The role of surface physicochemical properties in determining the distribution of the autochthonous microflora in mineral water bottles

Investigation of the distribution of the viable autochthonous microflora in three brands of 1-2-month-old bottled mineral water showed that 1.8 x 10(4) (S.E.M. 8.9 x 10(3), n = 5) to 1.2 x 10(5) (S.E.M. 1.3 x 10(4), n = 5) cfu ml-1 were planktonic cells while 11 (S.E.M. 4, n = 5)-632 (S.E.M. 176, n = 5) cfu cm-2 were found in the biofilm. The biofilm represented between 0.03 and 1.79% of the total viable microbial population in the 1.5 litre bottles studied. Scanning electron microscopy studies showed that the cells adhering to the polyethylene terephthalate (PET) bottles were predominantly rod-shaped, sparsely distributed over the surface. In contrast, the cells adhering to the high density polyethylene (HDPE) caps were found to be mainly clumps of coccoid cells, suggesting that the bottle may provide different microhabitats for different microfloras. Large-scale roughness, such as that observed as lettering inside the cap (average height (z) = 93 microns) was associated with a 46-fold increase in cell numbers. Increased small-scale roughness, as measured by atomic force microscopy on PET and HDPE surfaces (average roughness (Ra) = 5-551 (nm), showed no correlation with adhesion. Investigations of surface hydrophobicity by the sessile drop technique showed that contact angles (theta) were greater on the HDPE caps (theta = 89-96 degrees) than on the PET surfaces (theta = 69-80 degrees). However, no correlation was found between contact angle and attached cell numbers. Measurements of surface electrostatic charge by streaming potential showed that the PET carried an overall negative charge, measuring -15.9 to -16.6 mV in mineral water. No significant change in charge occurred when the monomer composition of the PET was altered. It was concluded that surface roughness, in particular the scale of surface topographical features, is the most important physicochemical surface characteristic determining the distribution of the autochthonous microflora in mineral water bottles.     

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.     

The filterability of leukocytes in undiluted blood

A filtrometer is described for measuring the flow of fluids through microfilters. The flow of Newtonian fluids through the filters can be predicted from the diameter, length and number of pores. There are no physical artefacts such as turbulent flow or a significant lag period before steady-state flow is achieved. The instrument has been used as a viscometer and has been used to record and analyse the flow of undiluted blood through 5 microns polycarbonate filters. The calculated viscosity of Newtonian fluids agrees well with those measured by a more conventional viscometer (Ostwald). Flow profiles of blood have been analysed to give both the numbers and the flow properties of a small population of slow leukocytes which equate numerically with the monocytes. They are subdivided into three distinct sub-populations, according to their rheological properties, and these are termed SL1, SL2 and PB. The concentration of these cells, in blood, are 0.12 +/- 0.02 x 10(6) ml-1, 0.11 +/- 0.02 x 10(6) ml-1, 0.09 +/- 0.02 x 10(6) ml-1 in young females aged about 25 years. The transit time of these cells, through 5 microns pores, is 34.8 +/- 1.4 s, 147.5 +/- 2.5 s and > 300 s, respectively. Analysis of blood from older men (53-79 years) gives essentially the same results although the concentration of SL1 is slightly higher at 0.19 +/- 0.09 x 10(6) ml-1.     

Lack of colonization of 1 day old chicks by viable, non-culturable Campylobacter jejuni.

Seven strains of Campylobacter jejuni, isolated from various sources [human (n = 2), chicken (n = 3), water (n = 2)], were studied under starvation conditions in filter-sterilized and pasteurized surface water by acridine orange direct count (AODC), viable count (DVC) and culture methods. Plate counts showed a rapid decline (2 log-units/day) for all strains under these conditions. Only one of the seven strains (14%) showed a (prolonged) viable, non-culturable 'state'. The ability of these viable, non-culturable cells to colonize the intestine was tested on day-old chicks. The infectious oral dose of freshly cultured cells of this model was 26-260 cfu; 1.8 x 10(5) viable, non-culturable C. jejuni were introduced to day-old chicks orally. Campylobacter jejuni was not isolated from the caeca of the chicks after incubation for 7 d. Also, passage through the allantoic fluid of embryonated eggs did not recover viable, non-culturable C. jejuni. These findings cast serious doubts on the significance of the viable, non-culturable 'state' in environmental transmission of C. jejuni.     

