Comparison of aerosol and bioaerosol collection on air filters

Air filters efficiency is usually determined by non-biological test aerosols, such as potassium chloride particles, Arizona dust or di-ethyl-hexyl-sebacate (DEHS) oily liquid. This research was undertaken to asses, if application of non-biological aerosols reflects air filters capacity to collect particles of biological origin. The collection efficiency for non-biological aerosol was tested with the PALAS set and ISO Fine Test Dust. Flow rate during the filtration process was 720 l/h, and particles size ranged 0.246–17.165 μm. The upstream and downstream concentration of the aerosol was measured with a laser particle counter PCS-2010. Tested bioaerosol contained 4 bacterial strains of different shape and size: Micrococcus luteus, Micrococcus varians, Pseudomonas putida and Bacillus subtilis. Number of the biological particles was estimated with a culture-based method. Results obtained with bioaerosol did not confirmed 100% filters efficiency noted for the mineral test dust of the same aerodynamic diameter. Maximum efficiency tested with bacterial cells was 99.8%. Additionally, cells reemission from filters into air was also studied. Bioaerosol contained 3 bacterial strains: Micrococcus varians, Pseudomonas putida and Bacillus subtilis. It was proved that the highest intensity of the reemission process was during the first 5 min. and reached maximum 0.63% of total number of bacteria retained in filters. Spherical cells adhered stronger to the filter fibres than cylindrical ones. It was concluded that non-biological aerosol containing particles of the same shape and surface characteristics (like DEHS spherical particles) can not give representative results for all particles present in the filtered air.     

Assessment of iodine‐treated filter media for removal and inactivation of MS2 bacteriophage aerosols

Aims: To investigate the performance of an iodine‐releasing filter medium for use as a protective device against airborne pathogens. Methods and Results: The filter’s physical and viable removal efficiencies (VRE) were investigated with challenges of MS2 bacteriophage aerosols, and the infectivity of MS2 collected on the filter was analysed. To test a proposed inactivation mechanism, media containing thiosulfate or bovine serum albumin (BSA) were put in impingers to quench and consume I₂ released from the filter. In direct plating experiments, treated filters presented significantly higher VREs than did untreated filters; however, collection in excess BSA decreased VRE by half and in thiosulfate the apparent VRE decreased drastically. No significant difference in infectivity of retained viruses on treated and untreated filters was observed at the same environmental condition. Conclusions: Evidence presented herein for competition by dissolved I₂ in infectivity assays supports a mechanism of induced displacement and capture of I_2. It also requires that dissociation of iodine from the filter and capture of iodine by MS2 aerosols as they pass through the filter be factored in the design of the assessment methodology. The filter’s strong retention capability minimizes reaerosolization but also makes it difficult to discriminate the antimicrobial effect at the surface. Significance and Impact of the Study: This study shows the direct plating assay method to be sensitive to interference by iodine‐releasing materials. This requires reevaluation of earlier reports of VRE measurements.     

Effect of Trophic Status on the Culturability and Activity of Bacteria from a Range of Lakes in the English Lake District

The bacterioplankton from a number of lakes that differed in nutrient status in the English Lake District was examined with a number of techniques for enumeration and activity assessment. Natural water samples showed a clear correlation between total counts and trophic status. Esterase activity measurements with Chemchrome B were able to distinguish high- and low-nutrient-status lakes, whereas tetrazolium salt (5-cyano-2,3-ditoyltetrazolium chloride) reduction, the direct viable count-cell elongation assay, and culturability measurements could not. Tetrazolium salt reduction and esterase activity measurements labeled a significant number of cells from water of all nutrient levels, whereas the direct viable count-cell elongation method was of use only in oligotrophic waters. Size fractionation of samples showed that the culturable cells were retained by the larger filters, especially in nutrient-rich waters. Esterase activity measurements also favored the larger cells. The differences observed between assays using water that differed in trophic status raise questions about the use of these tests as a definitive measure of viability.     

Effect of trophic status on the culturability and activity of bacteria from a range of lakes in the English Lake District

The bacterioplankton from a number of lakes that differed in nutrient status in the English Lake District was examined with a number of techniques for enumeration and activity assessment. Natural water samples showed a clear correlation between total counts and trophic status. Esterase activity measurements with Chemchrome B were able to distinguish high- and low-nutrient-status lakes, whereas tetrazolium salt (5-cyano-2,3-ditoyltetrazolium chloride) reduction, the direct viable count-cell elongation assay, and culturability measurements could not. Tetrazolium salt reduction and esterase activity measurements labeled a significant number of cells from water of all nutrient levels, whereas the direct viable count-cell elongation method was of use only in oligotrophic waters. Size fractionation of samples showed that the culturable cells were retained by the larger filters, especially in nutrient-rich waters. Esterase activity measurements also favored the larger cells. The differences observed between assays using water that differed in trophic status raise questions about the use of these tests as a definitive measure of viability.     

