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SCHMALZ, G. "Agar overlay method." International Endodontic Journal 21, no. 2 (September 25, 2007): 59–66. http://dx.doi.org/10.1111/j.1365-2591.1988.tb00956.x.
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YAN, ZHINONG, JOSHUA B. GURTLER, and JEFFREY L. KORNACKI. "A Solid Agar Overlay Method for Recovery of Heat-Injured Listeria monocytogenes." Journal of Food Protection 69, no. 2 (February 1, 2006): 428–31. http://dx.doi.org/10.4315/0362-028x-69.2.428.
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A solid agar overlay method was developed for recovery of heat-injured Listeria monocytogenes. Presolidified nonselective tryptic soy agar with 0.6% yeast extract (TSAYE, 2% agar) was overlaid on top of solidified modified Oxford agar (MOX). Heat injury of L. monocytogenes was conducted at 58°C for 6 min in a jacketed flask filled with tryptic soy broth. Both noninjured and heat-treated L. monocytogenes cells were plated onto TSAYE, MOX, and TSAYE-MOX plates. No significant differences (P > 0.05) in recovery were found among the three media for noninjured bacterial cells. Recovery of heat-injured L. monocytogenes cells on TSAYE-MOX overlay plates was equivalent to that on the nonselective TSAYE medium, whereas recovery on the selective MOX medium was significantly lower (P < 0.05) compared with both TSAYE and the overlay plates. There were no significant differences (P > 0.05) among the overlay plates prepared 0, 2, 4, 6, 8, 16, and 24 h prior to plating heat-injured bacterial cells. The TSAYE-MOX overlay also allowed differentiation of L. monocytogenes from a mixture of four other types of foodborne pathogens. This solid agar overlay method for recovery of heat-injured L. monocytogenes cells is less time-consuming and less complicated than the conventional overlay-underlay technique and the double overlay modification of the thin agar layer method and may allow for greater laboratory plating efficiencies.
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Parreira, Valéria Regina, Clarice Weis Arns, and Tomomasa Yano. "An agar-overlay method for detection of toxins produced byEscherichia coli." FEMS Microbiology Letters 120, no. 3 (July 1994): 303–6. http://dx.doi.org/10.1111/j.1574-6968.1994.tb07050.x.
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BACK, KYEONG-HWAN, SANG-OH KIM, KI-HWAN PARK, MYUNG-SUB CHUNG, and DONG-HYUN KANG. "Spray Method for Recovery of Heat-Injured Salmonella Typhimurium and Listeria monocytogenes." Journal of Food Protection 75, no. 10 (October 1, 2012): 1867–72. http://dx.doi.org/10.4315/0362-028x.jfp-11-512.
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Selective agar is inadequate for supporting recovery of injured cells. During risk assessment of certain foods, both injured and noninjured cells must be enumerated. In this study, a new method (agar spray method) for recovering sublethally heat-injured microorganisms was developed and used for recovery of heat-injured Salmonella Typhimurium and Listeria monocytogenes. Molten selective agar was applied as an overlay to presolidified nonselective tryptic soy agar (TSA) by spray application. Heat-injured cells (55°C for 10 min in 0.1% peptone water or 55°C for 15 min in sterilized skim milk) were inoculated directly onto solidified TSA. After a 2-h incubation period for cell repair, selective agar was applied to the TSA surface with a sprayer, and the plates were incubated. The recovery rate for heat-injured Salmonella Typhimurium and L. monocytogenes with the spray method was compared with the corresponding rates associated with TSA alone, selective media alone, and the conventional overlay method (selective agar poured on top of resuscitated cells grown on TSA and incubated for 2 h). No significant differences (P > 0.05) were found in pathogen recovery obtained with TSA, the overlay method, and the spray method. However, a lower recovery rate (P < 0.05) was obtained for isolation of injured cells on selective media. Overall, these results indicate that the agar spray method is an acceptable alternative to the conventional overlay method and is a simpler and more convenient approach to recovery and detection of injured cells.
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Drijber, R. A., and W. B. McGill. "A modification of the method using cellulose overlay agar to isolate and purify cellulolytic cytophagas from enrichment culture." Canadian Journal of Microbiology 38, no. 7 (July 1, 1992): 687–89. http://dx.doi.org/10.1139/m92-111.
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We report here on a modification of the cellulose overlay agar method for isolating and purifying cellulolytic cytophagas from enrichment cultures. We call it the "back-door" method, and it overcomes two existing problems. First, it prevents recontamination with organisms growing on the agar surface. Second, it permits purification of cellulolytic cytophagas that are unable to utilize glucose or that are accompanied by a rapidly growing contaminant with spreading habit. Pure cultures of cellulolytic cytophagas were obtained from five enrichment cultures of nine soil samples examined. Two limitations are that (i) the cytophaga must be able to penetrate the overlay and (ii) some noncellulolytic cytophagas may also penetrate the overlay to yield cocultures. In conclusion, the back-door method can be used both to isolate and purify cellulolytic cytophagas from soils, using only a cellulose-based medium. Key words: back-door method, cellulolytic cytophagas, cellulose overlay agar, cellulolysis.
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노태희, 이승연, Jeong Moon-Jin, and 이명화. "Cytotoxicity comparison of titanium by agar overlay method for various cell line applications." Korean Journal of Oral Anatomy 38, no. 1 (December 2017): 1–11. http://dx.doi.org/10.35607/kjoa.38.1.201712.001.
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Mackenzie, A. M., and R. L. Rivera-Calderon. "Agar overlay method to measure adherence of Staphylococcus epidermidis to four plastic surfaces." Applied and Environmental Microbiology 50, no. 5 (1985): 1322–24. http://dx.doi.org/10.1128/aem.50.5.1322-1324.1985.
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Nuthan, Bettadapura Rameshgowda, Devaraju Rakshith, Kuppuru Mallikarjunaiah Marulasiddaswamy, H. C. Yashavantha Rao, Kolathur Puttamadaiah Ramesha, Nagabhushana Chandra Mohana, Shiva Siddappa, Doreraj Darshan, Kigga Kaadappa Sampath Kumara, and Sreedharamurthy Satish. "Application of Optimized and Validated Agar Overlay TLC–Bioautography Assay for Detecting the Antimicrobial Metabolites of Pharmaceutical Interest." Journal of Chromatographic Science 58, no. 8 (August 7, 2020): 737–46. http://dx.doi.org/10.1093/chromsci/bmaa045.
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Abstract The agar overlay TLC–bioautography is one of the crucial methods for simultaneous in situ detection and separation of antimicrobial metabolites of pharmaceutical interest. The main focus of this research relies on the dereplication of an antimicrobial metabolite coriloxin derived from mycoendophytic Xylaria sp. NBRTSB-20 with a validation of agar overlay TLC–bioautography technique. This polyketide metabolite coriloxin was purified by column chromatography, and its purity was assessed by HPLC, UPLC-ESI-QTOF-MS, FT-IR and NMR spectral analysis. The antimicrobial capability of ethyl acetate extract and the purified compound coriloxin was determined by disc diffusion, minimal inhibitory concentration and agar overlay TLC–bioautography assay. The visible LOD of coriloxin antimicrobial activity was found at 10 μg for Escherichia coli and 20 μg for both Staphylococcus aureus and Fusarium oxysporum. Inter- and intra-day precision was determined as the relative standard deviation is less than 6.56%, which proved that this method was precise. The accuracy was expressed as recovery, and the values were found ranging from 91.18 to 108.73% with RSD values 0.94–2.30%, respectively. The overall findings of this investigation suggest that agar overlay TLC–bioautography assay is a suitable and acceptable method for the in situ determination of antimicrobial pharmaceuticals.
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Kang, D. H., and G. R. Siragusa. "Agar Underlay Method for Recovery of Sublethally Heat-Injured Bacteria." Applied and Environmental Microbiology 65, no. 12 (December 1, 1999): 5334–37. http://dx.doi.org/10.1128/aem.65.12.5334-5337.1999.
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ABSTRACT A method of recovering sublethally heat-injured bacteria was developed. The procedure (termed the agar underlay method) uses a nonselective agar underlaid with a selective medium. In a two-chambered petri dish, the Lutri plate (LP), a nonselective agar is inoculated with a population of sublethally heat-injured bacteria. After a 2-h repair incubation period, selective agar is added to the bottom chamber of the LP and incubated. By diffusing through the nonselective top agar, selective agents from the underlay medium impart selectivity to the system. By the agar underlay method, recovery rates of the heat-injured food-borne pathogens Escherichia coli O157:H7 and Salmonella typhimurium were not different (P > 0.05) from recovery rates determined with nonselective media. Sublethally heat-injured cells (60°C for 1.5 min in buffer or 80°C for 30 s on meat surfaces) grew and produced a typical colony morphology and color reaction when the agar underlay procedure was used with the appropriate respective selective agars. Unlike agar overlay methods for injury repair, the agar underlay procedure allows the typical selective-medium colony morphology to develop and allows colonies to be more easily picked for further characterization. Higher recovery rates of heat-injured fecal enterococci from bovine fecal samples and total coliforms from animal waste lagoons were obtained by the agar underlay method with selective agars than by direct plating on the respective selective media.
