Nitrocellulose Membrane Filters Research Articles

Generation of electric potential difference by chromatophores from photosynthetic bacteria in the presence of trehalose under continuous illumination

Measurement of the electrical potential difference (Δψ) in membrane vesicles (chromatophores) from the purple bacterium Rhodobacter sphaeroides, associated with the surface of a nitrocellulose membrane filter (MF), impregnated with phospholipid solution in decane, as well as immersed inside it, in the presence of exogenous mediators and disaccharide trehalose, demonstrated an increase in the amplitude and stabilization of the signal under continuous illumination. The ascorbate/N,N,N′N′-tetramethyl-p-phenylenediamine pair and ubiquinone-0 were used as mediators (electron donor and acceptor), respectively. Although the stabilization of photoelectric responses under long-term continuous illumination was observed in both cases of immobilization of chromatophores, only when the samples were immersed inside the MF, the functional activity of the reaction centers was maintained for a month when they were stored in the dark at room temperature. Stable photoresponses are probably associated with the preservation of the integrity of chromatophore proteins inside MF pores. The stabilizing effect of the bioprotector trehalose may be due to both its effect on RC proteins and on the phospholipid bilayer membrane. The results obtained will expand modern ideas about the use of semi-synthetic structures based on various intact photosynthetic systems capable of converting solar energy into an electrochemical form.

Generation of Electric Potential Difference by Chromatophores from Photosynthetic Bacteria in the Presence of Trehalose under Continuous Illumination.

Measurement of electrical potential difference (Δψ) in membrane vesicles (chromatophores) from the purple bacterium Rhodobacter sphaeroides associated with the surface of a nitrocellulose membrane filter (MF) impregnated with a phospholipid solution in decane or immersed into it in the presence of exogenous mediators and disaccharide trehalose demonstrated an increase in the amplitude and stabilization of the signal under continuous illumination. The mediators were the ascorbate/N,N,N'N'-tetramethyl-p-phenylenediamine pair and ubiquinone-0 (electron donor and acceptor, respectively). Although stabilization of photoelectric responses upon long-term continuous illumination was observed for both variants of chromatophore immobilization, only the samples immersed into the MF retained the functional activity of reaction centers (RCs) for a month when stored in the dark at room temperature, which might be due to the preservation of integrity of chromatophore proteins inside the MF pores. The stabilizing effect of the bioprotector trehalose could be related to its effect on both the RC proteins and the phospholipid bilayer membrane. The results obtained will expand current ideas on the use of semi-synthetic structures based on various intact photosynthetic systems capable of converting solar energy into its electrochemical form.

A novel technique to obtain polysaccharide gels that emulate encapsulation beads with size and shape suitable for rheological, mechanical and other measurements

In this work we built up and tested diffusion cells designed to obtain polysaccharide hydrogels by ionotropic gelation. The objective was to obtain gel samples that emulated the wall material of encapsulation beads but, unlike them, have appropriate size and shape for measurement of their structural properties, since these are of paramount importance for the protection, carry and delivery of bioactive compounds. Three polysaccharides widely used for encapsulation and with different gelation mechanisms were tested: kappa/iota-carrageenan (which gels mainly in the presence of K+ ions), and sodium alginate and low methoxyl pectin (which both gel in the presence of Ca2+ ions). The diffusion cells consisted of two nitrocellulose membrane filters supported between three rings made by 3D printing. They were filled with the polysaccharide solution, hermetically tightened with screws and nuts, and submerged into the saline solution. The membrane filters effectively avoided the loss of the polysaccharide solution but allowed the diffusion of the ions (either Ca2+ or K+) into the polysaccharide solutions to promote their gelation. Firm, self-standing gels were obtained, and their rheological and mechanical properties were successfully measured. Samples of different shapes and sizes could be obtained with this technique, for other types of measurement.

