteria.

Bacterial counts obtained by using a new Anopore inorganic membrane filter were 21 to 33% higher than those obtained by using a Nuclepore polycarbonate membrane filter. In addition, the inorganic filter had higher flow rates, permitting lower vacuum pressures to be used, while the intrinsically flat, rigid surface resulted in easier focusing and sharp definition of bacteria across the whole field of view.     

Comparison of a new inorganic membrane filter (Anopore) with a track-etched polycarbonate membrane filter (Nuclepore) for direct counting of bacteria

Bacterial counts obtained by using a new Anopore inorganic membrane filter were 21 to 33% higher than those obtained by using a Nuclepore polycarbonate membrane filter. In addition, the inorganic filter had higher flow rates, permitting lower vacuum pressures to be used, while the intrinsically flat, rigid surface resulted in easier focusing and sharp definition of bacteria across the whole field of view.     

A simple methodological approach for counting and identifying culturable viruses adsorbed to cellulose nitrate membrane filters

We identified conditions under which Buffalo green monkey cells grew on the surfaces of cellulose nitrate membrane filters in such a way that they covered the entire surface of each filter and penetrated through the pores. When such conditions were used, poliovirus that had previously been adsorbed on the membranes infected the cells and replicated. A plaque assay method and a quantal method (most probable number of cytopathic units) were used to detect and count the viruses adsorbed on the membrane filters. Polioviruses in aqueous suspensions were then concentrated by adsorption to cellulose membrane filters and were subsequently counted without elution, a step which is necessary when the commonly used methods are employed. The pore size of the membrane filter, the sample contents, and the sample volume were optimized for tap water, seawater, and a 0.25 M glycine buffer solution. The numbers of viruses recovered under the optimized conditions were more than 50% greater than the numbers counted by the standard plaque assay. When ceftazidime was added to the assay medium in addition to the antibiotics which are typically used, the method could be used to study natural samples with low and intermediate levels of microbial pollution without decontamination of the samples. This methodological approach also allowed plaque hybridization either directly on cellulose nitrate membranes or on Hybond N+ membranes after the preparations were transferred.