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Rahalison, L., M. Hamburger, K. Hostettmann, M. Monod, and E. Frenk. "A bioautographic agar overlay method for the detection of antifungal compounds from higher plants." Phytochemical Analysis 2, no. 5 (November 1991): 199–203. http://dx.doi.org/10.1002/pca.2800020503.
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HARRIS, P. L., S. L. CUPPETT, and L. B. BULLERMAN. "A Technique Comparison of Isolation of Lipolytic Bacteria." Journal of Food Protection 53, no. 2 (February 1, 1990): 176–77. http://dx.doi.org/10.4315/0362-028x-53.2.176.
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Three different agar diffusion screening methods, employing two growth media, standard plate count agar (SPCA) and 1% peptone enriched trypticase soy agar (TSA), were used to isolate psychrotrophic bacteria from raw milk which produce lipase. Clarified butterfat and nile blue sulfate served as the substrate and dye, respectively, in all methods. Although a double-overlay method yielded slightly greater numbers of lipolytic bacteria than a single-layered method with comparable media, better visual clarity was achieved with the single-layered method. Peptone enriched TSA medium supported better growth of lipolytic bacteria than SPCA.
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Bange, Jill, Emily Brumfield, and Alysha L. Ellison. "Recovery and Enumeration of Staphylococcus aureus by the Selective Agar." Fine Focus 2, no. 1 (January 1, 2016): 51–59. http://dx.doi.org/10.33043/ff.2.1.51-59.
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Staphylococcus aureus isan example of a commensal bacterium responsible for emesis, acute diarrheal syndrome, and sepsis. S. aureus often must be isolated from patient samples in a clinical setting or from food samples during food processing in an industrial setting, although these bacterial cells may be injured by the human immune system or by food processing measures. Therefore, injured cells may not be fully recovered on media selective for S. aureus and enumeration (e.g., CFU/mL) may not reflect the true concentration of the original sample. The objective of this study was to determine whether the selective agar overlay method of recovery is more sensitive, selective, and time-effective for enumeration of artificially injured S. aureus cultures when compared to more traditional techniques. The selective agar overlay method involves pour plating S. aureus in non-selective medium, allowing the sample to incubate for a four hour recovery period, and then overlaying selective medium over the non-selective medium. Artificial injury of S. aureus cells was accomplished by treatment with carvacrol, an extract from oil of oregano. Our results indicated that carvacrol-injured S. aureus cells were recovered by the selective agar overlay at the same concentration as recovery on non-selective media, and at a significantly higher concentration than recovery on selective media. This method allows for more rapid and accurate diagnoses, and may be more cost-effective due to the reduction or elimination of false negative results.
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CHARTERIS, WILLIAM P., PHILLIP M. KELLY, LORENZO MORELLI, and J. KEVIN COLLINS. "Gradient Diffusion Antibiotic Susceptibility Testing of Potentially Probiotic Lactobacilli." Journal of Food Protection 64, no. 12 (December 1, 2001): 2007–14. http://dx.doi.org/10.4315/0362-028x-64.12.2007.
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Minimum inhibitory contentrations (MICs) of selected inhibitors of cell wall synthesis (benzylpenicillin, ampicillin, and vancomycin), protein synthesis (gentamicin, streptomycin, tetracycline, chloramphenicol, and erythromycin), and nucleic acid synthesis (co-trimoxazole, rifampicin, and metronidazole) were determined by gradient diffusion (E test; AB Biodisk, Solna, Sweden) on deMan, Rogosa, Sharpe (MRS) agar for Lactobacillus strain GG and 11 closely related, rapidly growing, facultatively anaerobic, potentially probiotic Lactobacillus rhamnosus strains. All strains were resistant to vancomycin (MIC90 ≥256 μg/ml), co-trimoxazole (MIC90 ≥32 μg/ml), metronidazole (MIC90 ≥32 μg/ml), gentamicin (MIC90 ≥128 μg/ml), and streptomycin (MIC90 ≥256 μg/ml), and sensitive to pencillin G (MIC90 >0.375 μg/ml), ampicillin (MIC90 >0.750 μg/ml), rifampicin (MIC90 >0.375 μg/ml), tetracycline (MIC90 >1.5 μg/ml), chloramphenicol (MIC90 >8 μg/ml), and erythromycin (MIC90 >2 μg/ml). E test MICs were also determined for L. acidophilus National Collection of Food Bacteria (NCFB) 1748 and L. reuteri Deutsche Sammlung von Mikroorganismen 20016T by the inoculum application method recommended by the manufacturer (swabbing), with and without antibiotic prediffusion for 1 h at room temperature, and by an alternative inoculum application (agar overlay) method, without antibiotic prediffusion. Antibiotic prediffusion increased the MICs for penicillin G, ampicillin, tetracycline, and chloramphenicol by up to 2 log2 MIC dilutions without changing antibiotic susceptibility category. Agar overlay application also increased the MICs for these antibiotics as well as for gentamicin by up to 3 log2 MIC dilutions without changing antibiotic susceptibility category. Exact agreement between MICs determined by swab and agar overlay application without antibiotic prediffusion was strain dependent: 54.5% for strain DSM 20016T and 72.7% for strain NCFB 1748. The swab and agar overlay gradient diffusion methods provide a reliable basis for antibiotic susceptibility testing of rapidly growing, facultatively anaerobic lactobacilli, using MRS agar as test medium and are readily applicable for testing individual isolates as needed.
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KANG, DONG-HYUN, and DANIEL Y. C. FUNG. "Thin Agar Layer Method for Recovery of Heat-Injured Listeria monocytogenes." Journal of Food Protection 62, no. 11 (November 1, 1999): 1346–49. http://dx.doi.org/10.4315/0362-028x-62.11.1346.
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A thin agar layer (TAL) method was developed to recover heat-injured Listeria monocytogenes. Modified Oxford medium (MOX), a selective plating medium, inhibits heat-injured L. monocytogenes from growing, whereas tryptic soy agar (TSA), a nonselective medium, does not. In order to facilitate recovery of heat-injured L. monocytogenes cells while providing selectivity of isolation of L. monocytogenes from other bacteria in the sample, a unique TAL procedure was developed by overlaying 5 ml of nonselective medium (TSA) onto prepoured and solidified MOX medium in an 8.5-cm–diameter petri dish. The injured L. monocytogenes repaired and started to grow in the TSA during the first few hours after incubation of the plate. During the resuscitation of injured cells, the selective agents from MOX diffused to the TSA top layer to inhibit other microorganisms. L. monocytogenes showed a typical reaction (black colonies) on TAL after 24 h of incubation at 37°C. The recovery rate for heat-injured L. monocytogenes with the TAL method was compared with those rates associated with TSA, MOX, and the traditional overlay method (OV; pouring selective agar on top of resuscitated cells on TSA agar after 3 h incubation). Milk and 0.1% peptone water that were inoculated with L. monocytogenes (4 to 5 log CFU/ml) were heated for 15 min at 55°C. L. monocytogenes was enumerated on TSA, MOX, OV, and TAL media and procedures. No significant difference occurred among TSA, OV, and TAL (P > 0.05) in terms of enumeration of heat-injured L. monocytogenes, but these media recovered significantly higher numbers than did MOX agar (P < 0.05)—in both samples. The TAL method involves only one step, whereas OV is a more cumbersome two-step procedure.
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Fanani, Ahmad Zainal, Tisa Ayu Novitasari, Siti Choiriah Rofi’ah, Siti Nur Rukhoiyah Dewi, and Heru Pramono. "IDENTIFIKASI DAN UJI AKTIVITAS ANTIBAKTERI KAPANG LAUT DIISOLASI DARI KORAL LUNAK FAVITES SP." BIOLINK (Jurnal Biologi Lingkungan Industri Kesehatan) 6, no. 1 (May 21, 2019): 16. http://dx.doi.org/10.31289/biolink.v6i1.2253.
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The aims of this study was to investigate the antimicrobial activity of fungi associated with coral. The fungi was cultured by streak plate method on potato dextrose agar (PDA) and reisolated with the same medium and were stored in slant agar for identified with morphological approach. Antibacterial activity fungi associated with coral was tested using overlay method against Escherichia coli and Staphylococcus aureus. Based on morphological analysis, five fungi were identified as Candidasp., Fusariumsp., Cladophialophorasp., Phaeoacremoniumsp., and Trichophytonsp. The antibacterial assay showed that Phaeoacremoniumsp. and Trichophytonsp. has antibacterial activity and potential as source of antibacterial for futher study.
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Ballester, Nicola A., Justin H. Fontaine, and Aaron B. Margolin. "Occurrence and correlations between coliphages and anthropogenic viruses in the Massachusetts Bay using enrichment and ICC-nPCR." Journal of Water and Health 3, no. 1 (March 1, 2005): 59–68. http://dx.doi.org/10.2166/wh.2005.0006.