Rapid and reversible detection of trace amounts of H2S in air and packaged food using a biogenic bismuth oxide nanorod colorimetric sensor

A colorimetric reversible sensor has been developed for detection of trace amount of H2S gas in air and sealed meat packages. The active sensing area includes high surface area bismuth oxide (Bi2O3) nanorod prepared by green synthesis method. The as fabricated sensor demonstrates a rapid response in just 25 s for sensing 1 ppm H2S gas with distinct colour change in the active sensing area from white to brown. Sensing pellets were used to optimize the performance of the H2S sensor in terms of sensitivity and selectivity in air. Flexible nitrocellulose membrane filter papers coated with Bi2O3 were used to detect spoilage in packaged raw animal food products such as meat, poultry and fish. The sensor is reversible and can be recovered within a short cycle (3 min) of heat treatment at 300 °C. X-ray diffraction, Raman spectroscopy and band gap measurements confirmed partial conversion of Bi2O3 nanorods into Bi2S3 when exposed in the range of 1–100 ppm H2S gas.

Comparing the filtering efficiency of kombucha SCOBY and nitrocellulose membrane filter

Kombucha is a fermented tea made from tea broth 5-8% Sucrose (granulated sugar) and SCOBY (symbiotic culture of bacteria and yeast), it is an alcoholic drink with 0.5 -2.5% alcohol. The origin of kombucha tea is said to be from China. The kombucha tea takes about 7-10 days for its fermentation. The fermentation takes place in two stages, wherein the first stage takes about 7-10 days, and the pellicle is formed in the air-water interface and in the second stage the flavoring of the kombucha is done with the fruit pulp and it is left for 10 days. In the first stage of fermentation, it forms a polymeric cellulose pellicle (SCOBY) with the help of certain species of bacteria and yeast-like lactic acid bacteria, Acetic acid bacteria and Saccharomyces species. The consumption of kombucha is good for health. The SCOBY is a biofilm formed by the action of acetic acid bacteria, lactic acid bacteria, and yeast and it is made of cellulose polymeric compound which can trap the microorganisms to a certain extent and retain them on the filter pad (SCOBY). The increased use of nitrocellulose membrane filter is used for filtering microorganisms from the sample is used extensively as it gives out efficient results, but the continuous and increased use of nitrocellulose filter is not good for the environment and for the human population who live near the industries where it is made. This study focuses on the use of SCOBY as a filter pad on various samples and finding out its efficiency when compared to the nitrocellulose membrane filter pad.

Development of capillary-paper spray for small-molecule analysis in complex samples.

We develop a capillary-paper spray (CPS) ion source which allows for sample separation in the capillary and enables rapid and sensitive paper spray (PS) mass spectrometry (MS) analysis of biofluids. The CPS employs a glass capillary to load liquid analytes, vertically standing at the rear of the PS. To further reduce the matrix effect, a nitrocellulose filter membrane is placed between the glass tube and chromatography paper to absorb proteins and other macromolecules, which is beneficial for the detection of the small molecules. Compared with the normal PS method, the CPS method markedly improves spray stability and prolongs analysis duration, and also generates significantly better signal intensities during the analysis of drugs, thus indicating its potential for clinical use. As a proof of concept, quantitative analysis of drugs (metformin hydrochloride and berberine hydrochloride) in serum is performed.

How Human H1 Histone Recognizes DNA.

Linker H1 histone is one of the five main histone proteins (H1, H2A, H2B, H3, and H4), which are components of chromatin in eukaryotic cells. Here we have analyzed the patterns of DNA recognition by free H1 histone using a stepwise increase of the ligand complexity method; the affinity of H1 histone for various single- and double-stranded oligonucleotides (d(pN)n; n = 1–20) was evaluated using their competition with 12-mer [32P]labeled oligonucleotide and protein–oligonucleotide complex delaying on nitrocellulose membrane filters. It was shown that minimal ligands of H1 histone (like other DNA-dependent proteins and enzymes) are different mononucleotides (dNMPs; Kd = (1.30 ± 0.2) × 10−2 M). An increase in the length of single-stranded (ss) homo- and hetero-oligonucleotides (d(pA)n, d(pT)n, d(pC)n, and d(pN)n with different bases) by one nucleotide link regardless of their bases, leads to a monotonic increase in their affinity by a factor of f = 3.0 ± 0.2. This factor f corresponds to the Kd value = 1/f characterizing the affinity of one nucleotide of different ss d(pN)n for H1 at n = 2–6 (which are covered by this protein globule) is approximately 0.33 ± 0.02 M. The affinity of five out of six DNA nucleotide units is approximately 25 times lower than for one of the links. The affinity of duplexes of complementary homo- and hetero-d(pN)20 is only 1.3–3.3-fold higher in comparison with corresponding ss oligonucleotides. H1 histone forms mainly weak additive contacts with internucleoside phosphate groups of ssDNAs and one chain of double-stranded DNAs, but not with the bases.