Variables Influencing Extraction of Nucleic Acids from Microbial Plankton (Viruses,Bacteria, and Protists) Collected on Nanoporous Aluminum Oxide Filters

Anodic aluminum oxide (AAO) filters have high porosity and can be manufactured with a pore size that is small enough to quantitatively capture viruses. These properties make the filters potentially useful for harvesting total microbial communities from water samples for molecular analyses, but their performance for nucleic acid extraction has not been systematically or quantitatively evaluated. In this study, we characterized the flux of water through commercially produced nanoporous (0.02 μm) AAO filters (Anotop; Whatman) and used isolates (a virus, a bacterium, and a protist) and natural seawater samples to test variables that we expected would influence the efficiency with which nucleic acids are recovered from the filters. Extraction chemistry had a significant effect on DNA yield, and back flushing the filters during extraction was found to improve yields of high-molecular-weight DNA. Using the back-flush protocol, the mass of DNA recovered from microorganisms collected on AAO filters was ≥100% of that extracted from pellets of cells and viruses and 94% ± 9% of that obtained by direct extraction of a liquid bacterial culture. The latter is a minimum estimate of the relative recovery of microbial DNA, since liquid cultures include dissolved nucleic acids that are retained inefficiently by the filter. In conclusion, we demonstrate that nucleic acids can be extracted from microorganisms on AAO filters with an efficiency similar to that achievable by direct extraction of microbes in suspension or in pellets. These filters are therefore a convenient means by which to harvest total microbial communities from multiple aqueous samples in parallel for subsequent molecular analyses.     

Comparison of fluorescence microscopy and solid-phase cytometry methods for counting bacteria in water

Total direct counts of bacterial abundance are central in assessing the biomass and bacteriological quality of water in ecological and industrial applications. Several factors have been identified that contribute to the variability in bacterial abundance counts when using fluorescent microscopy, the most significant of which is retaining an adequate number of cells per filter to ensure an acceptable level of statistical confidence in the resulting data. Previous studies that have assessed the components of total-direct-count methods that contribute to this variance have attempted to maintain a bacterial cell abundance value per filter of approximately 10(6) cells filter(-1). In this study we have established the lower limit for the number of bacterial cells per filter at which the statistical reliability of the abundance estimate is no longer acceptable. Our results indicate that when the numbers of bacterial cells per filter were progressively reduced below 10(5), the microscopic methods increasingly overestimated the true bacterial abundance (range, 15.0 to 99.3%). The solid-phase cytometer only slightly overestimated the true bacterial abundances and was more consistent over the same range of bacterial abundances per filter (range, 8.9 to 12.5%). The solid-phase cytometer method for conducting total direct counts of bacteria was less biased and performed significantly better than any of the microscope methods. It was also found that microscopic count data from counting 5 fields on three separate filters were statistically equivalent to data from counting 20 fields on a single filter.     

Biotechnology applied to cultural heritage: biorestoration of frescoes using viable bacterial cells and enzymes

AIMS: To set up and employ, for the biorestoration of cultural heritage (altered frescoes), an advanced and innovative biotechnology method based on the sequential use of whole viable bacterial cells and specific enzymes. METHODS AND RESULTS: The bioremediation intervention consisted of the direct application onto an artwork surface of whole bacterial cells of the Pseudomonas stutzeri A29 strain (bioaugmentation), followed by, in a final step, a purified Protease enzyme. The bioremediation was performed on a Spinello Aretino fresco that had become altered by the animal glue residues of past restoration. For the reader's interest the fresco is the 14th century Conversione di S. Efisio e battaglia (Conversion of S. Efisio and battle), size 3.5 x 7.8 m at the Pisa Camposanto Monumentale, Italy. An assessment was made of the final costs of the biological tests (whole bacterial cells, enzymes) so as to compare them with other intervention techniques. CONCLUSIONS: A successful innovative biological approach to recover valuable frescoes was set up, and the best conditions for treatment efficiency were identified. Furthermore the cost of the biological cleaning using viable bacterial cells and enzymes (P. stutzeri, Protease, Collagenase, 1 : 3 : 10, ratio respectively) was much lower than that of other conventional methods, making this biotechnology not only very interesting but also very competitive. SIGNIFICANCE AND IMPACT OF THE STUDY: New biotechnologies with an innovative, soft approach to the 'biocleaning' and 'biorestoration' of cultural heritage are in constant demand, and our results are clear evidence that such an approach has been achieved; the technique could be of significant importance towards developing other goals.     