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We evaluated a two-step enrichment procedure to detect coliphages and an integrated cell culture-nested polymerase chain reaction (ICC-nPCR) to detect human astrovirus, enteroviruses, rotavirus and adenovirus type 40 and 41 in marine water samples collected by the Massachusetts Water Resource Authority (MWRA). MWRA has been monitoring its receiving waters for coliphages, anthropogenic viruses and indicator bacteria in order to evaluate the impact of Boston's Deer Island Sewage Treatment Plant discharge. Coliphages and enteric viruses were originally assayed using single agar overlay and most probable number cell culture (MPN) methods, respectively. Reanalysis of these samples for enteric viruses by ICC-nPCR demonstrated that 46% were positive for at least one virus compared with 23% with the MPN method. Use of the enrichment method showed a 47% increase in the detection of male specific and somatic coliphages compared with the single agar overlay method. Correlations between the presence of coliphages, enteric viruses and indicator bacteria were based on proximity to the treatment plant discharge, seasonal variations and site levels. The presence of enteric viruses was significantly correlated to coliphages but not to indicator bacteria. Preliminary comparative results demonstrate that effective and efficient monitoring of anthropogenic contamination can be achieved using these more sensitive and specific techniques.
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Müller, Rainer, Gerhard Gröger, Karl-Anton Hiller, Gottfried Schmalz, and Stefan Ruhl. "Fluorescence-Based Bacterial Overlay Method for Simultaneous In Situ Quantification of Surface-Attached Bacteria." Applied and Environmental Microbiology 73, no. 8 (February 16, 2007): 2653–60. http://dx.doi.org/10.1128/aem.02884-06.
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ABSTRACT For quantification of bacterial adherence to biomaterial surfaces or to other surfaces prone to biofouling, there is a need for methods that allow a comparative analysis of small material specimens. A new method for quantification of surface-attached biotinylated bacteria was established by in situ detection with fluorescence-labeled avidin-D. This method was evaluated utilizing a silicon wafer model system to monitor the influences of surface wettability and roughness on bacterial adhesion. Furthermore, the effects of protein preadsorption from serum, saliva, human serum albumin, and fibronectin were investigated. Streptococcus gordonii, Streptococcus mitis, and Staphylococcus aureus were chosen as model organisms because of their differing adhesion properties and their clinical relevance. To verify the results obtained by this new technique, scanning electron microscopy and agar replica plating were employed. Oxidized and poly(ethylene glycol)-modified silicon wafers were found to be more resistant to bacterial adhesion than wafers coated with hydrocarbon and fluorocarbon moieties. Roughening of the chemically modified surfaces resulted in an overall increase in bacterial attachment. Preadsorption of proteins affected bacterial adherence but did not fully abolish the influence of the original surface chemistry. However, in certain instances, mostly with saliva or serum, masking of the underlying surface chemistry became evident. The new bacterial overlay method allowed a reliable quantification of surface-attached bacteria and could hence be employed for measuring bacterial adherence on material specimens in a variety of applications.
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Shin, Michelle, Jung-Wei Chen, Chi-Yang Tsai, Raydolfo Aprecio, Wu Zhang, Ji Min Yochim, Naichia Teng, and Mahmoud Torabinejad. "Cytotoxicity and Antimicrobial Effects of a New Fast-Set MTA." BioMed Research International 2017 (2017): 1–6. http://dx.doi.org/10.1155/2017/2071247.
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Purpose. To compare the biocompatibility and antimicrobial effectiveness of the new Fast-Set MTA (FS-MTA) with ProRoot MTA (RS-MTA). Methods. The agar overlay method with neutral red dye was used. L929 mouse fibroblast cells were cultured. The liquid and oil extracts and solid test material were placed on the agar overlay, four samples for each material. Phenol was used as the positive control and cottonseed oil and MEM extracts were used as negative controls. Cytotoxicity was examined by measuring the zones of decolorization and evaluating cell lysis under an inverted microscope using the established criteria after 24 and 48 hours. The antimicrobial test was performed using the Kirby-Bauer disk-diffusion method against S. mutans, E. faecalis, F. nucleatum, P. gingivalis, and P. intermedia. The size of the zone of inhibition was measured in millimeters. Results. There was no zone of decolorization seen under or around the test materials for FS-MTA and RS-MTA at 24 and 48 hours. The antimicrobial test demonstrated no inhibitory effect of FS-MTA or RS-MTA on any bacterial species after 24 and 48 hours. Conclusions. There was no cytotoxicity or bacterial inhibition observed by the new Fast-Set MTA when compared to the ProRoot MTA after setting.
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Huys, G., K. D'Haene, and J. Swings. "Influence of the culture medium on antibiotic susceptibility testing of food-associated lactic acid bacteria with the agar overlay disc diffusion method." Letters in Applied Microbiology 34, no. 6 (June 2002): 402–6. http://dx.doi.org/10.1046/j.1472-765x.2002.01109.x.
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Bisha, Bledar, and Byron F. Brehm-Stecher. "Simple Adhesive-Tape-Based Sampling of Tomato Surfaces Combined with Rapid Fluorescence In Situ Hybridization for Salmonella Detection." Applied and Environmental Microbiology 75, no. 5 (January 5, 2009): 1450–55. http://dx.doi.org/10.1128/aem.01944-08.
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ABSTRACT A simple adhesive-tape-based method for sampling of tomato surfaces was combined with fluorescence in situ hybridization for rapid culture-independent detection of Salmonella strains. Tapes could also be placed face-down on selective agar for on-tape enrichment of captured Salmonella cells. Overlay of cell-charged tapes with small volumes of liquid enrichment media enabled subsequent detection of tape-captured Salmonella via flow cytometry.
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Sangha, Kamalpreet Kaur, B. V. Sunil Kumar, Ravi Kant Agrawal, Dipak Deka, and Ramneek Verma. "Proteomic Characterization of Lytic Bacteriophages of Staphylococcus aureus Isolated from Sewage Affluent of India." International Scholarly Research Notices 2014 (September 14, 2014): 1–6. http://dx.doi.org/10.1155/2014/265298.
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Staphylococcus aureus is a Gram-positive bacterium that causes a variety of diseases, including bovine mastitis, which has severe economic consequences. Standard antibiotic treatment results in selection of resistant strains, leading to need for an alternative treatment such as bacteriophage therapy. Present study describes isolation and characterization of a staphylococcal phage from sewage samples. S. aureus isolates obtained from microbial type culture collection (MTCC), Chandigarh, India, were used to screen staphylococcal phages. A phage designated as ΦMSP was isolated from sewage samples by soft agar overlay method. It produced clear plaques on tryptone soya agar overlaid with S. aureus. Transmission electron microscopy revealed that the phage had an icosahedral symmetry. It had 5 major proteins and possessed a peptidoglycan hydrolase corresponding to 70 kDa. ΦMSP infection induced 26 proteins to be uniquely expressed in S. aureus. This phage can be proposed as a candidate phage to treat staphylococcal infections.
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Miranda, Rosana Belchior, Sandra Rivera Fidel, and Maria Aparecida Affonso Boller. "L929 cell response to root perforation repair cements: an in vitro cytotoxicity assay." Brazilian Dental Journal 20, no. 1 (2009): 22–26. http://dx.doi.org/10.1590/s0103-64402009000100003.
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This study compared the cytotoxicity of an experimental epoxy-resin and calcium hydroxide-based cement (MBPc), gray mineral trioxide aggregate (MTA) and white mineral trioxide aggregate (WMTA) using the agar overlay method with neutral red dye. L929 cells were seeded into 6-well culture plates where 48-h set test materials were placed on the agar overlay, in triplicate. Teflon and natural rubber served as negative and positive controls. After an incubation period of 24 h at 37ºC in a humidified atmosphere of 5% CO2 in air, a discolored area around the samples and the positive controls could be observed and measured per quadrant. The mean values were compared and converted into grades to classify the results according to the table of cytotoxicity grades according to the Standard Operating Procedures (SOP) of the Oswaldo Cruz Foundation, Brazil. The nonviable cell areas and the morphological changes in the cells were observed with an inverted microscope. The results showed grade 1 (slight) for the two types of MTA (p>0.05) and grade 2 (mild) for the MBPc (p<0.001). All samples met the requirements of the test as none of the cultures showed reactivity higher than grade 2.
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Jacobson, Andrew P., Richard L. Thunberg, Mildred L. Johnson, Thomas S. Hammack, and Wallace H. Andrews. "Alternative Anaerobic Enrichments to the Bacteriological Analytical Manual Culture Method for Isolation of Shigella sonnei from Selected Types of Fresh Produce." Journal of AOAC INTERNATIONAL 87, no. 5 (September 1, 2004): 1115–22. http://dx.doi.org/10.1093/jaoac/87.5.1115.