A rapid and sensitive lateral flow immunoassay (LFIA) test for the on-site detection of banana bract mosaic virus in banana plants

Banana bract mosaic virus (BBrMV) is a serious pathogen threatening the cultivation of banana and plantain worldwide. This study reports the development of a practical, rapid, sensitive, specific and user-friendly lateral flow immunoassay (LFIA) test for the on-site detection of BBrMV. The BBrMV coat protein (CP) was expressed in Escherichia coli and purified and used to immunize rabbits to produce a polyclonal antiserum (anti-BBrMVCP). The test was based on a double-antibody sandwich format. Protein-A affinity column-purified anti-BBrMVCP Immunoglobulins (IgG) (16 μg/mL), conjugated to ∼30 nm gold nanoparticles, was applied onto the conjugate pad. The anti-BBrMVCP IgG and goat anti-rabbit IgG were printed on the surface of a nitrocellulose filter membrane as the test line and control line, respectively. A positive result could be confirmed visually by the presence of a pink band that developed on the LFIA strip within 5−10 min. The detection limit of the test was 10 ng of the expressed recombinant BBrMV CP (rBBrMVCP), and a 1:20 dilution of the BBrMV-infected crude extract. This LFIA test was validated using 114 banana leaf samples randomly collected from the field and the results indicated a very high diagnostic sensitivity (99.04 %) and specificity (100 %) for the test. A Cohen's kappa coefficient of 0.861 obtained also indicated a very good agreement between the LFIA developed in this study and ELISA. This assay could be adopted by farmers, tissue culture industries and quarantine departments for surveys and surveillance. This is the first report on the development of a LFIA-based test for BBrMV detection.

Isolation and identification of free-living amoeba from the hot springs and beaches of the Caspian Sea

Free-living amoeba (FLA) such as Acanthamoeba, Naegleria, Balamuthia, and Vermamoeba have been identified from both natural and human-made environments such as Hot springs and spa. Naegleria fowleri causes Primary Amoebic Meningoencephalitis (PAM), while Acanthamoeba and Balamuthia cause chronic granulomatous encephalitis. Acanthamoeba also can cause cutaneous lesions and Amoebic Keratitis (AK) that is associated with contact lens use or corneal trauma. FLA are known to serve as host of and vehicles for diverse intracellular organisms. This study aimed was to identify the presence of FLA in the hot springs and beaches of the Caspian Sea in Ramsar tourist town located in the northern part of Iran. Water samples were collected in sterile bottles and were transferred to the laboratory. One litre of each sample passed through the nitrocellulose membrane filter. Each filter insert was then placed in non-nutrient agar plates already seeded with lawn culture of Escherichia coli. Positive samples were analyzed by morphological keys and Polymerase chain reaction (PCR) using 18S rDNA gene and ITS region to identify amoeba isolates. A total of 81 water sampled were tasted. After identified using the morphological key and PCR assay, 54 (66.6%) of the samples were positive for FLA. Ten of the samples were identified as Acanthamoeba (belong to T3, T4, and T5 genotypes), three as Vermamoeba vermiformis, four as Naegleria (3 N.australiensis and 1 N.grubery). Only one sample was positive Vahlkampfia. The presence of thermotolerant FLA in the Hot springs and beaches of the Caspian Sea as places for recreational purposes or wellness may be a potential health risk.

