05_PREP of BACTERIAL Ags_111309

05_PREP of BACTERIAL Ags_111309 - Preparation of Bacterial...

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Preparation of Bacterial Antigens (Ags) LABORATORY EXERCISE #5 Rev: 07:14:39 Materials and Methods Bacterial Isolates and Media Specimens were obtained by the Flamingham Bored of Health (Flamingham MA) from food handlers (nasopharangeal swabs), animals found on the premises (oral swabs), food items, and patients (vomitis) with clear-cut diagnoses of infection. Bacteria were isolated using standard media: trypticase soy agar (TSA), phenylethyl alcohol agar, blood agar, and MacDConkey agar (Difco; Sparks,MD). Staphylocicci were tested for coagulase production (BBL Staphyloside ; Becton Dickinson Microbiological Systems, Cockysville MD) and identified using the Dade Behring Microscan PC-12 Panel ( Becton Dickinson). The strains were maintained on TSA supplemented with 1% dextrose (TSA/dex); they were subcultured weekly (grown on TSA/ dex for 18hr at 37°C, checked for purity, then sealed in plastic bags and stored at 4°C). Agar Plate-Dialysis Membrane Cultures were used to prepare whole-cell lysates and extracellular protein production. Extracellular proteins were obtained by growing bacteria overnight at 37 o C on a dialysis membrane (Spectra/Por 12-14,000 MWCO; Spectrum Medical Industries, Huston TX) overlaid onto a TSA plate. Cells and extracellular proteins were isolated by adding 100 µl of Tris-buffered saline (pH 7.2) to the membranes and scraping the growth into a microcentrifuge tube; the bacteria were separated from the extracellular proteins by centrifugation. Protein concentration was determined colormetrically. Preparation of Heat-Killed Bacterial Cells and Soluble Antigens - An Ag must have a molecular weight of at least 750 Da, and preferably over 10,000 Da, to be immunogenic (to elicit an immune response). Most particulate antigens or large complex macromolecules of protein and carbohydrate are good immunogens while nucleic acids and lipids are usually poor immunogens. - In this exercise, soluble bacterial antigens will be prepared for immunological assays; the preparations could also be used for immunizations. The antigen preparations will actually be mixtures of several protein and polysaccharide antigens. “Whole cells” can also be used to directly immunize animals and in assays. Cells are complex antigens with many antigenic sites (epitopes) on their surfaces, and can be administered without the use of an adjuvant. Many vaccines are actually preparations of whole bacteria which have either been killed or modified. 1
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Agar Plate-Dialysis Membrane Cultures - Materials : - 3 Tyrpticase soy agar (TSA) plates supplemented with 1% dextrose (TSA/dex) - 50-mL centrifuge tube - 5 mL of sterile TBS containing 0.005% thimerosal (2000x Stock = 10%) - Omit thimerosal if cells are to be used as a vaccine - 60°C water bath - 1 tube of thioglycollate broth - to test success of heat-killing, if to be used as a vaccine - Fresh, overnight (18hr/37°C) TSA/dex culture of bacteria Safety Tips : Staphylococcus pseudintermedius are canine pathogens, but can rarely cause human
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This note was uploaded on 03/09/2010 for the course BIOL 371 taught by Professor Eugenemuller during the Fall '09 term at Framingham State College.

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05_PREP of BACTERIAL Ags_111309 - Preparation of Bacterial...

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