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Abstract Alternative methods of reducing oxygen during anaerobic enrichment in the Bacteriological Analytical Manual (BAM) Shigella culture method were evaluated and compared to the current and less practical GasPak® method. The alternative anaerobic methods included the use of reducing agents in Shigella broth and reducing culture container headspace volume to minimize atmospheric effects on oxygen concentration in Shigella broth during enrichment. The reducing agents evaluated were sodium thioglycollate, L-cystine, L-cysteine, titanium(III) citrate, and dithiothreitol, each at concentrations of 0.1, 0.05, and 0.01%. The use of Oxyrase for Broth® with the enrichment medium (Shigella broth) was evaluated at concentrations of 10, 20 and 30 μL/mL. Recoveries of chill- and freeze-stressed S. sonnei strains 357 and 20143 were determined with each anaerobic method, including the GasPak method, using inoculation levels ranging from 100 to 103 cells. For each anaerobic method, strain, inoculation level, and stress type, 5 replicate enrichments were evaluated by streaking to MacConkey agar for isolation. The numbers of cultures with each method from which S. sonnei was isolated were used to compare the alternative anaerobic methods to the GasPak method. The alternative anaerobic method with which chill- and freeze-stressed S. sonnei strains 357 and 20143 were isolated most consistently was the use of Oxyrase for Broth in Shigella broth at a concentration of 20 μL/mL. This method was compared to the GasPak anaerobic method in evaluations on the recovery of S. sonnei strains 357 and 20143 from artificially contaminated test portions of parsley, cilantro, green onions, strawberries, carrots, and celery. A third anaerobic method included the use of 0.5 cm mineral oil overlay on cultures containing Oxyrase for Broth at concentrations of 20 μL/mL. Recovery rates of strain 357 were significantly greater (p < 0.05) with the GasPak method than with Oxyrase for Broth, with and without the 0.5 cm mineral oil overlay, for test portions of parsley, cilantro, and celery. When Oxyrase for Broth was used with Shigella broth, strain 357 was isolated at higher rates from all produce types, except cilantro, when 0.5 cm mineral oil overlay was applied to enrichment cultures. The use of mineral oil overlay with Oxyrase for Broth also improved recovery of strain 20143 from test portions of all produce types except green onion and strawberries. These differences were significant (p < 0.05) with parsley, carrots, and cilantro (1 of 2 evaluations). No statistically significant differences (p > 0.05) between the GasPak and Oxyrase for Broth anaerobic methods occurred when mineral oil overlay was used with Oxyrase for Broth. The use of Oxyrase for Broth with a 0.5 cm mineral oil overlay is a practical alternative for anaerobic enrichment with the BAM method in the analysis of some produce types. Differences in recovery among the different produce types and methods occurred between S. sonnei strains 357 and 20143, emphasizing the need for additional S. sonnei strains in future evaluations.
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Cutting, Jeffrey H., William M. Kiessling, Fred L. Bond, James E. Mccarron, Karen S. Kreuzer, Jeffrey A. Hurlbut, and John N. Sofos. "Agarose Gel Electrophoretic Detection of Six β-Lactam Antibiotic Residues in Milk." Journal of AOAC INTERNATIONAL 78, no. 3 (May 1, 1995): 663–67. http://dx.doi.org/10.1093/jaoac/78.3.663.
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Abstract An electrophoretic method coupled with bioautography was developed for detection and identification of penicillin G, ampicillin, amoxicillin, cloxacillin, cephapirin, and ceftiofur residues in milk. The method uses a 2% agarose gel for electrophoresis, an overlay of PM indicator agar seeded with Bacillus stearothermophilus var. calidolactis, and incubation at 55°C for 16–18 h. The new method separated and detected residues in milk at the levels of concern for the Food and Drug Administration (FDA) for penicillin G (5 ppb), cephapirin (20 ppb), and ceftiofur (50 ppb). The method also detected ampicillin, amoxicillin, and cloxacillin at 20,30, and 30 ppb, respectively, but these levels are above those of concern for FDA (10 ppb).
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Teramura, Hajime, Aya Ogura, Linda Everis, and Gail Betts. "MC-Media Pad CC for Enumeration of Total Coliforms in a Variety of Foods." Journal of AOAC INTERNATIONAL 102, no. 5 (September 1, 2019): 1492–501. http://dx.doi.org/10.1093/jaoac/102.5.1492.
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Abstract Background: Standard coliform count methods require the preparation of agar, the use of the pour-plate technique, the overlay of agar, and in some cases, the transfer of suspect colonies to broth medium for confirmation. The MC-Media Pad CC for the enumeration of coliforms is a ready-to-use dehydrated sheet medium with no agar preparation, no spreader, and no confirmation step required. Objective: Using a paired study design, the MC-Media Pad CC was compared to standard method ISO 4832:2006 for 10 matrixes including raw ground pork, raw chicken, cream, cream cheese, ready-to-cook vegetable mix, vegetable juice, cooked prawns, crab pâté, ham sandwiches, and cooked rice. Methods: Each matrix was tested at three levels of coliform contamination (approximately 102, 104, and 106 CFU/g). Five replicate 10 g test portions per level were tested in a paired comparison by the MC-Media Pad CC and ISO 4832:2006 methods. In addition, inclusivity/exclusivity, robustness, and product consistency and stability were evaluated. Results: The candidate and reference methods demonstrated SDs ranging from 0.027 to 0.264 and 0.025 to 0.157, respectively. The difference of means ranged from –0.015 to 0.381, showing no practical difference between the methods. The MC-Media Pad CC detected 58/62 inclusivity strains and correctly excluded 26/31 exclusivity organisms, similar to the reference method. Robustness testing demonstrated no significant change in results when small changes were made to sample volume, incubation temperature, and incubation time. The product consistency study demonstrated no significant difference between lots of product and supported the 1.5 year shelf life. Conclusions: The results support the conclusions that the MC-Media Pad CC is a suitable alternative to the ISO 4832:2006 reference method for the matrixes examined and the data support AOAC Performance Tested MethodSM certification.
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OH, SE-WOOK, GENISIS IRIS DANCER, and DONG-HYUN KANG. "Efficacy of Aerosolized Peroxyacetic Acid as a Sanitizer of Lettuce Leaves." Journal of Food Protection 68, no. 8 (August 1, 2005): 1743–47. http://dx.doi.org/10.4315/0362-028x-68.8.1743.
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Aerosolized sanitizer was investigated as a potential alternative to aqueous and gaseous sanitizers for produce. Peroxyacetic acid was aerosolized (5.42 to 11.42 μm particle diameter) by a commercially available nebulizer into a model cabinet. Iceberg lettuce leaves were inoculated with three strains each of Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella Typhimurium and then treated with aerosolized peroxyacetic acid for 10, 30, or 60 min in a model aerosol cabinet at room temperature (22 ± 2°C). After treatment, surviving healthy and injured bacterial cells were enumerated on appropriate selective agars or using the overlay agar method. Inoculated iceberg lettuce leaves exposed to aerosolized peroxyacetic acid for 10 min exhibited a 0.8-log reduction in E. coli O157:H7, a 0.3-log reduction in Salmonella Typhimurium, and a 2.5-log reduction in L. monocytogenes when compared with the control. After 30 min of treatment, the three pathogens were reduced by 2.2, 3.3, and 2.7 log, and after 60 min, the reductions were 3.4, 4.5, and 3.8 log, respectively. Aerosolization may be a new and convenient method for sanitizing produce for storage or shipping.
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Muangham, Supattra, Wasu Pathom-aree, and Kannika Duangmal. "Melanogenic actinomycetes from rhizosphere soil — antagonistic activity against Xanthomonas oryzae and plant-growth-promoting traits." Canadian Journal of Microbiology 61, no. 2 (February 2015): 164–70. http://dx.doi.org/10.1139/cjm-2014-0645.
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A total of 210 melanogenic actinomycetes were isolated from 75 rhizospheric soils using ISP6 and ISP7 agar supplemented with antifungal and antibacterial agents. Their morphological characteristics and the presence of ll-diaminopimelic acid in whole-cell hydrolyzates revealed that all isolates belonged to the genus Streptomyces. Their ability to inhibit the growth of 2 pathogenic rice bacteria, Xanthomonas oryzae pv. oryzae and Xanthomonas oryzae pv. oryzicola, was observed using the agar overlay method. The results indicated that 61.9% of the isolates could inhibit at least one of the tested rice pathogens. Among these, isolate TY68-3 showed the highest antibacterial activity and siderophore production. The 16S rRNA gene sequence analysis of 46 representative isolates revealed that isolates with high similarity to Streptomyces bungoensis were frequently found. The present study indicated the potential of melanogenic actinomycetes for use as biocontrol agents against X. oryzae as well as their diversity in rhizospheric soils.
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Amoroso, Ana M., and Gabriel O. Gutkind. "Chromogenic Detection of Aminoglycoside Phosphotransferases." Antimicrobial Agents and Chemotherapy 42, no. 2 (February 1, 1998): 228–30. http://dx.doi.org/10.1128/aac.42.2.228.
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ABSTRACT A coupled chromogenic reaction (based on an agar overlay combining NADH, pyruvate kinase, lactate dehydrogenase, phosphoenolpyruvate, ATP, and kanamycin sulfate with thiazolyl blue-phenazine methosulfate for detection of NADH consumption) was optimized for the detection of aminoglycoside phosphotransferases (APHs). When used after analytical isoelectrofocusing of bacterial extracts from APH-producing strains, this method revealed one band in each of two strains with a genetically confirmed APH (3′) I and two bands in another strain with both APH (3′) I and APH (3′) VI, whereas no bands were detected in susceptible control strains or in aminoglycoside-resistant microorganisms without APH genes.
