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Extraction and studying the effect of pH and temperature on hemolysin production by a local isolates of Staphylococcus aureus

Authors: Mohammed I. Nadir --- Hussain S. Al-Hasani --- Amer H. Al-Shammary
Journal: Karbala Journal of Medicine مجلة كربلاء الطبية ISSN: 19905483 Year: 2012 Volume: 5 no 1 Issue: 11 Pages: 1283-1294
Publisher: Kerbala University جامعة كربلاء

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Abstract

background: Staphylococcus aureus is a ubiquitous bacterium that is generating increasingly bad press coverage due to its propensity to adopt a pathogenic lifestyle in hospital and community settings. S. aureus colonies are found in approximately 30% of the general population. It colonizes the skin readily and can lead to a wide range of pathological conditions from skin lesions to osteomyelitis, endocarditis, and septicemia. Many bacteria produce substances that are cytolysins i.e they dissolve red blood cells (hemolysins) or kill tissue cells or leukocytes (leukocidins). The β-toxin degrades sphingomyelin and therefore is toxic for many kinds of cells, including human red blood cells. Hemolysins were extracted and the optimum conditions for their production were extensively studied including optimum pH, temperature, incubation period, and various manipulations of the culture media.Objectives1-Extraction of hemolysin from a local isolate of S. aureus.2-Studying the effects of pH and temperature on hemolysin production.Methods : Bacterial samples were identified by subjecting them to the standard laboratory procedures while semi quantitative screening on blood agar (containing 5% human blood) revealed that all isolates were hemolysin producer but in different efficiencies. Determination of the optimal conditions for hemolysin production including the optimum pH and temperature were also performed.Results : Bacterial samples were identified by subjecting them to the standard laboratory procedures and the results showed that forty isolates out of the total of 100 were identified as Staphylococcus aureus. Semi quantitative screening on blood agar (containing 5% human blood) revealed that all isolates were hemolysin producer but in different efficiencies. Depending on the semi-quantitative screening and hemolytic assays isolate SW-14 ofStaphylococcus aureus was the higher hemolysin producing isolate. Determination of the optimal conditions for hemolysin production including the optimum pH and temperature were performed, the results demonstrated that the best hemolysin production was in the pH near neutrality (pH 7-7.5) and in temperature of 35-40oC.Conclusions1.Conventional methods can be performed to extract hemolysins.2.Hemolysin was maximally produced when the pH was near neutrality and incubation temperature was 37oC and this conclusion indicates that hemolysin was produced when the conditions were similar to that of the host.


Article
Purification and characterization of hemolysin produced by a local isolates of Staphylococcus aureus

Authors: Mohammed I. Nadir --- Hussain S. Al-Hassani --- Amer H. Al-Shammary
Journal: Karbala Journal of Medicine مجلة كربلاء الطبية ISSN: 19905483 Year: 2012 Volume: 5 no 2 Issue: 12 Pages: 1455-1463
Publisher: Kerbala University جامعة كربلاء

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Abstract

background: Staphylococcus aureus is a ubiquitous bacterium that is generating increasingly bad press coverage due to its propensity to adopt a pathogenic lifestyle in hospital and community settings. S. aureus colonies are found in approximately 30% of the general population. It colonizes the skin readily and can lead to a wide range of pathological conditions from skin lesions to osteomyelitis, endocarditis, and septicemia. Hemolysins are extracellular toxic proteins which are produced by many gram negative (e.g. Escherichia coli, Serratia spp., Proteus spp., Vibrio spp., Pasteurella spp., Pseudomonas aeruginosa) and gram positive bacteria (e.g. Streptococcus spp., Staphylococcus aureus, Listeria spp., Bacillus cerius, Clostridium tetani), all of which possess a certain pathogenic potential. Hemolysins have been therefore always considered as virulence factors. Most hemolysins cause lysis of erythrocytes by forming pores of varying diameters in the membrane and are designated as such because they have the ability to lyse red blood cells (RBCs). Objectives1-Purification of hemolysin from a local isolate of S. aureus.2-Characterization of hemolysin produced by a local isolate of S. aureus.Methods: Bacterial samples were identified by subjecting them to the standard laboratory procedures while semi quantitative screening on blood agar (containing 5% human blood) revealed that all isolates were hemolysin producer but in different efficiencies. Hemolysin was extracted by cooling centrifugation and purified by many steps including: precipitation by ammonium sulphate, dialysis, ionic exchange chromatography by using DEAE-Cellulose, and gel filtration chromatography by using Sephadex G-100. The molecular weight of hemolysin was determined by gel filtration chromatography on Sephadex G-100 while the optimum pH and temperature for hemolysin stability were also determined.Results: The results showed that forty isolates out of 100 were identified as Staphylococcus aureus. Hemolysin was extracted by cooling centrifugation and purified by many steps including: precipitation by ammonium sulphate with 50-75% saturation percentage, dialysis, ionic exchange chromatography by using DEAE-Cellulose, and gel filtration chromatography by using Sephadex G-100. The results showed that hemolysin was purified 135 fold with a yield of 1.16%.The molecular weight of hemolysin determined by gel filtration chromatography on Sephadex G-100 column was about 35000 daltons, while the optimum pH for enzyme stability was 7 and the optimum temperature for enzyme stability was between 25-35oC.Conclusions1.Conventional methods can be performed to extract hemolysins.2.Hemolysin was maximally produced when the pH was near neutrality and incubation temperature was 37oC and this conclusion indicates that hemolysin was produced when the conditions were similar to that of the host.

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