Filter Paper Disk Assay Research Articles

Isolation, Screening, and Active Metabolites Identification of Anti-Vibrio Fungal Strains Derived From the Beibu Gulf Coral.

The Beibu Gulf harbors abundant underexplored marine microbial resources, which are rich in diversified secondary metabolites. The genera Vibrio is a well-known pathogenic bacterium of aquatic animals. In this study, 22 fungal strains were isolated and identified from the Beibu Gulf coral via the serial dilution method and internal transcribed spacer (ITS) sequence analysis, which were further divided into three branches by phylogenetic tree analysis. The crude extracts of them via small-scale fermentation were selected for the screening of inhibitory activity against Vibrio alginalyticus, Vibrio coralliilyticus, Vibrio harveyi, Vibrio parahaemolyticus, Vibrio owensii, and Vibrio shilonii. The results showed that eight fungal extracts displayed anti-Vibrio activity via the filter paper disk assay. Several of them showed strong inhibitory effects. Then, two tetramic acid alkaloids, equisetin (1) and 5′-epiequisetin (2), were identified from Fusarium equiseti BBG10 by bioassay-guided isolation, both of which inhibited the growth of Vibrio spp. with the MIC values of 86–132 μg/ml. The scanning electron microscope results showed that cell membranes of Vibrio became corrugated, distorted or ruptured after treatment with 1 and 2. Taken together, this study provided eight fungal isolates with anti-Vibrio potentials, and two alkaloid-type antibiotics were found with anti-Vibrio effects from the bioactive strain F. equiseti BBG10. Our findings highlight the importance of exploring promising microbes from the Beibu Gulf for the identification of anti-Vibrio for future antibiotic development.

Potential Assessment of Rumex spp. as a Source of Bioactive Compounds and Biological Activity

Secondary metabolites from the shoots and roots of three Rumex species collected from three different habitats were investigated (Rumex dentatus collected from cultivated land, R. pictus collected from the coastal desert and R. vesicarius collected from the inland desert) and tested for antioxidant activity as well as for anti-microbial activity against some human pathogenic bacteria. The present study indicated that the quantitative analysis of shoot and root extracts of three Rumex spp. were found to be rich in tannins and phenolics composition. The aerial parts of the three plants exhibited the highest significant values compared to the root parts. The MeOH extracts of Rumex species showed adequate antioxidant activity, wherein the IC50 values of the MeOH from the cultivated sample was 41.61 and 31.31 mg mL-1, coastal samples were 34.99 and 23.99 mg mL-1, while the sample of inland showed IC50 value of 41.59 and 31.67 mg mL-1, for root and shoot, respectively. Furthermore, using a filter paper disc assay, the MeOH extracts of the three Rumex species showed a substantial anti-microbial inhibitory effect on the growth of 10 pathogenic bacteria. According to sensitivity, the tested organisms could be sequenced as following: E. coli < K. pneumoniae ˂ S. typhi < P. aeruginosa for Gram-negative bacteria and B. subtilis < S. pneumoniae ˂ L. monocytoyenes < S. epidermis < S. aureus < B. cereus for Gram-positive bacteria. In addition, the antibacterial performance of R. dentatus root and R. vesicarius shoot MeOH extract is 100% broad spectrum against Gram-negative bacteria. A shoot of R. dentatus and R. pictus MeOH extract against Gram-positive bacteria is 83.3% broad spectrum. A further study is recommended for more characterization of the major compounds and assesses their efficiency and biosafety.