Purification of Chlamydia trachomatis by a simple and rapid filtration method

A simple method for filter purification of Chlamydia trachomatis from cell culture is described. Crude homogenates of chlamydiae-infected cells were passed through a glass prefilter and a 0.6 microns pore diameter polycarbonate filter. The filtrate was then passed through a 0.2 microns pore diameter filter on which the chlamydiae were trapped. This filter was then back-washed to collect the organisms. These procedures removed cell debris and soluble protein, and yielded particles with a narrow size distribution. The mean yield of viable chlamydiae purified by filtration was 64% when the filters were washed at each stage of the process.     

The validity of the cholesterol nucleation assay.

The validity of the cholesterol nucleation assay rests on the assumption that all cholesterol crystals are removed at the start of the assay so that de novo formation of crystals can be studied. In this paper we have tested the validity of this assumption. Cholesterol crystals were added to supersaturated model bile. Subsequently the mixtures were either filtered over a 0.22 micron filter or centrifuged at 37 degrees C for 2 h at 100,000 x g. After ultracentrifugation the isotropic interphase was collected. Using polarized light microscopy no crystals could be visualized in this fraction. However, the nucleation time of the isotropic interphase decreased from 6.8 +/- 1.1 days to 1.8 +/- 0.2 days (mean +/- S.E., P less than 0.01, n = 5) when 10-100 micrograms/ml crystals were added prior to centrifugation. Similar results were observed when instead of centrifugation the mixtures containing crystals were filtered. After filtration over a 0.22 micron filter no crystals could be detected in the filtrate. Yet the nucleation time of the filtrate decreased from 6.4 +/- 0.7 days to 3.1 +/- 0.5 days (mean +/- S.E.) when 10 micrograms/ml cholesterol crystals were added before filtration (n = 10, P less than 0.01). Since no cholesterol crystals could be detected at the start of the assay the reduction in nucleation time must have been brought about by cholesterol microcrystals that passed through the filter. Supplementation of cholesterol crystals to model bile did not accelerate the nucleation time when the samples were passed over a 0.02 micron filter, indicating that the size of the microcrystals was larger than 20 nm. The effect of addition of cholesterol crystals prior to filtration over a 0.22 micron filter was also tested in the crystal growth assay recently developed by Busch et al. ((1990) J. Lipid Res. 31, 1903-1909). Addition of crystals had only a minor effect on the assay. In conclusion, the reduced nucleation time of biles from gallstone patients is probably not only due to the presence of promoting or the absence of inhibiting proteins, but can be caused by the presence of small cholesterol crystals in these biles.     

The viability of bifidobacteria introduced into kimchi

The viability of bifidobacteria in mul-kimchi, a type of kimchi with added water, was investigated under various conditions. When a mul-kimchi preparation was inoculated with five strains of Bifidobacterium at a concentration of 10(7) cfu ml-1, Bif. longum JK-2 showed the highest viability, maintaining a population of 10(6) cfu ml-1 after 1 week at 4 degrees C. The influence of NaCl concentration and initial pH on viability was further investigated in mul-kimchi inoculated with Bif. longum JK-2; NaCl concentrations greater than 3% (w/w) reduced viability considerably. In kimchi started with an initial pH of 6.5, the cells showed the highest survival. When mul-kimchi containing 2% NaCl (w/w) was inoculated with 10(8) cfu ml-1 Bif. longum JK-2, there was a 10-fold reduction in viability during 10 d of incubation at 4 degrees C. These results demonstrate acceptable levels of the organism in the product, suggesting the possible use of selected strains of bifidobacteria in commercial kimchi production.     

