Automation of microbial enumeration: development of a disposable hydrophobic grid-membrane filter unit.

A disposable filter unit containing a hydrophobic grid-membrane filter (HGMF) was developed. The unit is liquid tight to serve as a specimen transport container and, by removal of the funnel extender (175- or 300-ml capacity), the unit becomes less than the height of two stacked petri plates to save space during in situ incubation. The polyethylene mesh which supports the HGMF facilitates rinse removal of any substance(s) that would interfere with microbial growth. The correlations between a pour plate, a conventional square HGMF, and a disposable filter unit on microbial enumeration were examined. Characteristics (e.g., clumping, spreading, etc.) of some microorganisms limit the linear counting range to less than 1,000 CFU per filter.     

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

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

COMPARISON OF MICROBIOLOGICAL METHODS FOR MONITORING CHICKEN CARCASS QUALITY

Many methods have been developed to determine microbial levels in food products and these include ATP bioluminescence, hydrophobic grid membrane filtration (HGMF), impediometry and turbidimetry. A comparison of these techniques for detecting microbial levels in chicken carcass rinses was conducted.
Only the ATP bioluminescence assay and the HGMF system showed a good correlation with plate counts (r = 0.82 and r = 0.83, respectively). The repeatability of these methods was acceptable. There was also a significant correlation between results obtained with two turbidimetric methods and HGMF as well as between HGMF and ATP bioluminescence data. However, only the ATP bioluminescence assay was able to provide results of microbial levels on a realtime basis (within 10 min). This would be beneficial for HACCP (Hazard Analysis of Critical Control Points) based programs.     

Enumeration of High Numbers of Bacteria Using Hydrophobic Grid-Membrane Filters

Printing a wax grid on a conventional membrane filter yields a device functioning as a most probable number apparatus (MPN), used at a single dilution but with a very large number of growth compartments (e.g., 3,650). By restraining the lateral spread and confluence of colonies, the hydrophobic grid-membrane filter (HGMF) allows growth- or colony-forming units (GU) to be resolved at levels far above those which produce an uncountable lawn on a conventional membrane filter. It also eliminates the size variation of normal bacterial colonies. As a result, the HGMF can give more accurate estimates of the concentration of GU. The method by which grid-cell count observations can be used to obtain MPN estimates of the number of GUs is described, and estimates obtained using the MPN method on the HGMF are compared with those resulting from conventional colony count procedures on membrane filters. A linear relation was observed between MPNGU and the number of GUs, at levels up to 30,000 GUs, for pure cultures of bacteria and for samples of natural waters. The HGMF has great potential for reducing the labor required in quantitative microbiology, since it allows, with one filter, enumeration of microorganisms over a very large concentration range and therefore reduces the need to make dilutions.     

Improved detection of coliforms and Escherichia coli in foods by a membrane filter method.

Analytical procedures based on filtration of homogenates through membrane filters, and particularly hydrophobic grid-membrane filters (HGMF), offer definite improvements in the enumeration of Escherichia coli and coliforms in foods. Whereas the counted specimen in pour plates may not usually be greater than 0.1 g, up to 1.0 g of ground beef, green beans, potato, cod, strawberries, or grapes could be filtered and counted on HGMF. Greatly improved limit of detection, reduced interference by noncoliforms, and complete removal of growth inhibitors such as polyphenols were demonstrated for HGMF, using violet red bile and mFC agars. In addition, counting on HGMF eliminated a false-positive reaction caused by sucrose in ice cream.     

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

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

疏水网格滤膜技术检测食源性致病菌的研究进展

疏水网格滤膜技术利用膜的疏水性黏附细菌细胞,通过膜的过滤作用截留细菌细胞,然后将膜进行选择性培养,达到检测鉴定待测菌的目的。主要概述了疏水网格滤膜技术的原理和特点,以及该技术与分子生物学技术、免疫学技术偶联在食源性致病菌检测中的应用。     

Improved detection of coliforms and Escherichia coli in foods by a membrane filter method.

Analytical procedures based on filtration of homogenates through membrane filters, and particularly hydrophobic grid-membrane filters (HGMF), offer definite improvements in the enumeration of Escherichia coli and coliforms in foods. Whereas the counted specimen in pour plates may not usually be greater than 0.1 g, up to 1.0 g of ground beef, green beans, potato, cod, strawberries, or grapes could be filtered and counted on HGMF. Greatly improved limit of detection, reduced interference by noncoliforms, and complete removal of growth inhibitors such as polyphenols were demonstrated for HGMF, using violet red bile and mFC agars. In addition, counting on HGMF eliminated a false-positive reaction caused by sucrose in ice cream.     