Comparison of methods for quantitative determinations of airborne bacteria and evaluation of total viable counts.

Three different methods of estimating airborne bacteria were compared. An Anderson sampler, a slit sampler, an impinger, and filter samplers with gelatine filters or membrane filters were tested for collection efficiency. The comparisons were made in laboratory experiments with an aerosol of Staphylococcus epidermidis or Serratia marcescens, in field experiments in two different industries, i.e., cotton mill and sewage plant, and in experiments with skin fragment sampling. Experiments were also performed estimating the total number of viable microorganisms on the airborne particles. The Andersen sampler gave the highest bacterial counts in all environments tested. The slit sampler gave statistically lower counts only in the aerosol experiments and cotton mill experiments, where the size of the majority of the particles carrying visible bacteria was 2 to 6 micrometers or smaller. In the sewage plant and skin fragment experiments, where the particles were mainly 5 micrometers or larger, the difference was not significant. The filters were efficient in sampling in skin fragment experiments only. With the agar impingement method, the total viable cell count showed a rising index value with increasing particle size. A mean of 13 bacteria was found per particle in the cotton mill, a mean of 24 in the sewage plant, and a mean of 147 in skin fragment experiments.     

Comparison of Fluorescence Microscopy and Solid-Phase Cytometry Methods for Counting Bacteria in Water

Total direct counts of bacterial abundance are central in assessing the biomass and bacteriological quality of water in ecological and industrial applications. Several factors have been identified that contribute to the variability in bacterial abundance counts when using fluorescent microscopy, the most significant of which is retaining an adequate number of cells per filter to ensure an acceptable level of statistical confidence in the resulting data. Previous studies that have assessed the components of total-direct-count methods that contribute to this variance have attempted to maintain a bacterial cell abundance value per filter of approximately 10⁶ cells filter⁻¹. In this study we have established the lower limit for the number of bacterial cells per filter at which the statistical reliability of the abundance estimate is no longer acceptable. Our results indicate that when the numbers of bacterial cells per filter were progressively reduced below 10⁵, the microscopic methods increasingly overestimated the true bacterial abundance (range, 15.0 to 99.3%). The solid-phase cytometer only slightly overestimated the true bacterial abundances and was more consistent over the same range of bacterial abundances per filter (range, 8.9 to 12.5%). The solid-phase cytometer method for conducting total direct counts of bacteria was less biased and performed significantly better than any of the microscope methods. It was also found that microscopic count data from counting 5 fields on three separate filters were statistically equivalent to data from counting 20 fields on a single filter.     

Microbial validation of vent filters

The Upjohn Company uses filtration to remove microorganisms and particulates from air and other gases which may come in contact with sterile products. To validate the microbial retentivity of these filters, they were challenged with an aerosol of Bacillus subtilis var niger spores. An aerosol challenge was used because it more closely simulated the use for which these filters were designed. The test apparatus was constructed of autoclavable components using a jet-type nebulizer and heated air mixing tube. Characterization of the aerosol particle size distribution with a particle size analyzer demonstrated that 80% of the particles had a diameter of x 3.0 times;m and that the particles had a mean mass diameter of 1.9 times;m with a geometric standard deviation of 1.8 times;m. Studies conducted with aerosols of Bacillus subtilis var niger spores demonstrated that the test apparatus could recover ca. 50% of the spores that were aerosolized. Hydrophobic filters from various manufactures were challenged with an aerosol of at least 10(8) spores of Bacillus subtilis. All filters tested could retain at least 10(9) spores when physical integrity of the filter was verfield.     

Efficacy of iodine-treated biocidal filter media against bacterial spore aerosols

Aims: To assess the effectiveness of iodine-treated biocidal filter media against bacterial spore aerosols. Methods and Results: Bacillus subtilis spores were aerosolized and introduced into a filtration system. Both treated and untreated filters exhibited high viable removal efficiency (>99·996%) with negligible variation in pressure drop during the entire experiment. The viability of collected spores on the filter was investigated by enumeration of spores extracted from the filter by vortexing. At room temperature and low relative humidity (RH), the survival fraction of the treated filter was significantly lower than that of the untreated filter (P-value < 0·05). Meanwhile, at room temperature and high RH and at high temperature and high RH, the survival fractions on the treated medium were statistically the same as the untreated control at room temperature and low RH. Conclusions: Both treated and untreated filters achieved excellent viable removal efficiency for spores. The pressure drop of the treated filter was not affected by the iodine treatment. The viability of collected bacterial spores was decreased because of the exertion of iodine disinfectant. Significance and Impact of the Study: The evaluation demonstrates that the iodine-treated filter is a viable medium for respiratory protection against infectious spore aerosols. The results warrant further evaluation of smaller biological agents, which exhibit higher penetration.     