Total counts, culturable and viable, and non-culturable microflora of a French mineral water: a case study

The changes in bacterial counts during the storage of a natural mineral water from a French spring were studied. Samples were taken from the spring and the bottling line. Viable cultivable (VC) bacteria were counted on R2A medium. Total counts, viable and dead bacteria were counted using the LIVE/DEAD Bac Light VIABILITY kit and epifluorescence microscopy. Viable but non-cultivable (VNC) bacteria were estimated by difference between viable and VC counts. Isolates were clustered by phenotype. The microflora in the spring water increased from < 10-3 x 10(5) bacteria ml-1 after 6 d in storage and then stabilized. Mechanical bottling increased the allochthonous bacteria in the water that stabilized at 10(5) bacteria ml-1. Maximal growth is controlled by the low concentration of nutrients in the mineral water and the lysis of dead cells. The allochthonous bacteria came from the aquifer and colonized the filling line. The changes in the VC and VNC populations showed that the bacteria used starvation-survival and entry into the VNC state to adapt to the bottling stress and the enclosed oligotrophic environment.     

A simple technique for supporting single cells in electron microscopic immunocytochemistry and embedding.

Cell suspensions of rat anterior pituitaries were filtered with a polycarbonate filter (pore size 3 micron) and fixed on the filter. After fixation the cells were adherent to the filter and immunocytochemical staining could be accomplished by simply dipping the filter into the different incubation media. The cells could be dehydrated and embedded in Epon 812 on the filter. After polymerization the embedded filter was sawn into small blocks and the cell layer was sectioned tangentially on an ultramicrotome. This method also seems to be applicable to other histochemical studies on single cells.     

Hippocampal slice (K+)e: depth profiles and changes induced by stimulation or anoxia

In an attempt to develop a technique which would allow early assessment of the functional state of explanted brain tissue, (K+)e was measured in the CA1 region of rat hippocampal slices using K+-selective microelectrodes. In slices (450 micron) maintained at the boundary between the incubation medium and 95% O2/5% CO2 atmosphere, (K+)e was highest (up to 25-30 mmol/l) immediately below the exposed surface and gradually decreased with depth to (K+) of the bathing fluid (5 mmol/l). (K+)e below the exposed surface remained high throughout the 2 h of incubation. In submersed slices, (K+)e was the highest in the center of the slice (200 micron, 10 mmol/l) and decreased towards both surfaces. During 2 h incubation, (K+)e decreased in the center of the slice to 6 mmol/l in viable preparations remaining high in the deteriorating ones. Electrical stimulation of Schaffer's collaterals (15 V; 0.2 ms; 10 Hz) increased (K+)e of viable slices 200 micron below the surface by 2-3 mmol/l. Similar but slower (K+)e changes were elicited by brief (3 min) anoxic episodes (perfusion with incubation medium equilibrated with 95% N2/5% CO2). It is concluded that submersed slices have a more uniform (K+)e profile as compared to the exposed ones and that low (K+)e in the early phase of incubation is a good predictor of slice viability.     

Comparison of conventional culture and PCR methods for the detection of Legionella pneumophila in water

A comparative assessment of conventional culture and nucleic acid techniques in the detection of Legionella pneumophila in seeded tap water samples was performed, using bacterial concentrations ranging from 994 to 0·015 cfu ml⁻¹. Different filtration and centrifugation protocols were evaluated. The results permitted the development of a tentative algorithm for the detection of legionellae in tap water. Samples should first be analysed using PCR methods. In the event of quantitative data and bacterial strains for epidemiologic typing being required, the same sample, or a greater volume of the sample, if positive with PCR, can be re-tested by filtration through polycarbonate membranes followed by plating a homogenate of the filter. If samples are found to be negative with PCR, they can be re-analysed in greater volumes by filtration through polycarbonate membranes followed by direct placing of the filter on culture media, to allow detection of very low numbers of bacteria. This protocol should be validated in the field before it can be routinely implemented.     