Antibacterial activity of Calotropis procera against some common human pathogens

Calotropis procera (Oshaar, family Asclepiadaceae) is a species of flowering plant used as a folk medicine for the treatment of various ailments. This study aimed to evaluate antibacterial and antioxidant activities of C. procera aerial parts collected from Eastern Desert of Egypt (Wadi Hagul). The different extracts of C. procera aerial parts showed a significant antimicrobial inhibitory effect (P < 0.05) on growth of nine pathogenic bacterial using filter paper disc assay. Qualitative and quantitative phytochemical screening was carried out on water and different extracts. The present study indicated that this plant has a good inhibitory activity against Klebsiella pneumonia (21.3 mm), Escherichia coli (22.5 mm), and Staphylococcus aureus (21.6 mm). Moreover, methanol extract of C. procera exhibited wide broad spectrum (100%) followed by petroleum ether and water extracts (60%, each). Activity index (AI) illustrated variable antibacterial activities, that support the folkloric usage of medicinal plants for treatment of infectious pathogenic microorganism. The crude extract of C. procera showed adequate antioxidant activity, wherein methanol, ethyl acetate and petroleum ether extracts have an IC50 value of 0.81, 0.88 and 1.09 mg mL-1, respectively. Therefore, phytochemicals from C. procera could be used as a raw material for producing cheaper pharmaceuticals, pesticides in addition to many more important commercialized products of public use.

Culture-dependent analysis of seed bacterial endophyte, Pseudomonas spp. EGN 1 against the stem rot disease (Sclerotium rolfsii Sacc.) in groundnut

BackgroundGroundnut, Arachis hypogaea L., crop is an important oil seed crop in India and is prone to attack by numerous fungal and viral diseases, among the soil-borne diseases, stem rot caused by Sclerotium rolfsii Sacc. is economically important.Main body of abstractIncidence (86.6%) of the stem rot disease was recorded in Coimbatore district, Tamil Nadu, India, during the roving survey. For its management, an attempt was made to isolate bacterial endophyte from peanut seeds of different groundnut cultivars by culture-dependent analysis. Totally, 16 bacterial endophytes (endophytes groundnut (EGN) 1 to EGN 16 along with standards TNAU-Pf1 and EPC 5) were obtained and characterized through morphological, biochemical and molecular studies and also phytostimulation activities were performed. Among the isolates, EGN 1 and EGN 4 showed positive results for indole acetic acid (IAA), siderophore, phosphate solubilization and protease tests in vitro. The dual culture analysis showed inhibition rates of 60.1% (dual plate assay), 68.23% (filter paper disc assay) and 100% (triangle method of streaking and culture filtrate assay) for EGN 1 against S. rolfsii. Further, the crude metabolite assay showed 97.7% inhibition in EGN 1, followed by 87.7% in EGN 4. The roll towel study showed a high vigour index of 4286.7 in EGN 1; hence, this isolate was chosen. Further, thin-layer chromatography (TLC) analysis showed various bands at 0.72 Rf, whereas GC-MS analysis indicated the prominent peaks of hexadecanoic acid and cis-vaccenic acid that may responsible for antifungal activity. In a molecular approach, the genomic DNA of EGN 1 strain was used to amplify a 1200 bp PCR-fragment and sequenced.Short conclusionThe overall outcome of this study showed that Pseudomonas spp. EGN 1 had a great potential as a bio-stimulant and biocontrol agent to manage effectively the stem rot in peanut.

Hydroxamate-based colorimetric method for direct screening of transglutaminase-producing bacteria

Microbial transglutaminase (MTGase) is a commercial enzyme that has been applied to many protein containing foods to improve their textural property. The screening of MTGase-producing microorganisms from various sources might lead to the discovery of a new MTGase with different characteristics. This report demonstrates the use of a direct detection method for MTGase-producing bacteria grown on an agar plate by filter paper disc (FPD) assay. The principle of the assay is the formation of a red burgundy color by the hydroxamate-ferric complex. The color developed intensity was linearly correlated by the concentration of hydroxamic acid in the range of 0.1-0.8 μM and was visually scored at 4 levels: 0, 1, 2 and 3. Streptoverticillium mobaraense DSM 40847, a positive MTGase-producer, was chosen for the verification and improving of the proposed method. The colonies grown on the nutrient agar plate at 37°C for 24 h were covered with FPDs and 30 μl of substrates (CBZ-Gln-Gly and hydroxylamine). After incubation, 10 μl of the ferric-TCA-HCl solution was placed on the FPD. The optimal time taken to catalyze the formation of CBZ-Gln-Gly-hydroxamic acid by the MTGase and the time taken for the hydroxamate-ferric complex to form color were 180 and 60 min, respectively. Using this assay, 30 of 189 colonies isolated from wastewater and floating-floc samples showed MTGase-positive colonies which were well correlated to the quantitative screening of MTGase activity (R(2) = 0.9758). The results revealed that the FPD assay could be used for the qualitative screening of MTGase-producing bacteria.