Use of selected excitation filters for enhancement of diaminobenzidine photomicroscopy

Enhancement of the diaminobenzidine (DAB) reaction product for light photomicroscopy was investigated using commercially available glass interference filters FITC-495, BG38, and BG12. The oxidized DAB transmission curve between 400-700 nm revealed a broad peak extending mostly through the yellow to red portions of the visible light spectrum, indicating that no single color predominates. Absorption spectra from the interference filters showed that FITC-495 gave total absorbance from 495-650 nm, with a smaller peak at 675 nm; BG38 transmitted at least a percentage of every wavelength up to 700 nm, whereas BG12 absorbed all light above 490 nm. To determine whether these filters could photographically increase DAB reaction product contrast, photographs were taken of corneal endothelial cells 24 hr after freeze injury. At this time, these cells demonstrate increased levels of laminin, as revealed by immunoperoxidase cytochemistry. When photography was performed using either no filter or a standard green filter, DAB contrast relative to background was minimal. However, when photographs were made using either the FITC-495 or the BG12 filter, DAB contrast increased sharply, although background density increased in the former case and decreased greatly in the latter. BG38 by itself did not increase DAB contrast. However, when used in combination with FITC-495 good DAB contrast was achieved and background density was lower than that seen using FITC-495 alone. Therefore, selective interference filters can photographically increase DAB contrast for studies using immunoperoxidase cytochemistry.     

Soft-agar-coated filter method for early detection of viable and thermostable direct hemolysin (TDH)- or TDH-related hemolysin-producing Vibrio parahaemolyticus in seafood

A novel method for detecting viable and thermostable direct hemolysin (TDH)-producing or TDH-related hemolysin (TRH)-producing Vibrio parahaemolyticus in seafood was developed. The method involved (i) enrichment culture, selective for viable, motile cells penetrating a soft-agar-coated filter paper, and (ii) a multiplex PCR assay targeting both the TDH gene (tdh) and TRH gene (trh) following DNase pretreatment on the test culture to eradicate any incidental DNAs that might have been released from dead cells of tdh- or trh-positive (tdh+ trh+) strains and penetrated the agar-coated filter. A set of preliminary laboratory tests performed on 190 ml of enrichment culture that had been inoculated simultaneously with ca. 100 viable cells of a strain of tdh+ trh+ V. parahaemolyticus and dense populations of a viable strain of tdh- and trh-negative V. parahaemolyticus or Vibrio alginolyticus indicated that the method detected the presence of viable tdh+ trh+ strains. Another set of preliminary tests on 190 ml of enrichment culture that had been initially inoculated with a large number of dead cells of the tdh+ trh+ strain together with dense populations of the tdh- and trh-negative strains confirmed that the method did not yield any false-positive results. Subsequent quasi-field tests using various seafood samples (ca. 20 g), each of which was experimentally contaminated with either or both hemolysin-producing strains at an initial density of ca. 5 to 10 viable cells per gram, demonstrated that contamination could be detected within 2 working days.     

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

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

Rapid and sensitive enumeration of viable diluted cells of members of the family enterobacteriaceae in freshwater and drinking water

Water quality assessment involves the specific, sensitive, and rapid detection of bacterial indicators and pathogens in water samples, including viable but nonculturable (VBNC) cells. This work evaluates the specificity and sensitivity of a new method which combines a fluorescent in situ hybridization (FISH) approach with a physiological assay (direct viable count [DVC]) for the direct enumeration, at the single-cell level, of highly diluted viable cells of members of the family Enterobacteriaceae in freshwater and drinking water after membrane filtration. The approach (DVC-FISH) uses a new direct detection device, the laser scanning cytometer (Scan RDI). Combining the DVC-FISH method on a membrane with Scan RDI detection makes it possible to detect as few as one targeted cell in approximately 10(8) nontargeted cells spread over the membrane. The ability of this new approach to detect and enumerate VBNC enterobacterial cells in freshwater and drinking water distribution systems was investigated and is discussed.     

Multilayer immobilized-enzyme filter reactors: urease bound to nylon fabric filters.