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Rönnqvist, Daniel, Ulla Forsgren-Brusk, Ulrika Husmark, and Eva Grahn-Håkansson. "Lactobacillus fermentum Ess-1 with unique growth inhibition of vulvo-vaginal candidiasis pathogens." Journal of Medical Microbiology 56, no. 11 (November 1, 2007): 1500–1504. http://dx.doi.org/10.1099/jmm.0.47226-0.
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The aim of this study was to characterize human isolates of Lactobacillus species for their capacity to interfere with the growth of different strains of Candida species in vitro in the search for a potential probiotic. Growth inhibition of Candida species was screened using an agar-overlay method. Inhibiting strains were selected to assay the effect of a cell-free Lactobacillus culture filtrate (LCF) on the growth of isolates of Candida albicans and Candida glabrata. A total of 126 human Lactobacillus isolates was investigated. Eighteen isolates significantly inhibited the growth of C. albicans on agar. The LCF of one of these strains showed strong inhibition of both C. albicans and C. glabrata. This strain was genetically identified as Lactobacillus fermentum and designated L. fermentum Ess-1. Further tests to evaluate the probiotic potential of this strain indicated that L. fermentum Ess-1 strain is a promising probiotic for use in clinical trials to treat and prevent vulvo-vaginal candidiasis.
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MONDRAGON, A., S. R. WILKINSON, M. C. TAYLOR, and J. M. KELLY. "Optimization of conditions for growth of wild-type and genetically transformed Trypanosoma cruzi on agarose plates." Parasitology 118, no. 5 (May 1999): 461–67. http://dx.doi.org/10.1017/s0031182099004230.
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Growth of Trypanosoma cruzi as colonies on solid medium has not been widely used as an experimental procedure. We therefore sought to establish a reliable and routine plating method. The optimal results were achieved with a matrix of 0·65% low melting point agarose onto which epimasigotes from the mid-to-late logarithmic phase of growth were spread. Colonies could be isolated after incubation for 21 days in a humidified 5% CO2 environment at 28°C. Plating efficiencies in the range of 40% were obtained by this method and clones could be recovered into liquid medium or onto blood-agar slopes with a high success rate. The procedure has also been adapted for the isolation of genetically transformed clones after electroporation of epimastigotes with either plasmid or cosmid vectors. This was best achieved by inclusion of the electroporated cell inoculum in a 0·6% agarose overlay containing G418 as the selective drug, on top of a 0·8% agar base. Transformation efficiencies were as high as 10−5 cells per μg of DNA. A reliable plating method for T. cruzi will have many applications and is a significant step towards the use of ‘shotgun transformation’ to generate libraries of T. cruzi recombinants.
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Easteal, Simon, and Ian A. Boussy. "A sensitive and efficient isoenzyme technique for small arthropods and other invertebrates." Bulletin of Entomological Research 77, no. 3 (September 1987): 407–15. http://dx.doi.org/10.1017/s0007485300011871.
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AbstractAn electrophoretic method for the study of enzyme variation, which uses cellulose acetate sheets with an agar overlay for staining, the use of a very good general purpose buffer (citric-aminopropyldiethanol amine) and the use of sodium azide as a bacteriocide to allow long term storage of chemicals as solutions are described. Tests are reported of the technique on Tetranychus urticae Koch, Aedes aegypti (L.) and several species of Drosophila. The results demonstrate that the technique offers sensitivity equal to or greater than starch or polyacrylamide gel electrophoresis and that it is applicable to very small organisms, allowing either the testing of single individuals for large numbers of enzymes or the testing of fewer enzymes under different electrophoretic conditions (i.e. to detect cryptic variation under a single condition). The technique is efficient of time and materials, and safer than conventional methods.
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Teramura, Hajime, Aya Ogura, Linda Everis, and Gail Betts. "MC-Media Pad EC for Enumeration of Escherichia coli and Coliforms in a Variety of Foods." Journal of AOAC INTERNATIONAL 102, no. 5 (September 1, 2019): 1502–15. http://dx.doi.org/10.1093/jaoac/102.5.1502.
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Abstract Background: Standard coliform count methods require preparation of agar, the use of pour-plate technique, the overlay of agar, and in some cases, the transfer of suspect colonies to broth medium for confirmation. The MC-Media Pad EC for enumeration of Escherichia coli and coliforms is a ready-to-use dehydrated sheet medium with no agar preparation, no spreader, and no confirmation step required. Objective: Using a paired study design, the MC-Media Pad EC was compared with standard method ISO 4832:2006. Ten matrixes including raw ground pork, raw chicken, cream, cream cheese, ready-to-cook vegetable mix, vegetable juice, cooked prawns, crab pâté, ham sandwiches, and cooked rice were evaluated in the study. Methods: Each matrix was tested at three levels of contamination (approximately 102, 104, and 106 CFU/g). Five replicate 10 g test portions per level were tested in a paired comparison by the MC-Media Pad EC, ISO 4832:2006, and ISO 16649-2:2001 (Part 2) methods. In addition, inclusivity/exclusivity, robustness, and product consistency and stability were evaluated. Results: The candidate and reference methods demonstrated standard deviations ranging from 0.034 to 0.188 and 0.028 to 0.181, respectively, for E. coli counts and 0.047–0.188 and 0.025–0.157, respectively, for total coliforms. The difference of means ranged from –0.025 to 0.331 for E. coli and from –0.037 to 0.372 for total coliforms, showing no practical difference between the methods. The MC-Media Pad EC detected 49/50 E. coli and 60/63 coliform inclusivity strains and correctly excluded 30/32 exclusivity organisms for E. coli and 24/31 exclusivity organisms for total coliforms, which was similar to the reference method. Robustness testing demonstrated no significant change in results when small changes were made to sample volume, incubation temperature, and incubation time. The product consistency study demonstrated no significant difference between lots of product and supported the 1.5 year shelf life. Conclusions: The results support the conclusions that the MC-Media Pad EC is a suitable alternative to the ISO 4832:2006 and ISO 16649-2:2001 reference methods for the matrixes examined and the data support AOAC Performance Tested MethodSM certification. Highlights: The MC-Media Pad EC was approved for Performance Tested Method certification No. 011901.
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Sayeed, Mohammed Abu, Altaf Hossen, Repon Saha, and Md Jakaria. "Antimicrobial activities of isolated probiotics and their metabolites against some pathogenic microorganisms." IIUC Studies 14, no. 1 (July 29, 2018): 21–28. http://dx.doi.org/10.3329/iiucs.v14i1.37652.
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The study was aimed to finding the antimicrobial activities among probiotics isolated from different yoghurts and their metabolites against some common bacterial pathogens. The nutrient agar media overlay method (Disc diffusion Method) was used to determine the presence of antibacterial effects among the isolated probiotics. Probiotics produced potential antibacterial activities against several pathogenic bacteria and fungi. The maximum antibacterial property (13.5 mm of zone of inhibition) of bacterial strain found against Salmonella paratyphi. Conversly, bacterial metabolites produced maximum effect (10.3 mm of zone of inhibition) against Staphylocuccos aureaus. The antibacterial effect is one of the most important criteria for probiotics selection, and the verified antibacterial activities of the probiotics supports the development of these functional foods as a key to the enhancement of health in the consuming public.IIUC Studies Vol.14(1) June 2017: 21-28
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Žėkaitė, G., V. Jaška, K. Poška, M. Andrulytė, and S. Grigiškis. "Microorganisms Producing Biosurfactant Selection and Characterization of New Discovered Bioemulsifier that will be Used to Create Ecological Heating Production Technology." Environment. Technology. Resources. Proceedings of the International Scientific and Practical Conference 1 (August 6, 2015): 222. http://dx.doi.org/10.17770/etr2013vol1.840.
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The chemical synthesis of surface active compounds is economically inefficient. It requires much energy expense, raw materials and harmful reagents. Biological biosynthesis of surface active substances happens in milder conditions without the use of dangerous chemical reagents. The main goal of this work was to select a microorganism strain capable of producing a bioemulsifier with an ability to create a stable water / fuel-oil emulsion that could be used to design a new ecological heating technology. To this end, 3 microorganism strains displaying a high emulsification activity were used. The new discovered surface active substance (SAS) was investigated with different methods (hydrocarbon overlay agar method, emulsification activity determination, microscopic observation). The production of bioemulsifier (BE) was studied by using soluble and insoluble carbon sources. It was found that Arthrobacter sp. Pr82 is the best bioemulsifier producer. Oleic acid was ascertained as the best carbon source for the production of discovered BE.
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Ulfah, Maria, Noer Kasanah, and Niken Satiti Nur Handayani. "Bioactivity and genetic screening of marine actinobacteria associated with red algae Gelidiella acerosa." Indonesian Journal of Biotechnology 22, no. 1 (January 18, 2018): 13. http://dx.doi.org/10.22146/ijbiotech.25920.