In vitro evaluation of solvent extracted compounds from edible macromycetes against phytopathogenic fungi

Extraction of antimicrobial compounds from medicinal mushrooms, namely, Auricularia polytricha (Jew's ear), Lentinulla edodes (Shiitake) and Volvariella volvacea (paddy straw), has been done with different solvent systems (chloroform, ethyl acetate, ether and methanol) and tested against a wide range of phytopathogens by filter paper disk assay. All the three basidiomycetes inhibited the phytopathogens tested so far. This enumeration was based on the number of organisms inhibited and the diameter of inhibitory zones produced. From the results obtained, it could be observed that ethyl acetate was the best solvent for extracting antimicrobial substances from these medicinal mushrooms. Thin layer chromatography revealed the presence of several compounds with different Retention Factor (RF) values. Sodium dodecyl sulphate–polyacrylamide gel electrophoresis profiling of V. volvacea showed the presence of several antimicrobial proteins with various molecular weights. Western blotting revealed the presence of thaumatin-like glycoproteins of molecular weight more than 45 kDa.

Antifungal leaf-surface metabolites correlate with fungal abundance in sagebrush populations.

A central component in understanding plant-enemy interactions is to determine whether plant enemies, such as herbivores and pathogens, mediate the evolution of plant secondary metabolites. Using 26 populations of a broadly distributed plant species, sagebrush (Artemisia tridentata), we examined whether sagebrush populations in habitats with a greater prevalence of fungi contained antifungal secondary metabolites on leaf surfaces that were more active and diverse than sagebrush populations in habitats less favorable to fungi. Because moisture and temperature play a key role in the epidemiology of most plant-pathogen interactions, we also examined the relationship between the antifungal activity of secondary metabolites and the climate of a site. We evaluated the antifungal activity of sagebrush secondary metabolites against two fungi, a wild Penicillium sp. and a laboratory yeast, Saccharomyces cerevisiae, using a filter-paper disk assay and bioautography. Comparing the 26 sagebrush populations, we found that fungal abundance was a good predictor of both the activity (r2 = 0.36 for Saccharomyces, r2 = 0.37 for Penicillium) and number (r2 = 0.34 for Saccharomyces) of antifungal secondary metabolites. This suggests that selection imposed by fungal pathogens has led to more effective antifungal secondary metabolites. We found that the antifungal activity of sagebrush secondary metabolites was negatively related to average vapor pressure deficit of the habitat (r2 = 0.60 for Saccharomyces, r2 = 0.61 for Penicillium). Differences in antifungal activity among populations were not due to the amount of secondary metabolites, but rather to qualitative differences in the composition of antifungal compounds. Although all populations in habitats with high fungal prevalence had secondary metabolites with high antifungal activity, different suites of compounds were responsible for this activity, suggesting independent outcomes of selection on plants by fungal pathogens. The location of antifungal secondary metabolites on the leaf surface is consistent with their putative defense role, and we found no evidence supporting other functions, such as protection from ultraviolet light or oxidation. That the antifungal activity of sagebrush secondary metabolites was similar for two different fungi provides support for broad antifungal defenses. The incidence and severity of fungal disease in the field (caused by Puccinia tanaceti) were similar in moist and dry habitats, possibly reflecting an equilibrium between plant defense and fungal attack, as sites with greater fungal abundance compensated with more effective secondary metabolites. The geographic correlation between fungal abundance and antifungal secondary metabolites of sagebrush, coupled with our other data showing heritable variation in these metabolites, suggests that pathogenic fungi have selected for antifungal secondary metabolites in sagebrush.