แผ่นกรองต้านแบคทีเรียจากผืนเส้นใยนาโนผสมระหว่างพอลิไวนิลลิดีนฟลูออไรด์กับ พอลิไวนิลแอลกอฮอล์

Antibacterial Poly (vinylidene fluoride) (PVDF) nanofibers can be used to produce filter with the property of liquid filtering and bacteriostatic effect. However, its present efficiency is still dissatisfied due to the low absorption of PVDF causing the limitation of antibacterial substances diffusion. In this study, the researcher introduced hydrophilic membrane by spinning with Poly (vinyl alcohol) nanofiber (PVA) to produce FA filter. The contact angle of FA filter has been reduced from 126 deg to 78 deg with the water absorption duration only of 1 minute and 20 seconds. The FA 8M antibacterial filter has the thickness of 60±3 micrometers, a pore size of 0.40 micrometer, and the porosity of 70±3%. The FA 8M antibacterial filter was compared with the commercial nitrocellulose membrane filter with the thickness of 125 micrometers and a pore size of 0.45 micrometers showing that the FA 8M antibacterial filter can effectively separate the bacteria from the water and still contain bacteriostatic effect though AgNO3 and BKC (Benzalkonium Chloride) added only 0.1% of the polymer weight.

Development of nitrocellulose membrane filters impregnated with different biosynthesized silver nanoparticles applied to water purification

Bactericidal water filters were developed. For this purpose, nitrocellulose membrane filters were impregnated with different biosynthesized silver nanoparticles. Silver nanoparticles (AgNPs) from Aspergillus niger (AgNPs-Asp), Cryptococcus laurentii (AgNPs-Cry) and Rhodotorula glutinis (AgNPs-Rho) were used for impregnating nitrocellulose filters. The bactericidal properties of these nanoparticles against Escherichia coli, Enterococcus faecalis and Pseudomona aeruginosa were successfully demonstrated. The higher antimicrobial effect was observed for AgNPs-Rho. This fact would be related not only to the smallest particles, but also to polysaccharides groups that surrounding these particles. Moreover, in this study, complete inhibition of bacterial growth was observed on nitrocellulose membrane filters impregnated with 1mgL−1 of biosynthesized AgNPs. This concentration was able to reduce the bacteria colony count by over 5 orders of magnitude, doing suitable for a water purification device.

Selection of cholesterol esterase aptamers using a dual-partitioning approach.

Non-systematic evolution of ligands by exponential enrichment and other capillary-based methods have grown in popularity for selection of aptamers since they provide a fast and efficient partitioning method when compared to classical techniques. Despite promising developments in these techniques, a major obstacle needs to be overcome for capillary-based selections to be widely accepted. Due to the small injection volumes associated with CE, only a small proportion of the nucleic acid library can be partitioned at any one time. In this paper, we propose a new two-step method for the selection of aptamers which firstly incorporates a nitrocellulose membrane filter followed by CE. This technique allows for nonbinding sequences to be eliminated, reducing the library size before subsequent capillary-based partitioning, while still reducing the time taken for aptamers to be selected. We demonstrated this technique on the selection of aptamers for cholesterol esterase and the highest binding truncated aptamer CES 4T displayed a K(D) of 203 ± 14 nM. In addition, an increase in the number of sequences partitioned was estimated using spectrophotometry and capillary injection volumes. The results suggested that for successful selection a two-step approach is needed. This hybrid technique could be used to select aptamers that bind to targets both in solution and immobilized onto a stationary phase, allowing the aptamers to be used in different binding environments.

Activation of the NOTCH signaling pathway stimulates migration of human b-lymphocytes

The NOTCH signaling pathway is required for embryonic development, cell differentiation, and for maintenance of tissue homeostasis and stem cell function. Consequently, dysfunction is associated with several genetic diseases or cancer development. Engagement in inflammatory disorders is under investigation. We explored whether the NOTCH ligands Jagged-1 and Delta-like-4 might affect migration of human B-lymphocytes in vitro. B-lymphocytes were isolated from EDTA-anticoagulated venous blood of healthy volunteers. For migration assays 5µm pore sized nitrocellulose membrane filters and a modified 48well microchemotaxis chamber were used. After an incubation period of 90 min., the distances cells migrated were measured microscopically. The “Chemotactic Index” was defined as the ratio between the distance cells migrated towards the NOTCH ligands and that towards control medium. Additionally, migration behavior was investigated after preincubation of cells with the γ-secretase inhibitor DAPT and ADAM17. We report for the first time that the NOTCH ligands Jagged-1 and Delta-like-4 significantly stimulate the migration of human B-lymphocytes in vitro. The observed effects were comparable to the maximal effects elicited by the chemoattractant fMLP. Furthermore, preincubation of cells with DAPT and ADAM17 diminished migration. Checkerboard assays showed that B-lymphocytes migrate both, in a chemokinetic and chemotactic manner in response to the NOTCH ligands tested. In conclusion this study shows that activation of the NOTCH signaling pathway can stimulate migration of B-lymphocytes and thus may serve as a new target in inflammatory pulmonary diseases.