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Bacterial resistance to existing antibiotics has driven a search for new antibiotics from marine actinobacteria. Bioactivity and genetic screening of actinobacteria associated with red algae Gelidiella acerosa were conducted to discover new antibacterial compounds against Vibrio alginolyticus. A total of 14 actinobacteria isolates were obtained from G. acerosa. The isolates were subjected to genetic screening for nrps (non-ribosomal peptide synthetase) and FADH2-dependent halogenase genes. The isolates’ ability to produce secondary metabolites was examined by fermentation in various media in a six-well mini plate. The bioactivity of the secondary metabolites was screened using a microtiter assay and the agar overlay method. The results showed that all 14 isolates had the nrps gene, whereas none had the halogenase gene. Meanwhile, eight of the actinobacteria isolates showed antibacterial activity against V. alginolyticus.
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ROY, DENIS, and PIERRE WARD. "Rapid Detection of Bifidobacterium dentium by Enzymatic Hydrolysis of β-Glucuronide Substrates." Journal of Food Protection 55, no. 4 (April 1, 1992): 291–95. http://dx.doi.org/10.4315/0362-028x-55.4.291.
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Enzyme profiles and fermentation patterns of bifidobacteria were studied to determine phenotypic characteristics that allow the rapid detection of Bifidobacterium dentium and its differentiation from Bifidobacterium adolescentis, Bifidobacterium angulatum, Bifidobacterium catenulatum, and Bifidobacterium pseudocatenulatum. Among 43 bifidobacterial strains tested, the production of β-glucuronidase was limited to six strains of B. dentium. The presence of B. dentium on a selective medium may be rapidly confirmed by the detection of β-glucuronidase activity. Columbia agar containing propionic acid was chosen to enumerate bifidobacteria previously cultivated in MRS medium. After 48 h of incubation, β-glucuronidase activity was determined by using a plate staining procedure. B. dentium strains gave positive results for β-glucuronidase activity after application of the overlay solution of β-glucuronide substrate. The β-glucuronidase assay is a rapid screening method for B. dentium. This method might be useful for selection of nonpathogenic strains or detection of fecal contamination from human origin.
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EIJLANDER, ROBYN T., FRANZISKA BREITENWIESER, ROSANNE de GROOT, ERIK HOORNSTRA, HENRI KAMPHUIS, MICHIEL KOKKEN, ANGELINA KUIJPERS, et al. "Enumeration and Identification of Bacterial Spores in Cocoa Powders." Journal of Food Protection 83, no. 9 (August 21, 2020): 1530–39. http://dx.doi.org/10.4315/jfp-20-071.
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ABSTRACT The presence of bacterial spores in cocoa powders is inevitable due to the cocoa bean fermentation process, during which members of the genera Bacillus and Geobacillus are typically present. Spores are a concern in heat-treated foods when they survive heat treatments and the finished product supports germination, growth, and potentially toxin production. In this study, available methods for the enumeration of total mesophilic and thermophilic spores (TMS and TTS, respectively) were evaluated, leading to the recommendation of one global method specifically for cocoa powders. The proposed method was validated during a ring test on seven selected cocoa powders and applied during routine analyses on commercial powders. The method includes dilution of cocoa powder using buffered peptone water, heating at 80°C for 10 min for TMS and TTS counts, and heating at 100°C for 30 min for a heat-resistant (HR) spore count. Tryptic soy agar is used as a recovery medium with a maximal concentration of cocoa powder of 2.5 mg/mL (to prevent growth inhibition) and a nonnutrient agar overlay to prevent swarming of bacteria. Plates are incubated for at least 72 h at 30°C for recovery of mesophilic bacteria and 55°C for thermophilic bacteria. Suitable alternatives to specific method parameters are provided. Median values of total spore concentrations are low (<400 CFU/g for TMS and <75 CFU/g for TTS), and concentrations of HR spores are very low (<5 CFU/g). Importantly, the relation between concentrations of HR spores in cocoa powder and incidence of spoilage of heat-treated beverages containing cocoa is currently unclear. In the powders included in this study, Bacillus subtilis and Bacillus licheniformis were the predominant spore-forming species identified (49 and 39%, respectively). Both species are known for high variability in spore heat resistance. The development of reliable and sensitive molecular methods is therefore required to assess the risk of spoilage caused by spores present in cocoa powders. HIGHLIGHTS
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Jeganathan, P., K. M. Rajasekaran, N. K. Asha Devi, and S. Karuppusamy. "Antimicrobial activity and Characterization of Marine bacteria." Indian Journal of Pharmaceutical and Biological Research 1, no. 04 (December 31, 2013): 38–44. http://dx.doi.org/10.30750/ijpbr.1.4.8.
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Marine bacteria were isolated from seawater was collected from different coastal areas of the Tamilnadu Sea. The antimicrobial activities of these bacteria were investigated. Ethyl acetate extracts of marine bacterial fermentation were screened for antimicrobial activities using the method of agar diffusion. The results showed that 25 strains of the isolates have antimicrobial activity. The proportion of active bacteria associated with isolated from seawater. The active marine bacteria were assigned to the genera Alteromonas, Pseudomonas, Bacillus and Marinobacter. The TLC autobiographic overlay assay implied that the antimicrobial metabolites produced by four strains with wide antimicrobial spectrum were different. These marine bacteria were expected to be potential resources of natural antibiotic products. It can be concluded that isolation of Marine bacterial samples can offer a numbers of microbial strains for sources of new biomolecules from Marine sources.
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Khattab, Ahmed I., Eltahir H. Babiker, and Humodi A. Saeed. "Streptomyces: isolation, optimization of culture conditions and extraction of secondary metabolites." International Current Pharmaceutical Journal 5, no. 3 (February 6, 2016): 27–32. http://dx.doi.org/10.3329/icpj.v5i3.26695.
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The objectives of this study were to isolate and identify Streptomyces from soil sediments as well as to optimize cultural growth conditions for maximum antibacterial productivity. A total of fifty soil sediments were collected from Red Sea, Sudan. The soil sediments were pretreated and cultivated on agar medium. Promising Streptomyces spp. were isolated by agar overlay method using indicator organisms. Optimization of chemical and physical culture conditions was carried out. The later was judged by assessment of antibacterial activity. Ethyl acetate was used to extract the secondary metabolite compounds. The separation of the active ingredients was performed using both thin layer chromatography (TLC) and gas chromatography-mass spectrometer (GC-MS). The results revealed nine strains of Streptomyces. Of them two (PS1 and PS28) isolates exhibited high activity against pathogenic bacteria. The optimum growth conditions were pH 7.5, temperature at 30°C, soyabean concentration 2.5 g/l, incubation period in 7 days, MgSO4.7H2O conc. 1g/l and K2HPO4 conc. 2.5g/l. TLC test showed three and two fragments from metabolites of PS1 and PS28 respectively, while the GC-MS analysis revealed eight and eleven compounds with antibacterial activity of PS1 and PS28 respectively. It is concluded that marine is promising source of secondary metabolites.Khattab et al., International Current Pharmaceutical Journal, February 2016, 5(3): 27-32
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Brahami, Anissa, Annie Castonguay, and Éric Déziel. "Novel ‘Bacteriospray’ Method Facilitates the Functional Screening of Metagenomic Libraries for Antimicrobial Activity." Methods and Protocols 2, no. 1 (January 7, 2019): 4. http://dx.doi.org/10.3390/mps2010004.
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Metagenomic techniques, notably the cloning of environmental DNA (eDNA) into surrogate hosts, have given access to the genome of uncultured bacteria. However, the determination of gene functions based on DNA sequences alone remains a significant challenge. The functional screening of metagenomic libraries represents an interesting approach in the discovery of microbial metabolites. We describe here an optimized screening approach that facilitates the identification of new antimicrobials among large metagenomic libraries. Notably, we report a detailed genomic library construction protocol using Escherichia coli DH10B as a surrogate host, and demonstrate how vector/genomic DNA dephosphorylation, ligase inactivation, dialysis of the ligation product and vector/genomic DNA ratio greatly influence clone recovery. Furthermore, we describe the use of an airbrush device to screen E. coli metagenomic libraries for their antibacterial activity against Staphylococcus aureus, a method we called bacteriospray. This bacterial spraying tool greatly facilitates and improves the functional screening of large genomic libraries, as it conveniently allows the production of a thinner and more uniform layer of target bacteria compared to the commonly used overlay method, resulting in the screening of 5–10 times more clones per agar plate. Using the Burkholderia thailandensis E264 genomic DNA as a proof of concept, four clones out of 70,000 inhibited the growth of S. aureus and were found to each contain a DNA insert. Analysis of these chromosomic fragments revealed genomic regions never previously reported to be responsible for the production of antimicrobials, nor predicted by bioinformatics tools.
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Liu, Peter Yuk-Fong, Jai-Chin Tung, Se-Chin Ke, and Shun-Liang Chen. "Molecular Epidemiology of Extended-Spectrum β-Lactamase-Producing Klebsiella pneumoniae Isolates in a District Hospital in Taiwan." Journal of Clinical Microbiology 36, no. 9 (1998): 2759–62. http://dx.doi.org/10.1128/jcm.36.9.2759-2762.1998.