In vitro inhibition of Candida albicans growth by plant extracts and essential oils

The opportunistic Candida albicans yeast strain ATCC 10261 grows at 37 °C and gives germ tubes after 3 h on corn meal agar and blood plasma. It produces chlamydospores and assimilates sucrose, dextrose, galactose, maltose, trehalose and xylose among the tested carbon sources. Other growth characters were also investigated. The agar diffusion cut plug technique revealed that 200 μl of Foeniculum vulgare Miller (fennel), Mentha piperita L. (peppermint) and Citrus limon (lemon) essential oils showed inhibitory actions. Fumigation test of the three essential oils had complete inhibition on the growth. The essential oil of Eucalyptus occidentalis End1 (eucalyptus, eucalypt) had no influence on C. albicans growth by the two tests. Meanwhile, methanol extracts of both leaves and male cones of the conifer Thuja orientalis (eastern thuya) had an inhibitory influence on the growth by two tests (cut plug and filter paper disc assay). In comparative tests the antibiotics mycostatin and dermatin had an inhibitory activity on C. albicans at the concentrations of 50–80 μg per hole. Crude methanol extract of leaves of eastern thuya (10–80 μg/ml) reduced C. albicans growth and intercellular protein nitrogen on liquid media.

Detection of human immunodeficiency virus-1 nucleic acid on inactivated filter paper disks by polymerase chain reaction and microtiter plate assay.

Human immunodeficiency virus type 1 (HIV-1) in cultured cells, peripheral blood samples and sera were adsorbed on filter paper disks and inactivated by heat or ethanol. Two procedures, the polymerase chain reaction (PCR) and microtiter plate assay (HMPA) were used to detect the nucleic acid. The sensitivity after different heat treatments with nested PCR for HIV-1 DNA (or nested reverse transcription-PCR for HIV-1 RNA) was identical regardless of whether the samples were examined immediately or one month later. Inactivation by ethanol treatment resulted in a slight loss of sensitivity. The HMPA proved to be as reliable and specific as the conventional PCR technique. We conclude that the heat-treated filter paper disk assay is suitable for identifying HIV nucleic acid in clinical samples sent to the laboratory from a distance, e.g. in an envelope.

Antimicrobial screening of medicinal plants from Baja California Sur, Mexico

The ethanolic extracts of 72 plants belonging to 35 different families, and used in traditional medicine in Baja California Sur (México), were tested for antimicrobial activity in vitro using the filter paper disk assay method. Activity against Staphylococcus aureus. Bacillus subtilis. Streptococcus faecalis (Gram-positive microorganisms), Escherichia colt (Gram-negative microorganisms) and Candida albicans (yeast) is discussed.

The Analysis of Plasmid-Mediated Streptomycin Resistance inErwinia amylovora

Streptomycin-resistant mutants of Erwinia amylovora were isolated from an apple orchard in Michigan and from crabapple trees adjacent to the same orchard in 1990. Isolates that grew on King's medium B amended with 100 μg/ml of streptomycin sulfate were considered to be resistant strains, whereas isolates that failed to grow on this medium were considered to be sensitive strains. Growth of the resistant strains was not inhibited in a filter-paper disk assay (0.06-5 μg of streptomycin sulfate), but growth of sensitive strains was inhibited at concentrations as low as 0.06 μg of streptomycin sulfate (...)

Preliminary screening of far-eastern ethnomedicinal plants for antibacterial activity

The results of antibacterial screening of 308 ethanol extracts from 250 wild far-eastern plants belonging to 77 families are reported. Antibacterial activity of plant extracts was tested in vitro by the filter-paper disc assay method. The plants screened were reported to be used by the population of the Soviet Far East against some bacterial infections. Activity against grampositive as well as gram-negative groups of microorganisms was tested.

Inhibition of Pathogenic Bacteria by Camel's Milk: Relation to Whey Lysozyme and Stage of Lactation