유연한 CNT Nanosheet 기판을 이용한 생체연료전지 Roll 제작

The most promising application of the biofuel cells is implantable devices, so the biofuel cells should have an appropriate shape for the vascular vessel. We demonstrated the biofuel cell roll for using in tubes. MWNTs were aggregated by vacuum filtration on a nitrocellulose membrane filter, which was biocompatible and flexible. The MWNT aggregated nitrocellulose membrane used the electrodes of the biofuel cells because it was conductive as well as nanostuructured. Then, the membrane was rolled into the roll shape. The maximum power density of the biofuel cell roll was 7.9 μW/cm2 at 153mV and 50 mM glucose. Also, the power density is expected to increase in its practical application if there is flow in the tube, which makes the transportation of fuel easy. The biofuel cell roll contacts with the wall of the tube, so flow in the tube does not disturb. Also, the biofuel cell roll has multi-layers offering more electroactive area.

P66 * AN INVESTIGATION INTO SUBSTRATE EFFECTS AND TISSUE PRE-PROCESSING IN SPECTRAL HISTOPATHOLOGY USING RAMAN SPECTROSCOPY

INTRODUCTION: Raman spectroscopy is a low cost non-destructive and non-invasive technique showing great potential for the diagnosis of cancer. Biobanks are generally comprised of formalin fixed paraffin preserved (FFPP) tissue used in spectroscopic investigations. In order to carry out spectroscopic investigations the tissue needs to undergo a dewaxing process which removes and reduces paraffin contributions in the spectra. The ineffective removal of paraffin from the tissue and substrate contributions can affect the results and quality of the spectra. The aim of this study was to investigate the efficacy of tissue pre-processing steps used in spectral histopathology and the influence of commonly used substrates. METHOD: Nitrocellulose membrane filters of varying density, 8.0 µM, 5.0 µM and 1.2 µM were used to model human tissue. Filters underwent histological processing as clinical biopsies. Sections from each filter were cut at 10 microns and placed on three substrates, CaF2, Low-E and Glass which were subsequently dewaxed using two clearing agents, Histoclear and Xylene in three different bath times, 5 min, 10 min and 15 min. Spectra was collected from three biological replicates of each filter using Raman Spectroscopy with a 785 nm laser. RESULTS: CaF2 had the least background noise; however Glass and Low-E retained the least amount of paraffin whilst Xylene completely removed paraffin under the same bath conditions as Histoclear. CONCLUSION:Glass and Low-E are relatively cheaper and also provide reproducible results. These substrates can allow us to use standard clinical preparation procedures. Whilst Xylene is more effective in paraffin removal it is a hazardous chemical.

An antibody–magnetic nanoparticle conjugate-based selective filtration method for the rapid colorimetric detection of Listeria monocytogenes

