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Morphology Cl. tetani Cl. perfringens Cl. botulinum Culture Cl. tetani Cl. perfringens Cl. botulinum Culture ch’: •O2 obligate anaerobe •CO2 0.03% •Temp. 37°C •PH 7.4 __________________________________________________________________________________ Media •Ordinary media: grow •Blood agar: haemolysis •Robrtson cooked meat medium Biochemical Reactions Cl. tetani Cl. perfringens Sugar: -ve GMMLS indole +ve LM: acidified+clotted Cl. botulinum GML Methods of anaerobiosis 1)Deep agar 2)Media containing reducing compounds 3)Absorption of O2 by Na-pyrogallate (Buchner`s tube or McLeod’s plate) 4) Replacement of Oxygen with hydrogen Gas-bag jar Patient treated on a clinical basis without waiting for laboratory data. Samples: Tissues from wounds Direct smear: • Gram positive bacilli • terminal spherical spores (Drum stick appearance), • motile with peritrichate flagella, • non-capsulated. Culture • Culture ch: O2: Obligate anaerobic, CO2 Temp. 37°C. • Media On blood agar: complete haemolysis swarming, BR: • Indole: +ve • Suger fermantation: -ve. of C.tetani must be confirmed by production of toxin and its neutralization by specific antitoxin. Isolation Patient should be treated without waiting for laboratory confirmation. Specimens: Material from wounds, • Pus, • Tissue, • Exudates. Direct smear stained with Gram stain: • Gram-positive, • Sporulated: subterminal, not bulging • non motile • capsulated bacilli. Culture: Culture ch.: • O2: Obligate anaerobe. CO2: … Temp.: … PH: …. Media: • On blood agar complete haemolysis. double-zone haemolysis,(When examined with transmitted light), (inner clear zone &outer hazy zone) • egg yolk-glucose agar or serum agar, colonies are surrounded by opaque white precipitate due to production of lecithinase (Nagler ‘s reaction). Nagler’s reaction: Use: Identification of Clostridium perfringens. Principle: • Clostridium perfringens produces opalescence in human serum or egg yolk media due to the production of lecithinase C (phospholipase). Biochemical reactions • Suger fermentation: C perfringens ferments GMLS large amounts of gases & acids • litmus milk stormy clot. Litmus milk: Use: differentiate bacteria based on various reactions they produce in milk supplemented with a litmus pH indicator Principle: Milk is a complex nutritional source contains proteins (mainly casein), lactose and minerals. Reactions on litmus milk Reaction Color of litmus milk Interpretation Acid production Indicator pink Glucose and lactose utilised Acid clot Indicator pink, medium clotted. Big amount of gas may be produced which splits the clotted milk into pieces (stormy clot) Casein precipitated by acid. This type of clot will dissolve if treated with NaOH Reaction Indicator blue Ammonia liberated Confirmation of initial clinical diagnosis rests on demonstrating toxin in the patient’s feces, serum, or vomitus. Fecal samples are the best specimens for detecting toxin in food poisoning or infant botulism. Toxin is usually detected by • lethal effect in mice • neutralization by specific antisera. Diarrhea associated with antibiotic therapy in the preceding 4 to 6 weeks. isolation of C difficile and the presence of toxin A and/or toxin B in the stool. The Toxins can be detected • Cellular cytotoxicity test: • Latex agglutination test • ELISA by: