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Microbial Physiology and Genetics

Microbial Physiology and Genetics. 微生物及免疫學研究所 何漣漪. Microbial Physiology. Nutritional factors for microbial growth Environmental factors for microbial growth Pure culture Culture of microorganisms Culture media Dependence on oxygen Bacterial growth in laboratory conditions

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Microbial Physiology and Genetics

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  1. Microbial Physiology and Genetics 微生物及免疫學研究所 何漣漪

  2. Microbial Physiology Nutritional factors for microbial growth Environmental factors for microbial growth Pure culture Culture of microorganisms Culture media Dependence on oxygen Bacterial growth in laboratory conditions Growth curve Microbial metabolism

  3. Growth of microorganisms Nutritional factors Required elements C, H, O sources (amino acids, lipids, nucleic acids, sugars) N source (amino acids and nucleic acids) S source (amino acids) P source (nucleic acids, membrane lipids, ATP) K, Mg, Ca, Fe (enzyme cofactors, etc.) Growth factors Compounds that bacteria require but cannot synthesize Energy sources Sunlight for phototrophs Oxidation of chemical compounds for chemotrophs • Nutritional diversity (concerning the energy source and carbon source) • Photoautotrophs • (primary producers) • Photoheterotrophs • Chemoautotrophs • Chemoheterotrophs

  4. Nitrogen source Ammonium (NH4+) is used as the sole N source by most microorganisms. Ammonium could be produced from N2 by nitrogen fixation, or from reduction of nitrate and nitrite. Sulfur source Most microorganisms can use sulfate (SO42-) as the S source. Phosphorus source Phosphate (PO43-) is usually used as the P source. Mineral source For most microorganisms, it is necessary to provide sources of K+, Mg2+, Ca2+, Fe2+, Na+ and Cl-. Many other minerals (e.g., Mn2+ , Mo2+, Co2+, Cu2+ and Zn2+) can be provided in tap water or as contaminants of other medium ingredients.

  5. Environmental factors for microbial growth Temperature Psychrophile (15 oC - 20 oC) Mesophile (30 oC - 37 oC) Thermophile (50 oC - 60 oC) pH Neutrophile (pH 6 - 8) Acidophile (pH 1-5) Alkaliphile (pH 9-11) Oxygen availability Obligate aerobe Obligate anaerobe Facultative anaerobe Microaerophile (5-10% O2) Water availability Osmophile Halophile

  6. Obtaining a pure culture Cultivating bacteria on a solid medium (bacterial isolation) A solid medium is required for obtaining a pure culture of microorganism. Agar:an algae extract, polysaccharide in nature, which very few bacteria can degrade. The agar plate contains 1.5% of agar. Colony:population of bacterial cells arising from a single cell.

  7. Streak-plate method

  8. Pour plate method

  9. Culture of microorganisms Culture media Differential media Substances that certain bacteria change in a recognizable way are added. Complex (rich) media nutrient agar or broth; blood agar or chocolate agar for more fastidious bacteria. Chemically defined (minimal media) Selective media Inhibitors for organisms other than the one being sought are added. Nutrient broth Glucose-salt Peptone Glucose Meat extract Dipotassium phosphate Water Monopotassium phosphate Magnesium sulfate Ammonium sulfate Calcium chloride Iron sulfate Water

  10. Bacterial growth in laboratory conditions Principles of bacterial growth Bacteria multiply by binary fission. Microbial growth is defined as an increase in the number of cells in a population. Doubling (generation) time E. coli: 20 min M. tuberculosis: 12-24 h Bacterial growth curve

  11. The doubling time is measured during this period. The bacteria are most susceptible to antibiotics during this time. Bacteria stop growing due to decrease of nutrients and O2 supply, and accumulation of toxic metabolites. Bacteria synthesize macromolecules required for multiplication. The length of lag phase depends on the conditions in the original culture and the medium into which they are transferred. A balance between slow loss of cells through death and the formation of new cells through growth and division.

  12. Bacterial Metabolism Assimulation (anabolism): energy-requiring Dissimulation (catabolism): energy-acquiring Glycolysis Pentose phosphate pathway TCA cycle Respiration (aerobic and anaerobic) Fermentation Focal metabolites: metabolic intermediates that link anabolic and catabolic pathways.

  13. Glycolysis(the Embden-Meyerhoff-Parnas pathway) Substrate-level phosphorylation

  14. Oxidative phosphorylation The electron transport chain water

  15. ATP synthesis by proton motive force

  16. Fermentation:a metabolic process in which the final electron acceptor is an organic compound. The only ATP-yielding reactions of fermentation are those of glycolysis, and involve substrate-level phosphorylation.

  17. Saccharomycetes Clostridium E. coli Propionebacterium Enterobacter Streptococcus Lactobacillus

  18. Bacterial Genetics Gene mutation Spontaneous mutation Base substitution; addition and deletion of nucleotides; transposition Induced mutation Chemical mutagens; transposition; radiation Gene transfer Transformation Natural and artificial competence Transduction Conjugation F and R plasmids

  19. Gene mutation Base substitution Removal or addition of nucleotides (insertion and deletion; frame shift and chain termination) Transposable elements Sources of diversity in microorganisms: Gene mutation and gene transfer.

  20. Mechanisms of gene transfer Transformation:uptake of naked exogenous DNA by living cells. Conjugation:mediated by self-transmissible plasmids. Transduction:phage-mediated genetic recombination.

  21. Demonstration of transformation Avery, MacLeod, and McCarty (1944)

  22. Transformation Natural competence

  23. Transformation Artificial competence or plasmid Plasmid Electroporation

  24. Transduction

  25. Conjugation Transfer of plasmid F plasmid R plasmid F+ cell (donor) F-cell (recipient) F+ cell F+ cell

  26. R plasmid R: drug resistance RTF: transfer of R plasmid

  27. Microaerophile Obligate aerobe Facultative anaerobe Obligate anaerobe

  28. Culture methods Anaerobic Anaerobic jar; anaerobic chamber; reducing agents Increased CO2 (for capnophils) Candle jar; CO2 incubator Microaerophilic Back

  29. MacConkey agar plate Blood agar plate Back

  30. Back

  31. Back Caused by tautomeric shift

  32. Back

  33. Transposition by transposable elements(Insertion sequences and transposons) Transposable element:gene that moves from one DNA molecule to another within the same cell or from one site on a DNA molecule to another site on the same molecule Back

  34. Enrichment cultures Isolating an organism from natural sources

  35. Maintaining stock cultures Agar slant Store agar slant cultures in a refrigerator. Stock at –70 oC Store a pure culture in the presence of 17% glycerol. Lyophilization (freeze drying) Dry a pure culture with a lyophilizer. This can be stored at room temperature for years.

  36. Methods to detect and measure bacterial growth Direct cell count Count under a microscope; cell-counting instrument Measuring biomass Turbidity; total weight; chemical constituents Viable cell count Plate counts; membrane filtration; Detecting cell products

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