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Thirty-one of 104 clinical isolates of Klebsiella pneumoniae collected over a period of 8 months were found to be putative extended-spectrum β-lactamase (ESBL) producers. Isoelectric focusing and an iodine overlay agar method were used for preliminary identification of the ESBLs. They were further identified by DNA sequencing. Seventy-one percent of the isolates were found to produce SHV-5. The variation in the ESBL patterns of these isolates was slight, with only five patterns being identified. The strains were typed by pulsed-field gel electrophoresis (PFGE), and 16 different genotypes were identified. When the PFGE patterns were analyzed by the algorithmic clustering method called the unweighted-pair group method using arithmetic averages, five clusters were found. However, significant genetic variations were found among 11 isolates and between each cluster. A plasmid of 36 kb was found in all clinical isolates and in the transconjugants. Our results indicate that the increase in the number of ESBL-producing K. pneumoniae isolates in this hospital is due mainly to the dissemination of a resistance plasmid rather than to the clonal spread of a few epidemic strains.
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Čipinytė, Vilma, Saulius Grigiškis, Dovilė Šapokaitė, and Egidijus Baškys. "Production of Biosurfactants By Arthrobacter Sp . N3 , a Hydrocarbon Degrading Bacterium." Environment. Technology. Resources. Proceedings of the International Scientific and Practical Conference 1 (August 5, 2015): 68. http://dx.doi.org/10.17770/etr2011vol1.888.
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Different screening methods, such as emulsification capacity and oil spreading assays, hydrocarbon overlay agar and modified drop collapse methods were used to detect biosurfactant production by hydrocarbon degrading Arthrobacter sp N3 strain. It was indicated that oil spreading assay was the most reliable method to detect biosurfactant production. To investigate biosurfactant production, batch cultivation of Arthrobacter sp N3 was carried out in a fermenter with complex nutrient medium supplemented by sunflower oil as a carbon source. The highest oil displacement activity was achieved when Arthrobacter sp N3 strain was cultivated in two stages (with aeration for cell production and without aeration for biosurfactant synthesis). Then, two forms of the biosurfactant (crude preparation and partially purified biosurfactant) were recovered from the culture liquid. Furthermore, the biosurfactant produced by Arthrobacter sp N3 strain was analyzed by thin layer chromatography and it was estimated that even a few compounds have surface activity. The effect of temperature and pH on biosurfactant activity was also studied. It was observed that no appreciable changes in biosurfactant activity occurred at temperature and pH values ranges of 4–125 ºC and 5–10, respectively.
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Thaweboon, Sroisiri, Boonyanit Thaweboon, Surachai Dechkunakorn, Passiri Nisalak, and Rattiporn Kaypetch. "Anticandidal Activity of Cratoxylum formosum Gum and its Cytotoxicity." Advanced Materials Research 974 (June 2014): 394–97. http://dx.doi.org/10.4028/www.scientific.net/amr.974.394.
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Cratoxylumformosumis a plant widely distributed in mountainous area of various Asian countries. The extract prepared from the burnt bark has been used among the local people as a varnish to prevent tooth decay and other oral diseases. The aim of this study was to examine antifungal activity ofC. formosumgum againstCandidaalbicansand to evaluate its cytotoxicity. The gum prepared from the extract ofC.formosumwas investigated for antimicrobial activity against 3 strains ofC.albicans. Inhibition of microbial growth was primarily tested by agar diffusion method. A two-fold broth dilution method was then used to determine the minimum inhibitory concentration (MIC) of the gum. Based on the MIC value, cytotoxicity test was performed on mouse fibroblasts (ATCC clone 929) using agar overlay technique. Inhibitory effect of the gum was seen againstC. albicanswith zones of inhibition ranging from 8.0 to 9.3 mm. MIC values were between 0.50 and 1.25 mg/mL. In term of cytotoxicity,C. formosumgum at the concentration of 20 MIC (25 mg/mL) was classified as grade 3 (moderate cytotoxicity) whereas those of 10 MIC and 1 MIC were grade 1 (slight cytotoxicity). In conclusion, the gum prepared fromC. formosumextract exhibited antimicrobial activities against all the test strains ofC. albicans. From the present study, it can be suggested that this plant can be used as a novel antifungal agent, effective againstC.albicansinfections, due to its inhibitory effects onC. albicansand acceptable biocompatibility. Furtherin vitro/in vivostudies should be conducted to understand the mechanisms of action and to establish the safe profile of this gum for clinical usage.
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Silva, Ethige Isuru P., Pathmakumara Jayasingha, Saman Senanayake, Anura Dandeniya, and Dona Helani Munasinghe. "Microbiological study in a gneissic cave from Sri Lanka, with special focus on potential antimicrobial activities." International Journal of Speleology 50, no. 1 (March 2021): 41–51. http://dx.doi.org/10.5038/1827-806x.50.1.2343.
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The emergence of antibiotic resistance is a global health crisis, thus the search for novel antimicrobial compounds has become a continuous necessity. Underexplored and extreme environments, such as cave ecosystems, have been identified as a promising potential source for the discovery of novel microorganisms with novel antimicrobial compounds (AMC). This study presents the first cave microbiological investigation in Sri Lanka, with a special preference for bioprospecting of novel AMC. The cave sediment characterization demonstrated the presence of close to strong acidic conditions (pH 3.1 – 3.3) and thus indicates the possibility of isolating acidophilic microorganisms. Eight cave wall/ceiling fungal strains were isolated from Sthreepura Cave - Kuruwita and identified using both morphological and ribosomal Internal Transcribed Spacer (ITS) region sequence analysis. Interestingly, four fungal isolates (Penicillium panissanguineum, P. cremeogriseum, Aspergillus bertholletius and Trichoderma yunnanense) were found to be the first records in Sri Lanka. Of these eight isolates, three showed antimicrobial activity (AMAs) against at least one of the five tested human pathogens in preliminary screening, while A. fumigatus (SKW 404) strain showed the highest AMA against Staphylococcus aureus (ATCC 11778) assessed by agar culture plug method on Muller Hinton Agar (MHA). Crude Ethyl Acetate (EtOAc) fraction of both mycelial and Potato Dextrose Broth (PDB) extracts of A. fumigatus demonstrated similar bioactive metabolic profiles with four corresponding chemical fractions [Rf = 0.47, 0.56, 0.65, 0.82; EtOAc: Hexane (4:1, v/v)] in TLC: agar overlay bioassay. The present study indicates that there is potential for discovering novel Sri Lankan deep cave microorganisms and bioprospecting of their novel bioactive compounds. Hence, further island-wide in-depth cave microbiological investigations are required for a better understanding of the Sri Lankan cave microbiology.
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GONZÁLEZ-RODRÍGUEZ, NIEVES, JESÚS A. SANTOS, ANDRÉS OTERO, and MARÍA-LUISA GARCÍA-LÓPEZ. "Cell-Associated Hemolytic Activity in Environmental Strains of Plesiomonas shigelloides Expressing Cell-Free, Iron-Influenced Extracellular Hemolysin." Journal of Food Protection 70, no. 4 (April 1, 2007): 885–90. http://dx.doi.org/10.4315/0362-028x-70.4.885.
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Hemolysis is a means of providing pathogenic bacteria with heme iron in vivo. In a previous work, iron-influenced hemolytic activity against sheep erythrocytes was detected in cell-free supernatants, but not in the cell fraction of two environmental Plesiomonas shigelloides strains incubated without shaking. Both strains have the hugA gene, which encodes an outer membrane receptor required for heme iron utilization. The present study was undertaken to investigate the expression of a second hemolytic activity detected during aerated incubation in normal and iron-depleted tryptone soya broth (id-TSB). An agar overlay procedure and doubling dilution titrations were employed to detect the hemolytic activity against several erythrocyte species. The kinetics of growth and hemolytic activity were assayed at 35°C in aerated normal and id-TSB and salmon extract. Overlaid colonies showed a cell-associated beta-hemolytic activity within 4 h. For aerated cell-free supernatants, titers above 16 were not attained until 30 to 48 h of incubation; the best activity was noted with dog and mouse erythrocytes. After 24 h of aerated incubation, sonicated cells yielded high hemolytic activity against dog erythrocytes without activity in supernatants, but after 48 h, only 28 to 30% of the total activity remained cell associated. The hemolytic factor was released in broths during the death phase. Hemolytic activity was not detected in fish extract. This and other studies suggest that P. shigelloides may produce at least two hemolytic factors, their expression and detection being influenced by environmental growth conditions and testing procedures. The overlay assay appears to be the best routine method for detecting hemolytic activity in P. shigelloides.
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KYCIA, KATARZYNA, ANNA BZDUCHA-WRÓBEL, KAROLINA KRAŚNIEWSKA, ANNA CHLEBOWSKA-ŚMIGIEL, and MAŁGORZATA GNIEWOSZ. "Effect of Magnesium Acetate on the Antimold Activity of Lactobacillus." Journal of Food Protection 80, no. 1 (December 21, 2016): 96–103. http://dx.doi.org/10.4315/0362-028x.jfp-16-125.