In the present study, a method for the rapid colorimetric detection of Listeria monocytogenes based on selective filtration was developed by using monoclonal antibody (MAb)–magnetic nanoparticle (MNP) conjugates with a nitrocellulose filter membrane possessing a pore size of 1.2 μm. The conjugates bound and unbound to L. monocytogenes after being isolated on a magnet were directly passed through a nitrocellulose filter membrane by vacuum pressure, and the color signals from conjugate-bacteria that remain on the membrane are measured, reflecting the amount of bacteria in a sample. The capture efficiencies of the conjugates were confirmed to be 48% to 89% for 103, 102, and 101 cells per mL of L. monocytogenes. After optimization, 1.2 × 101 cells of L. monocytogenes were detectable in a pure pathogen solution from the standard curve, and the cut-off value for artificially inoculated vegetables by the method was 1 × 102 cells per g. Although weak cross-reactivity to Listeria spp. and Staphylococcus aureus was observed, the method was confirmed to be highly specific to L. monocytogenes. The method can be concluded within 35 min, yielding results in a shorter or similar period to recently reported antibody-immobilized MNP-based methods without additional steps such as dissociation of the target bacteria from MNP–MAb or enrichment. Therefore, the method could be used as a rapid, sensitive, and cost-effective tool for the detection of L. monocytogenes in various vegetables at a minimum detection level of 1 × 102 cells per g.

Comparison of ZetaPlus 60S and nitrocellulose membrane filters for the simultaneous concentration of F-RNA coliphages, porcine teschovirus and porcine adenovirus from river water

Increasing attention is being paid to the impact of agricultural activities on water quality to understand the impact on public health. F-RNA coliphages have been proposed as viral indicators of fecal contamination while porcine teschovirus (PTV) and porcine adenovirus (PAdV) are proposed indicators of fecal contamination of swine origin. Viruses and coliphages are present in water in very low concentrations and must be concentrated to permit their detection. There is little information comparing the effectiveness of the methods for concentrating F-RNA coliphages with concentration methods for other viruses and vice versa. The objective of this study was to compare 5 current published methods for recovering F-RNA coliphages, PTV and PAdV from river water samples concentrated by electronegative nitrocellulose membrane filters (methods A and B) or electropositive Zeta Plus 60S filters (methods C–E). Method A is used routinely for the detection of coliphages (Méndez et al., 2004) and method C (Brassard et al., 2005) is the official method in Health Canada's compendium for the detection of viruses in bottled mineral or spring water. When river water was inoculated with stocks of F-RNA MS2, PAdV, and PTV to final concentrations of 1×106PFU/100mL, 1×105gc/100mL and 3×105gc/100mL, respectively, a significantly higher recovery for each virus was consistently obtained for method A with recoveries of 52% for MS2, 95% for PAdV, and 1.5% for PTV. When method A was compared with method C for the detection of F-coliphages, PAdV and PTV in river water samples, viruses were detected with higher frequencies and at higher mean numbers with method A than with method C. With method A, F-coliphages were detected in 11/12 samples (5–154PFU/100mL), PTV in 12/12 samples (397–10,951gc/100mL), PAdV in 1/12 samples (15gc/100mL), and F-RNA GIII in 1/12 samples (750gc/100mL) while F-RNA genotypes I, II, and IV were not detected by qRT-PCR.

Effect of Gallium on Growth of Streptococcus mutans NCTC 10449 and Dental Tissues

Gallium-doped phosphate-based glasses (Ga-PBG) were assessed for their impact on Streptococcus mutans and dental mineralisation, firstly by disc diffusion assays followed by biofilms grown on nitrocellulose filter membrane (NFM) and constant-depth film fermentor (CDFF). Short-time exposure (10 min) effects of Ga-PBG on S. mutans biofilm were compared with that of 0.2% chlorhexidine. The effects of Ga-PBG on bovine enamel (which was investigated under pH-cycling condition) and dentine were analysed using transverse microradiography (TMR), profilometry and inductively coupled plasma optical-emission spectrometry (ICP-OES). The disc diffusion assays showed inhibition zones of 24.5 ± 0.5 mm for Ga-PBG compared with controls (C-PBG). Ga-PBG showed statistically significant growth inhibition of S. mutans biofilms on NFM (p = 0.001) and CDFF (p < 0.046) compared with hydroxyapatite (HA) and C-PBG. The CDFF assay revealed a maximum of 2.11 log colony-forming unit (CFU) reduction at 48 h, but short-time exposure effects were comparable with that of 0.2% chlorhexidine only on older biofilms (maximum of 0.59 vs. 0.69 log CFU reduction at 120 h). TMR analyses of the enamel revealed non-significant mineral loss (p = 0.37) only in the case of Ga-PBG samples compared with controls including sodium fluoride. ICP-OES analyses indicated transient gallium adsorption into dentine by calcium displacement. The results confirmed that gallium inhibited S. mutans growth and appears to have the potential to protect the enamel surface under conditions representative of the oral environment. Further work is needed to establish whether it has an application in daily oral hygiene procedures to prevent or reduce caries.