Production of Aspergillus xylanase by lignocellulosic waste fermentation and its application

Strains of Aspergillus terreus and A. niger, known to produce xylanase with undetectable amounts of cellulase, were studied for xylanase (EC 3.2.1.8) production on various lignocellulosic substrates using solid state fermentation. Of the lignocellulosic substrates used, wheat bran was the best for xylanase production. The effects of various parameters, such as moistening agent, level of initial moisture content, temperature of incubation, inoculum size and incubation time, on xylanase production were studied. The best medium for A. terreus was wheat bran moistened with 1:5 Mandels and Strenberg mineral solution containing 0.1% tryptone, at 35 degrees C, and at inoculum concentration 2x107-2x108 spores 5 g-1 substrate; for A. niger, the best medium was wheat bran moistened with 1:5 Mandels and Strenberg mineral solution containing 0.1% yeast extract, at 35 degrees C, and at an inoculum concentration of 2x107-2x108 spores 5 g-1 substrate. Under these conditions, A. terreus produced 68.9 IU ml-1 of xylanase, and A. niger, 74.5 IU ml-1, after 4 d of incubation. A crude culture filtrate of the two Aspergillus strains was used for the hydrolysis of various lignocellulosic materials. Xylanase preparations from the two strains selectively removed the hemicellulose fraction from all lignocellulosic materials tested.     

A filtration method for measuring cellular uptake of [14C]methylamine and other highly permeant solutes

A filtration technique has been developed for study of the uptake of [14C]methylamine by Azotobacter vinelandii. This dual filter arrangement requires a precision microporous polycarbonate film which overlays a paper filter. Cellular uptake of radioactivity is terminated by vacuum filtration of the reaction mixture onto the polycarbonate filter without dilution or washing. Filtration was complete in 0.7 s with retention of less than 0.2% of the extracellular radioactivity. The dual filter method gave 20-fold higher levels for intracellular methylamine than filtration followed by washing. Without washing, nitrocellulose filters retained 18 times more extracellular [3H]sorbitol and 80 times more extracellular [14C]methylamine than polycarbonate filters. Use of an underlying paper filter did not significantly improve the performance of nitrocellulose filters. However, addition of a paper filter reduced extracellular methylamine and sorbitol retention on polycarbonate filters by 77 and 86%, respectively. This method is generally applicable to measurement of the uptake of highly permeant molecules by cells and subcellular organelles.     

Effects of cellulose on growth,enzyme production,and ultrastructure of a Cellulomonas species

Cellulomonas sp. (NRCC 2406) was grown on complex medium (peptone-tryptone-yeast extract) alone, or with the addition of different celluloses (solka floc, avicel, CF 11 cellulose or Whatman No. 1 filter paper) and/or glucose. Cultures growing on the complex medium without cellulose produced low levels of endo- and exo-cellulases and very little -glucosidase. Adding cellulose stimulated growth, as measured by cellular protein or by viable counts, and also stimulated production of cellulases. Adding glucose in the prescene of cellulose inhibited growth and cellulose breakdown. Glucose also inhibited attachment of growing cells to cellulose fibres. Electron microscope studies showed that Cellulomonas sp. adhered to the cellulose fibers. In the presence of cellulose in the media, the cells developed a thicker outer layer which probably helps in the adhesion process.Abbreviations PTYE peptone, tryptone, yeast extract medium - DNS dinitrosalicylic acid - CMC carboxymethyl cellulose - cfu/ml colony-forming units per ml     

Flow characteristics of red cell containing fluids through pores. The effect of filter plugging, a mathematical model

A mathematical model was developed that describes the effects of filter plugging on flow through 3 micron pore polycarbonate filters as a function of time, pressure, and cell concentration, both under stirring and nonstirring conditions. The mathematical constants for the model were derived from experimental data generated with a filtration apparatus, and were tested by using various concentrations of cells that are able to plug filter pores. A computer simulation program was written to test the model over a wide range of nonfilterable cell concentrations.     

Growth and survival of E. coli O157:H7 during the manufacture and ripening of a smear-ripened cheese produced from raw milk

AIMS: The behaviour of Escherichia coli O157:H7 was studied during the manufacture and ripening of a smear-ripened cheese produced from raw milk. METHODS AND RESULTS: Cheese was manufactured on a laboratory scale using milk (20 l) inoculated with E. coli O157:H7, and enumeration was carried out using CT-SMAC. From an initial level of 1.52 +/- 0.03 log cfu ml-1 in the milk (34 +/- 2 cfu ml-1), the numbers increased to 3.4 +/- 0.05 log cfu g-1 in the cheese at day 1. During ripening, the numbers decreased to <1 cfu g-1 and <10 cfu g-1 in the rind and core, respectively, after 21 days, although viable cells were detected by enrichment after 90 days. The presence of E. coli O157:H7 in the cheese was confirmed by latex agglutination and by multiplex PCR. CONCLUSION: The results indicate that the manufacturing procedure encouraged substantial growth of E. coli O157:H7 to levels that permitted survival during ripening and extended storage. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of low numbers of E. coli O157:H7 in milk, destined for raw milk cheese manufacture, could constitute a threat to the consumer.     