ICMSF methods study. XVII. An international comparative study of the direct plate and hydrophobic grid-membrane filter methods for enumeration of Escherichia coli in foods. International Commission on Microbiological Specifications for Foods

Eight laboratories compared counts of Escherichia coli from naturally or artificially contaminated ground beef, other meats and poultry, vegetables, fish and shellfish, cheese, and diverse sources such as swabs, by the Anderson-Baird-Parker direct plate (DP) and a hydrophobic grid-membrane filter (HGMF) method. For five of the eight laboratories overall counts by HGMF were significantly low (51-83%) compared with those by DP. Counts by HGMF tended to be lower for naturally contaminated samples; several possible causes were investigated. In a subsidiary study, analyst variation in counting HGMF ranged from 0.8-7.3%, with little evidence of effects from counting positive versus negative grid cells or from the fullness of growth or staining intensity.     

Rapid detection of Salmonella spp. in food by use of the ISO-GRID hydrophobic grid membrane filter.

A rapid hydrophobic grid-membrane filter (HGMF) method was developed and compared with the Health Protection Branch cultural method for the detection of Salmonella spp. in 798 spiked samples and 265 naturally contaminated samples of food. With the HGMF method, Salmonella spp. were isolated from 618 of the spiked samples and 190 of the naturally contaminated samples. The conventional method recovered Salmonella spp. from 622 spiked samples and 204 unspiked samples. The isolation rates from Salmonella-positive samples for the two methods were not significantly different (94.6% overall for the HGMF method and 96.7% for the conventional approach), but the HGMF results were available in only 2 to 3 days after sample receipt compared with 3 to 4 days by the conventional method.     

Development of a novel disposable filter bag for separation of biomolecules with functionalized magnetic particles

The integration of disposable magnetic filters in combination with functionalized magnetic particles represents a fast and cost‐effective alternative for enzyme purification in comparison to solid/liquid separation by means of centrifugation followed by chromatographic purification. The main advantage of the particle‐based process is the solid/solid/liquid separation in one step combined with disposable equipment. Furthermore this combination provides the possibility to also process biocatalytic reactions in cell‐containing media into disposable equipment with preimmobilized enzymes onto the magnetic particles. The focus of the presented study is on the design and performance of a disposable filtration unit consisting of a plastic bag with an inlet and outlet and a stainless steel filter matrix. During magnetic separation, the magnetic particles selectively retard at the filter matrix due to the magnetic force, which counteracts the drag force. It was found that the length of a lengthwise aligned filter matrix should be longer than the magnetic pole surfaces in fluid flow direction. Hereby, a filtration capacity of 5.6 g magnetic particles was measured with a loss of below 0.5%. Introducing a two‐phase flow optimizes the cleaning of the bag after a magnetic filtration. The procedure offered a high cleaning efficiency. Herewith, the cleaned filter unit could be discarded with minimum losses of product and magnet particles.     

Improved Aerobic Colony Count Technique for Hydrophobic Grid Membrane Filters

The AOAC International official action procedure for performing aerobic colony counts on hydrophobic grid membrane filters (HGMFs) uses Trypticase soy-fast green FCF agar (FGA) incubated for 48 h. Microbial growths are various shades of green on a pale green background, which can cause problems for automated as well as manual counting. HGMFs which had been incubated 24 or 48 h at 35°C on Trypticase soy agar were flooded underneath with 1 to 2 ml of 0.1% triphenyltetrazolium chloride (TTC) solution by simply lifting one corner of the filter while it was still on the agar and adding the reagent. Microbial growths on HGMFs were counted after color had been allowed to develop for 15 min at room temperature. With representative foods, virtually all colonies stained pink to red. Automated electronic counts made by using the MI-100 HGMF Interpreter were easier and more reliable than control HGMF counts made by the AOAC International official action procedure. Manual counting was easier as well because of increased visibility of the microbial growths. Except in the case of dairy products, 24-h TTC counts did not differ significantly from 48-h FGA counts, whereas the FGA counts at 24 h were always significantly lower, indicating that for many food products the HGMF TTC flooding method permits aerobic colony counts to be made after 24 h.     

