Cultivation of Microorganisms Department of Microbiology Harbin Medical
Cultivation of Microorganisms Department of Microbiology, Harbin Medical University
KEY TERMS Obligate aerobe Obligate anaerobe Aerotolerant anaerobe Facultative anaerobe Microaerophilic Mesophile Thermophile Psychrophile Halophilic Osmophilic Medium Department of Microbiology, Harbin Medical University
Bacterial Requirements for Growth • • • Nutrients Oxygen (or absence) Energy Optimal temperature Optimal p. H Department of Microbiology, Harbin Medical University
SOURCES OF METABOLIC ENERGY n The three major mechanisms for generating metabolic energy are u Fermentation u Respiration u Photosynthesis n At least one of these mechanisms must be employed if an organism is to grow Department of Microbiology, Harbin Medical University
Fermentation n n The formation of ATP in fermentation is not coupled to the transfer of electrons Fermentation is a substrate phosphorylation, an enzymatic process in which a pyrophosphate bond is donated directly to ADP (adenosine diphosphate) by a phosphorylated metabolic intermediate The phosphorylated intermediates are formed by metabolic rearrangement of a fermentable substrate such as glucose, lactose, or arginine Example: fermentation of a molecule of glucose (C 6 H 12 O 6) yields a net gain of two pyrophosphate bonds in ATP and produces two molecules of pyruvic acid (C 3 H 6 O 3) Department of Microbiology, Harbin Medical University
Fermentation Reaction Department of Microbiology, Harbin Medical University
Respiration n n Respiration is chemical reduction of an oxidant (electron acceptor) through a specific series of electron carriers in the membrane establishes the proton motive force across the bacterial membrane Reductants (electron donor) u n may be organic or inorganic: For example, lactic acid serves as a reductant for some organisms, and hydrogen gas is a reductant for other organisms Oxidants u Gaseous oxygen (O 2) often is employed as an oxidant, but alternative oxidants that are employed by some organisms include carbon dioxide (CO 2), sulfate (SO 42–), and nitrate (NO 3−) Department of Microbiology, Harbin Medical University
Respiration in Eukaryotic Cell 36 ATP Department of Microbiology, Harbin Medical University
Respiration in Eukaryotic Cell Department of Microbiology, Harbin Medical University
Photosynthesis n n Photosynthesis is similar to respiration in that the reduction of an oxidant via a specific series of electron carriers establishes the proton motive force Difference in the two processes u in photosynthesis the reductant and oxidant are created photochemically by light energy absorbed by pigments in the membrane n Photosynthesis can continue only as long as there is a source of light energy Department of Microbiology, Harbin Medical University
Photosynthesis Department of Microbiology, Harbin Medical University
Environmental Factors Affecting Growth Nutrients n Hydrogen ion concentration (p. H) n Temperature n Aeration n Ionic strength & osmotic pressure n Department of Microbiology, Harbin Medical University
Nutrient Requirements • • • Carbon Nitrogen Sulfur Phosphorus Metal ions (e. g. iron) Growth factors Department of Microbiology, Harbin Medical University
Carbon Source Autotroph Heterotroph CO 2 + Sunlight Photosynthesis CO 2+H+SO 42 - Chemolithotroph Organic carbon eg. , glucose Fermentation Respiration Department of Microbiology, Harbin Medical University
Nitrogen Source n The end products are NH 4+ or NH 3 Department of Microbiology, Harbin Medical University
Sulfur Source Most microorganisms can use sulfate as a sulfur source, reducing the sulfate to the level of hydrogen sulfide (H 2 S) n Some microorganisms can assimilate H 2 S directly from the growth medium, but this compound can be toxic to many organisms n Department of Microbiology, Harbin Medical University
Phosphorus Source n n n Phosphate (PO 43−) is required as a component of ATP, nucleic acids, and such coenzymes as NAD, NADP, and flavins Many metabolites, lipids (phospholipids, lipid A), cell wall components (teichoic acid), some capsular polysaccharides, and some proteins are phosphorylated Phosphate is always assimilated as free inorganic phosphate (Pi) Department of Microbiology, Harbin Medical University
Mineral Sources n n In formulating a medium for the cultivation of most microorganisms, it is necessary to provide sources of potassium, magnesium, calcium, and iron, usually as their ions (K+, Mg 2+, Ca 2+, and Fe 2+) Many other minerals (eg, Mn 2+, Mo 2+, Cu 2+, and Zn 2+) are required; these frequently can be provided in tap water or as contaminants of other medium ingredients Department of Microbiology, Harbin Medical University