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ABSTRACT The antimold activity of lactic acid bacteria (LAB) is used in food biopreservation. The aim of this study was to evaluate the effect of magnesium acetate added to de Man Rogosa Sharpe (MRS) medium on the antimold activity of three LAB strains (Lactobacillus plantarum, Lactobacillus brevis, and Lactobacillus fermentum) against molds contaminating food (Aspergillus oryzae, Aspergillus niger, Penicillium chrysogenum, Fusarium avenaceum, and Rhizopus arrhizus) and their ability to produce organic acids (acetic acid, lactic acid, and phenyllactic acid). The antimold activity of LAB strains was evaluated using the overlay method, and the concentration of the organic acids was determined with the gas chromatography technique. Changes in viable cell counts and the pH of LAB culture also were monitored over a 48-h period. The results show that the growth inhibition of all the molds (except R. arrhizus) was higher in LAB strain cultures on MRS with magnesium acetate agar than on MRS agar, and inhibition increased over the 48 h. Magnesium acetate added to MRS broth stimulated the production of acetic acid by all LAB strains in the first 8 h and slightly stimulated the production of lactic acid by L. plantarum during the first 24 h. No adverse effect of magnesium acetate on growth of LAB strains was noted. The results confirm that magnesium acetate enhances the antimold activity of LAB strains.
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Muhialdin, Belal J., Zaiton Hassan, Mohamed Muftah Ahmed Imdakim, Fredy Kesnawan Shah Abdul Kahar, and Mohamed Mustafa Aween. "Malaysian Isolates of Lactic Acid Bacteria with Antibacterial Activity against Gram-Positive and Gram-Negative Pathogenic Bacteria." Journal of Food Research 1, no. 1 (January 31, 2012): 110. http://dx.doi.org/10.5539/jfr.v1n1p110.
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<p>Contamination of foodstuff with foodborne and pathogenic bacteria are global issue and it is serious hazard for the health of the human. Lactic acid bacteria are well known for their health properties and their antimicrobial activity against spoilage and pathogenic bacteria. In this study, three isolates <em>Lactobacillus fermentum </em>Te007,<em> Pediococcus pentosaceus </em>Te010, <em>L. pentosus </em>G004 isolated from Malaysian fermented foods and fruits such as (tempeh, tempoyak, guava and banana) were evaluated for their antibacterial activity and antibiotic resistant against Gram-positive and Gram-negative bacteria by dual agar overlay method. The three isolates inhibited the growth of indicator bacteria and the activity was varied between weak and strong. All the isolates were resistant to the antibiotic nalidixic acid and vancomycin. The tested bacteria can be added to food as antibacterial agents to prevent the growth of harmful microorganisms.</p>
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ZHANG, GUODONG, LI MA, and MICHAEL P. DOYLE. "Potential Competitive Exclusion Bacteria from Poultry Inhibitory to Campylobacter jejuni and Salmonella." Journal of Food Protection 70, no. 4 (April 1, 2007): 867–73. http://dx.doi.org/10.4315/0362-028x-70.4.867.
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The objective of this study was to isolate from chickens potential competitive exclusion bacteria (CE) that are inhibitory to Campylobacter jejuni or Salmonella, or to both, for subsequent development of a defined CE product for use in poultry. Adult chickens from family farms, commercial farms, and broiler chicken research centers were sampled to identify and select C. jejuni–free donor chickens. A challenge treatment, which included administering perorally 106 CFU C. jejuni per chicken and determining undetectable cecal shedding of campylobacters at 4 weeks, was important for identifying the best CE donor chickens. Screening of bacterial colonies obtained from nine donor chickens by using selective and nonselective media yielded 636 isolates inhibitory to six C. jejuni strains in vitro, with 194 isolates being strongly inhibitory. Of the 194 isolates, 145 were from ceca, and 117 were facultative anaerobic bacteria. One hundred forty-three isolates were inhibitory to six strains of Salmonella (including five different serotypes) in vitro. Of these, 41 were strongly inhibitory to all C. jejuni and Salmonella strains evaluated, and most were Lactobacillus salivarius. A direct overlay method, which involved directly applying soft agar on plates with discrete colonies from mucus scrapings of gastrointestinal tracts, was more effective in isolating CE than was the frequently practiced isolation method of picking and transferring discrete colonies and then overlaying them with soft agar. The best approach for obtaining bacteria highly inhibitory to Salmonella and C. jejuni from chickens was to isolate bacteria from ceca under anaerobic conditions. Free-range chickens from family farms were better donors of potential CE strongly inhibitory to both Salmonella and Campylobacter than were chickens from commercial farms and broiler chicken research centers.
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Kartika, Ary Giri Dwi. "Penapisan antibakteri pada Bakteri Simbion Sinularia sp terhadap Escherichia coli." Rekayasa 10, no. 2 (October 2, 2017): 87. http://dx.doi.org/10.21107/rekayasa.v10i2.3609.
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<p>karang lunak) memiliki metabolit sekunder dengan konsentrasi yang rendah (10-6% dari berat basa invertebrata). Oleh karena itu dibutuhkan cara yang lebih konservatif dalam pemanfaatan metabolit sekunder untuk menjaga keseimbangan ekosistem.Tujuan dari penelitian ini adalah mengisolasi bakterisimbion <em>Sinularia</em> sp dan melakukan penapisan aktivitas antibakteri terhadap <em>Escherichia coli</em>. Isolasi bakteri simbion Sinularia sp dilakukan dengan menggunakan metode pengenceran dan sebar (<em>spread</em>). Purifikasi bakteri menggunakan metode streak, kemudian, uji antibakteri dilakukan dengan menggunakan metode overlay dan difusi agar. Sebanyak 5 isolat bakteri didapatkan dari hasil isoasi sampel. Hasil uji aktivitas antibakteri menunjukkan sebanyak 4 isolat (Isolat L2.2, L2.3, L2.4, dan L2.5) memiliki aktivitas antibakteri terhadap bakteri <em>Escherichia coli.</em> Isolat L2.5 memiliki diameter zona hambat terbesar yaitu sebesar 2,207 ± 0,401 cm.</p><p><em>Kata Kunci: Sinularia </em>sp, Antibakteri, <em>Escherichia coli</em>, Gili Labak<em>.</em></p><p>Skrinning Antibacterial from Sinularia sp Symbiont Bacteria Againts <em>Escherichia coli</em><strong></strong></p><p><strong><em>ABSTRACT</em></strong></p><p>Several studies have shown that invertebrates (including soft corals) contain secondary metabolites in low concentration (10-6% by wet weight of invertebrate). Therefore a more conservative approach is needed in the utilization of secondary metabolites to maintain the balance of ecosystems. The purposes of this study was to isolate the bacterium Sinularia sp and to screen antibacterial activity against Escherichia coli. Isolation of the bacteria symbione of Sinularia sp was performed by dilution method and spread. Purification of bacteria was performed by streak metho then, antibacterial test was done by using overlay method and agar disk-diffusion. A total of 5 bacterial isolates were obtained from the result of the isoation of the sample. The result of antibacterial activity test showed 4 isolates (Isolates L2.2, L2.3, L2.4, and L2.5) had antibacterial activity against Escherichia coli. Isolates L2.5 has the largest inhibitory zone diameter of 2.207 ± 0.401 cm.</p><p><em>Keywords: Sinularia sp, Antibactery, Escherichia coli, Gili Labak.</em></p>
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LEE, SUN-YOUNG, MICHAEL COSTELLO, and DONG-HYUN KANG. "Efficacy of Chlorine Dioxide Gas as a Sanitizer of Lettuce Leaves." Journal of Food Protection 67, no. 7 (July 1, 2004): 1371–76. http://dx.doi.org/10.4315/0362-028x-67.7.1371.
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Aqueous solutions of sodium hypochlorite or hypochlorous acid are typically used to sanitize fresh fruits and vegetables. However, pathogenic organisms occasionally survive aqueous sanitization in sufficient numbers to cause disease outbreaks. Chlorine dioxide (ClO2) gas generated by a dry chemical sachet was tested against foodborne pathogens on lettuce leaves. Lettuce leaves were inoculated with cocktail of three strains each of Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella Typhimurium and treated with ClO2 gas for 30 min, 1 h, and 3 h in a model gas cabinet at room temperature (22 ± 2°C). After treatment, surviving cells, including injured cells, were enumerated on appropriate selective agar or using the overlay agar method, respectively. Total ClO2 generated by the gas packs was 4.3, 6.7, and 8.7 mg after 30 min, 1 h, and 3 h of treatment, respectively. Inoculated lettuce leaves exposed to ClO2 gas for 30 min experienced a 3.4-log reduction in E. coli, a 4.3-log reduction in Salmonella Typhimurium, and a 5.0-log reduction in L. monocytogenes when compared with the control. After 1 h, the three pathogens were reduced in number of CFU by 4.4, 5.3, and 5.2 log, respectively. After 3 h, the reductions were 6.9, 5.4, and 5.4 log, respectively. A similar pattern emerged when injured cells were enumerated. The ClO2 gas sachet was effective at killing pathogens on lettuce without deteriorating visual quality. Therefore, this product can be used during storage and transport of lettuce to improve its microbial safety.