Twenty (10%) of 200 milk samples collected from different individual camels inhibited growth of one or more of six pathogenic test organisms in a filter-paper disk assay. The milk samples with inhibitory properties scored zero in the California Mastitis Test. The percentages of milk samples which inhibited Clostridium perfringens, Staphylococcus aureus, Shigella dysenteriae and Salmonella typhimurium were 7.5, 4.0, 2.0 and 1%, respectively. None of the 200 samples inhibited Bacillus cereus or Escherichia coli. Five milk samples inhibited growth of two test organisms and two samples inhibited growth of three organisms. Inhibition zone diameters ranged from 13.5mm to 22.0mm (average of 15 mm) using a disk of 12.5mm in diameter. The average amount of lysozyme in the whey of 58 samples of camel's milk was 288 μg/100 ml. The average lysozyme content of the 20 samples showing growth inhibition of one or more test organisms was 648 μg/100 ml, significantly higher (P<0.01) than the average in the 38 samples (62.6 μg/100 ml) that had no inhibitory effect. There was a negative correlation (r= −0.669) between lysozyme content in the whey of milk samples (y) and the stage of lactation of the camels (x) with the following regression equation: y = −2.86x + 665.8.

A semiautomated assay procedure for the determination of aminoacyl-tRNA synthetase activity

A semiautomated assay procedure for the determination of aminoacyl-tRNA synthetase activity is described. The assay system employs a Technicon AutoAnalyzer with a continuous filter paper attachment. A larger number of samples can be measured with greater accuracy, reproducibility, and more rapidly than the manual filter paper disk assay system.

An improved filter-disc assay for 3H-DNA and serum deoxyribonucleases

1. The filter-paper disc assay for tritium-labeled nucleic acids was modified to reduce washing and manipulation. Evaluation of the procedure indicated greatly enhanced counting efficiency for tritium, complete removal of nuclease digestion products and retention of even nanogram levels of radioactive DNA on the discs in the presence of protein. The modified assay was quite sensitive and had a coefficient of variation of 5.2%. Quite low concentrations of DNase I and endogenous human serum nuclease activity were readily measured by the improved technique, 22 sera yielding a mean value equivalent to about 0.5 ng DNase I/ml serum. We are now using the disc assay to follow serum DNA and DNase levels in patients with autoimmune disorders and various types of cancers.

A rapid filter paper disk assay for picomole amounts of cyclic AMP using a cyclic AMP dependent protein kinase.

A rapid filter paper disk assay for cyclic AMP based on the activation of a cyclic AMP dependent protein kinase from rabbit skeletal muscle is described. This procedure permits determination of as little as 0.2 picomoles of cyclic AMP. In addition this procedure employs ATP-γ-33P instead of ATP- γ-32P.

Potential of wild plant Artemisia judaica L. as sustainable source of antioxidant and antimicrobial compounds

Artemisia judaica L. (Shih Balady, family Asteraceae), is a medicinal plant that grows in the desert, commonly used as tea by population in Egypt Sinai and of Saudi Arabia. The present study aims to evaluate antimicrobial and antioxidant activities of aerial parts of A. judaica collected from the inland desert (Wadi Hagoul) of Egypt. Total phenolics, tannins, alkaloids, flavonoids, and saponins were measured in the plant of interest. According to the reduction of DPPH the anti-oxidant activity was measured. Different extracts of A. judaica were screened for their antimicrobial activity against nine pathogenic microorganisms using filter paper disc assay. The secondary metabolites of A. judaica were examined and proved that it was rich in tannins and phenolics (13.29 and 7.62 mg/g dw). The IC50 values of A. judaica extract was 1.78 mg.ml-1 compared to standard catechol (0.15 mg.ml-1). In the present study, the extracts of A. judaica showed unusual activity depending on the type of bacteria and fungi. Also, ethyl acetate and methyl alcohol extracts showed considerable broad spectrum (77.78%, each) against both bacteria and fungi, followed by acetone, then methylene chloride and petroleum ether extracts. This study showed that A. judaica extracts could be used as antimicrobial substances in pharmaceutical and food preservation systems.

Use of a filter-paper disk assay in the measurement of lipid biosynthesis

The precipitation by trichloroacetic acid (TCA) of radioactive lipids on disks of filter-paper, followed by extraction with TCA and water to remove soluble radioactive precursors, provides the means for a convenient assay for lipid biosynthesis. Large numbers of disks can be washed in the same vessel, dried, and assayed for radioactivity with liquid scintillation counting equipment. Extraction of disks with lipid solvents provides a convenient control for the possible presence of TCA- and water-insoluble, nonlipid products. The application of the method to the assay of cyclopropane fatty acid synthetase is shown.