Urease was bound to commercially available nonwoven nylon fabric filters. Multilayer immobilized-enzyme filter reactors were constructed by packing varying numbers of urease-nylon filters in a column. Owing to the relatively open structure and high mechanical strength of the filter fabric, compaction and pressure drop effects were minimal. The reactors could be operated in a wide range of substrate concentrations and flow rates under conditions where mass-transfer limitations could be neglected. The kinetic behavior of the immobilized-enzyme filter reactors could be described by a linear form of the integrated Michaelis-Menten equation using a model based on the sequential action of the enzyme filters.     

Testing Air-Filtering Systems: I. Procedure for Testing High-Efficiency Air Filters on Exhaust Systems

A procedure was developed for evaluating high-efficiency filters mounted in exhaust ducts at the National Animal Disease Laboratory. An aerosol of the test organism, Escherichia coli B T₃ bacteriophage, was generated in a chamber attached to a ceiling exhaust register in concentrations of at least 1000 viable organisms per ft³ of air. Samples were collected from both the pre- and postfilter areas, and the number of organisms per ft³ of air was determined. The efficiency of the filter was calculated from these figures. A total of 269 high-efficiency filters were tested. Of these, 249 had efficiencies of 98% or greater. The remaining 20, with efficiencies of less than 98%, were repaired and retested. No filter was accepted with an efficiency of less than 98%.     

The direct epifluorescent filter technique (DEFT): increased selectivity, sensitivity and rapidity

With the direct epifluorescent filter technique (DEFT), differentiation of bacteria was achieved by a modified Gram-staining procedure using acridine orange as the counterstain. The method enumerated viable Gram-negative and all Gram-positive bacteria. Counts of clumps of orange fluorescent cells (Gram-negative DEFT count) correlated well with colony counts of Gram-negative bacteria in samples of raw milk (r = 0.94). The use of stainless steel membrane filter supports and the addition of citrate-NaOH buffer (0.1 M, pH 3.0) during filtration enabled 10 ml samples of milk to be filtered, thereby increasing the sensitivity of the DEFT five-fold. The relationship between colony and DEFT counts with 10 ml samples was better (r = 0.90) than that using standard 2 ml samples (r = 0.88). Alternatively, these modifications in procedure allowed the preincubation time for 2 ml milk samples to be reduced from 10 to 2 min. Sonication was successful in dispersing bacterial clumps in both pure cultures and in raw milk samples to yield a bacterial count by DEFT which should give a better indication of the hygienic status and keeping quality of a product, than counts of colony forming units.     

Efficacy of filter types for detecting Campylobacter jejuni and Campylobacter coli in environmental water samples by polymerase chain reaction.

A previously developed polymerase chain reaction (PCR) amplification of a target region in the flaA Campylobacter flagellin gene was evaluated and adapted for use with environmental water samples. The ability to detect Campylobacter jejuni or Campylobacter coli in seeded water samples was tested with various filters after concentration and freeze-thaw lysis of the bacterial cells. A nonradioactive probe for the amplified flagellin gene fragment detected as little as 1 to 10 fg of genomic DNA and as few as 10 to 100 viable C. jejuni cells per 100 ml of water filtered onto Fluoropore (Millipore Corp.) filters. No amplification was obtained with cellulose acetate filters, most likely because of binding of the DNA to the filter. Concentration and lysis of target cells on Fluoropore and Durapore (Millipore Corp.) filters allowed PCR to be performed in the same reaction tube without removing the filters. This methodology was then adapted for use with environmental water samples. The water supply to a broiler chicken production farm was suspected as the source of C. jejuni known to be endemic in grow-out flocks at the farm, despite the inability to culture the organisms by standard methods. The filtration-PCR method detected Campylobacter DNA in more than half of the farm water samples examined. Amplified campylobacter DNA was not detected in small volumes of regional surface water samples collected on a single occasion in February. The filtration-PCR amplification method provided a basis for detection of C. jejuni and C. coli in environmental waters with a high degree of specificity and sensitivity.     

Miniaturized direct on air sampling filter quantification of pollen allergens

A recently in this journal reported luminescence immunoassay for the direct quantification of birch and grass pollen allergens on air sampling filters. DOSIS, has been miniaturized. By means of a commercially available chlorinated analogue of the previously used 1,2 dioxetane phosphate derivative as enzyme substrate, the air sampling filter diameter could be reduced from 25 mm to 13 mm. The procedure leads to a more than twenty times reduction of the previously reported limit of quantification for the grass pollen allergen.