A simple technique to concentrate the protozoan Mikrocytos mackini, causative agent of Denman Island Disease in oysters

This report describes a simple filtration technique to isolate the parasite Mikrocytos mackini from oyster tissue. The technique is based on successive filtration through filter papers and polycarbonate membrane filters of decreasing mesh using a low vacuum (<8 cm Hg). This technique allows for the recovery of about 1 x 10(8) parasites (microcells) from about 2 g of heavily infected oyster tissue. About 99% of the particulate material in the final preparation consisted of intact M. mackini.     

Na, k, and nonspecific solute requirements for induction and function of galactose active transport in an antarctic psychrophilic marine bacterium

Strong adherence of bacteria, yeast, erythrocytes, leukocytes, platelets, spores, and polystyrene spheres to membrane filter materials was noted during filtration through membranes with pore size diameters much larger than the particles themselves. Quantitative recovery on the membrane filters of these particles from low-concentration suspensions was achieved during gravity- or vacuum-assisted filtration through membranes with pore diameters as much as 30 times that of the filtered particles. Mechanical sieving was not responsible. The phenomenon was judged to be electrostatic. It could be partially blocked by pretreating the filter with a nonionic surfactant (Tween 20), and elution of adherent particles was achieved with 0.05% Tween 20. Gram-positive cocci were removed from suspension more efficiently than gram-negative rods. The commonly used cellulose membranes adsorbed more bacteria, blood cells, and other particles than did polycarbonate filters. Of lesser adsorptive capacity were vinyl acetate, nylon, acrylic, and Teflon membranes. Backwashing with saline, serum, 6% NaCl, dextran solutions, or phosphate buffers of varying molality and pH removed only a fraction of adherent particles. Tween 20 (0.05%) eluted up to 45% of adherent particles in a single back-filtration. Selected filters quantitatively removed the particles tested, which then could be washed and subjected to reagents for a variety of purposes. It is important to anticipate the removal of particles during membrane filtration, since it is not a simple mechanical event.     

Mechanistic study of membrane concentration and recovery of Listeria monocytogenes

Detection of the foodborne pathogen Listeria monocytogenes requires that food samples be processed to remove proteins and lipids, concentrate microorganisms to a detectable concentration, and recover the concentrated cells in a small volume compatible with micron-scale biochips. Mechanistic considerations addressed in this research include the roles of membrane structure, pore size, and detergents in maximizing recovery of cells from a complex biological fluid. The fluid in this case was a food sample (hotdog extract) inoculated with L. monocytogenes. This study showed how membrane filtration using a syringe filter is able to concentrate L. monocytogenes by 95x with up to 95% recovery of living microorganisms by concentrating 50 mL of food sample into a volume of 500 microL. Tween 20 was added to the sample to prevent irreversible adsorption of the microorganism to the membrane and thereby help to ensure high recovery. Comparison of polycarbonate, mixed cellulose, nylon, and PVDF membranes with 0.2 to 0.45 microm pores showed the 0.2 microm polycarbonate membrane with straight through, mono-radial pores gives the highest recovery of living microorganisms. The mixed cellulose, nylon, and PVDF membranes have a fibrous structure whose characteristic openings are much larger than their effective pore size cut-offs of 0.22 or 0.45 microm. We define conditions for rapid membrane-based cell concentration and recovery that has the potential to supplant enrichment steps that require a day or more. This approach has the added benefit of facilitating examination of a large amount of fluid volume by reducing its volume to a range that is compatible with the microliter scales of biochip or other biosensor detection systems.