Magnetophoretic induction of curvature in coleoptiles and hypocotyls

Coleoptiles of barley (Hordeum vulgare) were positioned in a high gradient magnetic field (HGMF, dynamic factor gradient of H(2)/2 of 10(9)-10(10) Oe2 cm-1), generated by a ferromagnetic wedge in a uniform magnetic field and rotated on a 1 rpm clinostat. After 4 h 90% of coleoptiles had curved toward the HGMF. The cells affected by HGMF showed clear intracellular displacement of amyloplasts. Coleoptiles in a magnetic field next to a non-ferromagnetic wedge showed no preferential curvature. The small size of the area of nonuniformity of the HGMF allowed mapping of the sensitivity of the coleoptiles by varying the initial position of the wedge relative to the coleoptile apex. When the ferromagnetic wedge was placed 1 mm below the coleoptile tip only 58% of the coleoptiles curved toward the wedge indicating that the cells most sensitive to intracellular displacement of amyloplasts and thus gravity sensing are confined to the top 1 mm portion of barley coleoptiles. Similar experiments with tomato hypocotyls (Lycopersicum esculentum) also resulted in curvature toward the HGMF. The data strongly support the amyloplast-based gravity-sensing system in higher plants and the usefulness of HGMF to substitute gravity in shoots.     

Optical microbial biosensor for detection of methyl parathion pesticide using Flavobacterium sp. whole cells adsorbed on glass fiber filters as disposable biocomponent

An optical microbial biosensor was described for the detection of methyl parathion pesticide. Whole cells of Flavobacterium sp. were immobilized by trapping in glass fiber filter and were used as biocomponent along with optic fiber system. Flavobacterium sp. has the organophosphorus hydrolase enzyme, which hydrolyzes the methyl parathion into detectable product p-nitrophenol. The immobilized microbial biocomponent was disposable, cost-effective and showed high reproducibility and uniformity. The detection of methyl parathion by the use of disposable microbial biocomponent with optical biosensor was simple, single step and direct measurement of very low quantity of the sample. The home made reaction vessel was small and needed only 75 microl of sample. A lower detection limit 0.3 microM methyl parathion was estimated from the linear range (4-80 microM) of calibration plot of organophosphorus hydrolase enzymatic assay. The applicability to synthetic methyl parathion spiked samples was demonstrated.     

A closed system for the filtration of Mycobacterium bovis liquid cultures using disposable capsule filters

A filtration system was designed to sterilize large volumes of Mycobacterium bovis BCG Tokyo culture safely, needed to purify protein antigens for immunodiagnosis of bovine tuberculosis. A closed system consists of culture bottles connected to three disposable filter capsules of decreasing pore size in series : a depth prefilter over a 1·2 μm filter ; a 0·8 μm prefilter over a 0·45 μm filter ; and a 0·2 μm sterile filter. Low air pressure (3 psi) forces liquid from below the bacillary pellicle. The system features a stainless steel clamp to hold rubber stoppers on the culture bottles, pleated filters to exclude bacillary clumps, a quick disconnector to minimize aerosols, and a closed system with plastic disposable filters that can be autoclaved as a unit without dismantling.     

Altered development of Xenopus embryos in a hypogeomagnetic field

The hypogeomagnetic field (HGMF; magnetic fields <200 nT) is one of the fundamental environmental factors of space. However, the effect of HGMF exposure on living systems remains unclear. In this article, we examine the biological effects of HGMF on the embryonic development of Xenopus laevis (African clawed frog). A decrease in horizontal third cleavage furrows and abnormal morphogenesis were observed in Xenopus embryos growing in the HGMF. HGMF exposure at the two‐cell stage, but no later than the four‐cell stage, is enough to alter the third cleavage geometry pattern. Immunofluorescent staining for α‐tubulin showed reorientation of the spindle of four‐cell stage blastomeres. These results indicate that a brief (2‐h) exposure to HGMF is sufficient to interfere with the development of Xenopus embryos at cleavage stages. Also, the mitotic spindle could be an early sensor to the deprivation of the geomagnetic field, which provides a clue to the molecular mechanism underlying the morphological and other changes observed in the developing and/or developed embryos. Bioelectromagnetics 33:238–246, 2012. © 2011 Wiley Periodicals, Inc.     

An evaluation of a water purification system for use in animal facilities

A commercially available water purification system was evaluated for its ability to minimize chemical and microbial contaminants. The reduction or removal of these impurities from the drinking water of experimental animals would reduce experimental variability. 3 strains of bacteria were collected from the processed water. An increase in the total number of bacteria was observed the longer the filters remained in use. Determinations of heavy metals in water samples before and after processing were made for lead, zinc, copper, nickel, manganese, iron, arsenic and mercury. Calcium and magnesium levels were also determined. The concentrations of these inorganic chemicals were reduced by the purification process except at 2 time points in which desorption of the chemical could have occurred. Bacterial colonization and desorption of these chemicals were controlled by installing new filter cartridges. Volatile halocarbon concentrations were determined for water samples before and after purification. All volatile halocarbons analyzed were less than 10 ppb before and after purification at all time points. Other organic chemicals were greatly reduced by the purification process. In a study of contaminants associated with installation of the unit, it was found that flushing the unit for 8 days reduced lead and methyl ethyl ketone concentrations to insignificant levels. The purification system was found to be effective in providing high quality drinking water as verified by a microbial and chemical testing program.     

