2120 Lab Week 5 Experiments 7 & 10 Bacterial Endospores Inert, resting, cell modification produced by some Gram positive genera: Clostridium, Bacillus. Hardiest of all life forms Withstands extremes in heat, drying, freezing, radiation, and chemicals Not a means of reproduction Endospores Thick coat, Dehydrated, metabolically inactive Longevity verges on immortality, 250 million years Resistant to ordinary cleaning methods, heat, chemicals, radiation Pressurized steam at 120oC for 20-30 minutes will destroy them Microbiology. 2017. Openstax, Rice University Sporulation cycle Sporulation - formation of endospores Germination - return to vegetative growth Microbiology. 2017. Openstax, Rice University Have a 2-phase life cycle: Vegetative cell and Endospore 1) Vegetative cell – metabolically active and growing 2) Endospore – when exposed to adverse environmental conditions; metabolically inert, capable of high resistance and very long-term survival Endospore classification- location in cell Name of bacteria Location Shape Bacillus anthracis Central Ovoid Clostridium tetani Terminal Spherical Clostridium botulinum Sub-terminal Spherical Clostridium perfringens Sub-terminal Ovoid Clostridium difficle Sub-terminal Ovoid Central, ovoid Terminal, spherical Sub-terminal, spherical Sub-terminal, ovoid J Bacteriol. 1968 Nov; 96(5): 1818–1834 Clinical significance- Endospore Not all bacteria have ability to form endospores. Endospore forming Gram positive bacteria: Bacillus sp. & Clostridium sp. Such pathogens are difficult to treat because their endospores are hard to kill. Name of bacteria Disease Bacillus anthracis Anthrax Clostridium tetani Tetanus Clostridium botulinum Botulism Clostridium perfringens Gas gangarene Clostridium difficle Pseudomembranous colitis Clinical significance- Endospore Tetanus Evolution of an anthrax eschar in a 4-year-old boy (A&B) the lesion when first seen (day 0). Note the arm swollen from the characteristic edema. (C) Day 6. (D) Day 10. (E) Day 15. Although penicillin Gas gangrene treatment was begun immediately and the lesion was sterile by about 24 hours, it continued to evolve and resolve as seen. Medical Microbiology. 1996. 4th edition. Baron S, editor. Endospore staining Microbiology. 2017. Openstax, Rice University Endospore staining 1 2 3 Make a smear. Cover the smear Steam the slide for 5 Air dry and heat fix the with malachite minutes. smear. Place a small green. piece of paper over the smear. 4 5 6 Rinse the smear Stain with Saffranin Rinse the smear gently with water. for 1 minute. gently with water. Different types of media Synthetic – contains pure organic and inorganic compounds in an exact chemical formula Complex or non-synthetic – contains at least one ingredient that is not chemically definable General purpose media – grows a broad range of microbes, usually non-synthetic Enriched media – contains complex organic substances such as blood, serum, hemoglobin, or special growth factors required by fastidious microbes Differential media: Differentiation of colonies of desired microbes from others Selective media: contains one or more agents that inhibit growth of some microbes and encourage growth of the desired microbes Why do you need different types of media? Bacterial pathogens are often in very small numbers compared to normal flora in the body. Bacterial pathogens may be overgrown by the normal flora. Many bacterial pathogens are fastidious (remember the meaning of this word). To grow and identify potential pathogens, different media have been developed. Types of microbial growth media: Experiment 10 Some media can be both Selective & Differential Mannitol salt agar Selective agent: High Salt concentration (keeps most Gram negative bacteria from growing. Selects for Gram positive bacteria). Differential agent: Mannitol (Colonies of bacteria that ferment mannitol reduce the pH and turn media yellow). Some media can be both Selective & Differential MacConkey agar Selective agent: Bile Salts (keeps most Gram positive bacteria from growing. Selects for Gram negative bacteria especially of family Enterobacteriaceae also called Enterics) Differential agent: Lactose (Colonies of bacteria that ferment lactose reduce the pH and turn colonies pink). Some media can be both Enriched & Differential Blood agar Chocolate blood agar Enriched agent: Blood Differential agent: Type of hemolysis Types of hemolysis on Blood agar Beta hemolysis (β hemolysis) : complete breakdown of blood cells Large, creamy white, beta hemolytic colonies typical of Staphylococcus aureus. (Rebecca Buxton, University of Utah, Salt Lake City, UT) Types of hemolysis on Blood agar Alpha hemolysis (α hemolysis) : partial breakdown of blood cells Streptococcus mutans Streptococcus pneumoniae (Rebecca Buxton, University of Utah, Salt Lake City, UT) (Rebecca Buxton, University of Utah, Salt Lake City, UT) Types of hemolysis on Blood agar Gamma hemolysis (γ hemolysis) : No breakdown of blood cells Nonhemolytic Staphylococcus epidermidis. (Rebecca Buxton, University of Utah, Salt Lake City, UT).