ANAEROBES Ananerobes • Anaerobic bacteria do not have cytochrome system for oxygen metabolism and hence are unable to neutralize toxic oxygen metabolites • Classification • Obligate anaerobes: Cannot tolerate O2. Completely lack superoxide dismutase and catalase enzymes • Aerotolerant anaerobes: Do not utilize oxygen forgrowth, but tolerate its presence. Possess small amounts of superoxide dismutase and peroxidase (but lack catalase) Special requirements to grow Anaerobes Anaerobic condition: - McIntosh and Filde’s anaerobic jar - GasPak system - Anoxomat system - Anaerobic glove box - Pre-reduced anaerobically sterilized (PRAS) media. Medium with low redox potential: By adding reducing substances -unsaturated fatty acid, ascorbic acid, glutathione, cysteine, glucose, sulfites and metallic iron CLOSTRIDIUM CLOSTRIDIUM • Gram-positive bacilli, having bulging spores • Saprophytes found in soil, fresh water, marine water, decaying vegetation, animal matter and sewage • Harbored in intestine of vertebrates and invertebrates • Human Pathogens: - C. perfringens: Gas gangrene - C. tetani: Tetanus - C. botulinum: Causes botulism • C. difficile: Causes pseudomembranous colitis. Spore of Clostridia • Wider than the vegetative bacteria  swollen or spindleshaped appearance • Most of the clostridia bear a sub-terminal spores except - C.bifermentans – Central & oval - C.perfringens – subterminal & oval - C.tetani – terminal & spherical (drumstick) - C.tertium – terminal & oval (tennis racket) Spores of Clostridia Cultivation • Clostridia grow well in common anaerobic media • Robertson’s cooked meat (RCM) broth - Chopped meat particles  glutathione and unsaturated fatty acids which take up oxygen • Proteolytic - turn the meat black and produce foul odor, e.g. C. tetani, C. botulinum A, B and F. • Saccharolytic species - turn the meat pink, e.g. C. perfringens, C. difficile and C. botulinum C, D and E. Robertson cooked meat broth: A. Uninoculated; B. Pink and turbid (C. perfringens); C. Black and turbid (C. tetani)