Purification of equine chorionic gonadotropin (eCG) using magnetic ion exchange adsorbents in combination with high‐gradient magnetic separation

Current purification of the glycoprotein equine chorionic gonadotropin (eCG) from horse serum includes consecutive precipitation steps beginning with metaphosphoric acid pH fractionation, two ethanol precipitation steps, and dialysis followed by a numerous of fixed‐bed chromatography steps up to the specific activity required. A promising procedure for a more economic purification procedure represents a simplified precipitation process requiring only onethird of the solvent, followed by the usage of magnetic ion exchange adsorbents employed together with a newly designed ‘rotor‐stator’ type High Gradient Magnetic Fishing (HGMF) system for large‐scale application, currently up to 100 g of magnetic adsorbents. Initially, the separation process design was optimized for binding and elution conditions for the target protein in mL scale. Subsequently, the magnetic filter for particle separation was characterized. Based on these results, a purification process for eCG was designed consisting of (i) pretreatment of the horse serum; (ii) binding of the target protein to magnetic ion exchange adsorbents in a batch reactor; (iii) recovery of loaded functionalized adsorbents from the pretreated solution using HGMF; (iv) washing of loaded adsorbents to remove unbound proteins; (v) elution of the target protein. Finally, the complete HGMF process was automated and conducted with either multiple single‐cycles or multicycle operation of four sequential cycles, using batches of pretreated serum of up to 20 L. eCG purification with yields of approximately 53% from single HGMF cycles and up to 80% from multicycle experiments were reached, with purification and concentration factors of around 2,500 and 6.7, respectively. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:78–89, 2015     

A novel UASB–MFC–BAF integrated system for high strength molasses wastewater treatment and bioelectricity generation

An up-flow anaerobic sludge blanket reactor–microbial fuel cell–biological aerated filter (UASB–MFC–BAF) system was developed for simultaneous bioelectricity generation and molasses wastewater treatment in this study. The maximum power density of 1410.2 mW/m² was obtained with a current density of 4947.9 mA/m² when the high strength molasses wastewater with chemical oxygen demand (COD) of 127,500 mg/l was employed as the influent. The total COD, sulfate and color removal efficiencies of the proposed system were achieved of 53.2%, 52.7% and 41.1%, respectively. Each unit of this system had respective function and performed well when integrated together. The UASB reactor unit was mainly responsible for COD removal and sulfate reduction, while the MFC unit was used for the oxidation of generated sulfide with electricity generation. The BAF unit dominated color removal and phenol derivatives degradation. This study is a beneficial attempt to combine MFC technology with conventional anaerobic–aerobic processes for actual wastewater treatment.     

Retention and removal of the fish pathogenic bacterium Yersinia ruckeri in biological sand filters

AIMS: To investigate the retention and removal of the fish pathogenic bacterium Yersinia ruckeri in biological sand filters and effects on the microbial community composition. METHODS AND RESULTS: Sand filter columns were loaded (70 mm day(-1)) with fish farm wastewater and a suspension (10(8) CFU ml(-1)) of Y. ruckeri. Bacterial numbers and protozoan numbers were determined by plate counts and epifluorescence microscopy, respectively, and microbial biomass and community composition were assessed by phospholipid fatty acids (PLFA) analysis. Concentrations of Y. ruckeri in the filter effluent decreased from 10(8) to 10(3)-10(5) CFU ml(-1) during the experiment. Numbers of Y. ruckeri in the sand decreased from 10(6) CFU g(-1) dry weight (DW) sand to 10(4) CFU g(-1) DW sand. In contrast, microbial biomass determined with plate counts and total PLFA increased during the whole experiment. Principal component analysis (PCA) revealed a change in microbial community composition with time, with the most pronounced change in surface layers and towards the end of the experiment. Protozoan numbers increased from ca 0-600 cells g(-1) DW sand, indicating the establishment of a moderate population of bacterial grazers. CONCLUSIONS: The removal of Y. ruckeri improved during the experiment. Introduction of Y. ruckeri to the sand filter columns stimulated growth of other micro-organisms, which in turn caused a shift in the microbial community composition in the sand. SIGNIFICANCE AND IMPACT OF THE STUDY: This study increases the understanding of the dynamics of sand filters subjected to a high loading of a pathogenic bacterium and can therefore be used in future work were the overall aim is to provide a more reliable and efficient removal of pathogenic bacteria in biological sand filter systems.