Fluorescent Brighteners with Nitrocellulose, Nylon, and Polyvinyl Chloride Membrane Filters for In Situ Staining of Fungi in Cultures

Objective: To test the fluorescent brightening (FB) staining method, in which fungi are grown on cellulose nitrate filter membranes, dried, and rendered transparent with oil after staining with lactophenol cotton blue and to encourage use of FBs to eliminate the need for drying and clearing the membranes, therefore simplifying the procedure and reducing the time to perform it. Methods: Uvitex 2B (U2B) and 9 calcofluor white (CFW) reagents were used with 25 genera or species of fungi grown on nitrocellulose (NC), nylon (N), and polyvinyl chloride (PVC) filter membranes placed on a variety of growth media. For microscopic examination, membranes were removed from the surface of the growth medium and placed on microscope slides, with a drop of FB added to each; the preparations were then coverslipped and examined via violet light epi-illumination. Results: With many of the fungi tested, if growth was not completely inhibited, it was restricted on the PVC compared to that on the NC and N membranes and directly on a medium surface. The fluorescence of fungal structures and their contrast with the background were favorable with all 3 types of membranes and all the FBs tested. For staining yeasts on membranes from media containing polysorbate 80 (TWEEN 80), U2B is preferable to CFW because TWEEN 80 interferes with CFW staining. Conclusion: FB staining of fungi grown on NC or N membrane filters provides a rapid and simple method to examine fungal growth, especially young cultures, with minimal disturbance. * LPCB : lactophenol cotton blue; FBs : fluorescent brighteners; CFW : calcofluor white; U2B : uvitex 2B; NC : nitrocellulose; N : nylon; PVC : polyvinyl chloride; PBS : phosphate-buffered saline; cSt : centistokes

Identification of a Bacterium Isolated from Soil of Ahvaz Contaminated by Oil and Determination of its Protease Stability in Organic Solvents

Background: Proteases are often used as catalysts for peptide synthesis. To shift the thermodynamic equilibrium in favor of the peptide synthesis, reaction media should contain organic solvent. However, known enzymes are usually inactivated by adding organic solvents to reaction media. Objectives: In this study, we reported a bacterium isolated from soil of Ahvaz, Iran contaminated by crude oil producing an organic solvent-stable protease, and the effect of organic solvents on proteolytic activity was investigated. Materials and Methods: Isolated bacterium was cultured in mineral salt medium containing glucose and peptone. After 48 hours incubation at 35 C and 130 rpm, culture media were centrifuged and resulted supernatant filtered using 0.22 ?m nitrocellulose membrane filter. Proteolytic activity of supernatant was determined by Keay and Wildi method (1970) by using casein as substrate. The effects of different concentrations of various organic solvents including acetone, ethanol, pentanol, cyclohexane, benzene, n-hexane, and n-decane and, also, the effects of temperature and pH on protease stability and activity were examined. Results: According to 16SrDNA sequencing, strain ISA9 was identified as a new strain of Bacillus licheniformis. This strain was able to produce an extracellular organic solvent- tolerant protease. After 30 minutes incubation at 37 C, caseinolytic activity of crude protease was increased in 25 and 50% of acetone, ethanol, benzene, cyclohexane, and hexane compared to non-solvent control. The enzyme was also activated 1.64, 1.23, and 1.17 times by 75% (v/v) of benzene, decane, and hexane, respectively. The protease was active in a broad range of pH (from 6 to 10) with the optimum pH 10. The optimum temperature for the activity of this protease was 70 C and the enzyme remained active after incubation at 30-50 C for 30 minutes. Conclusions: In this study, we isolated B. licheniformis producing an organic solvent-stable protease from oil-contaminated soil. The protease was stable and active in various organic solvents. By purification, the protease could be used as a biocatalyst for organic solvent-based enzymatic synthesis.