Growth Factors n A growth factor is an organic compound which a cell must contain in order to grow but which it is unable to synthesize u Some amino acids u Some purines and pyrimidines u Vitamins n Different species of bacteria needs different growth factors Department of Microbiology, Harbin Medical University
Nutrient Requirements • • • Carbon Nitrogen Sulfur Phosphorus Metal ions (e. g. iron) Growth factors Department of Microbiology, Harbin Medical University
Environmental Factors Affecting Growth Nutrients n Hydrogen ion concentration (p. H) n Temperature n Aeration n Ionic strength & osmotic pressure n Department of Microbiology, Harbin Medical University
Hydrogen Ion Concentration (p. H) • Many grow best at neutral p. H • Some can survive/grow - Acid - Alkali Department of Microbiology, Harbin Medical University
Hydrogen Ion Concentration (p. H) n Most organisms (neutralophiles) grow best at a p. H of 6. 0 – 8. 0, although some forms (acidophils) have optima as low as p. H 3. 0 and others (alkaliphiles) have optima as high as p. H 10. 5 n Microorganisms regulate their internal p. H over a wide range of external p. H values u u u Acidophiles maintain an internal p. H of about 6. 5 over an external range of 1. 0– 5. 0 Neutralophiles maintain an internal p. H of about 7. 5 over an external range of 5. 5– 8. 5 Alkaliphiles maintain an internal p. H of about 9. 5 over an external range of 9. 0– 11. 0 Department of Microbiology, Harbin Medical University
Temperature Psychrophilic forms grow best at low temperatures (15– 20 o. C) n Mesophilic forms grow best at 30– 37 o. C n u Most organisms are mesophilic F 30 o. C is optimal for many free-living forms, and the body temperature of the host is optimal for symbionts of warm-blooded animals n Thermophilic forms grow best at 50– 60 o. C Department of Microbiology, Harbin Medical University
Optimal Growth Temperature n n Mesophile u human body temperature * pathogens * opportunists Pyschrophile u close n to freezing Thermophile u close to boiling Department of Microbiology, Harbin Medical University
Heat Shock / Cold Shock n Microorganisms share with plants and animals the heatshock response, a transient synthesis of a set of “heatshock proteins, ” (HSP) when exposed to a sudden rise in temperature above the growth optimum u n HSPs appear to be unusually heat-resistant and to stabilize the heat -sensitive proteins of the cell Bacteria also exhibit a phenomenon called cold shock: the killing of cells by rapid cooling u u For example, the rapid cooling of Escherichia coli from 37 o. C to 5 o. C can kill 90% of the cells A number of compounds protect cells from either freezing or cold shock: glycerol, dimethyl sulfoxide (DMSO) Department of Microbiology, Harbin Medical University
Aeration n Obligate aerobe u specifically n Facultative anaerobe u able n requiring oxygen as hydrogen acceptor to live aerobically or anaerobically Obligate anaerobe u requiring a substance other than oxygen as hydrogen acceptor and being sensitive to oxygen inhibition Department of Microbiology, Harbin Medical University
Obligate Aerobes • Grow in presence of oxygen • No fermentation • Oxidative phosphorylation Department of Microbiology, Harbin Medical University
Obligate Anaerobes • Fermentation • No oxidative phosphorylation • Killed by oxygen • The natural by-products of aerobic metabolism are the reactive compounds hydrogen peroxide (H 2 O 2) and superoxide (O 2 -) • Lack certain enzymes superoxide dismutase O 2 -+2 H+ H 2 O 2 catalase H 2 O 2 H 20 + O 2 peroxidase H 2 O 2 H 2 O /NAD NADH Department of Microbiology, Harbin Medical University
Facultative Anaerobes • • • Fermentation Aerobic respiration Survive in oxygen Department of Microbiology, Harbin Medical University
Microaerophilic Bacteria • Grow – • low oxygen Killed – high oxygen Department of Microbiology, Harbin Medical University
Ionic Strength and Osmotic Pressure n For most organisms, the properties of ordinary media are satisfactory; however, for marine forms and organisms adapted to growth in strong sugar solutions, these factors must be considered u Halophilic Organism: requires high salt concentrations u Osmophilic Organism: requires high osmotic pressures Department of Microbiology, Harbin Medical University
Cultivation Methods n Two problems will be considered u Media: the choice of a suitable medium u Cultivation Methods: the isolation of a bacterial organism in pure culture Department of Microbiology, Harbin Medical University
Cultivation Medium: Based on Purpose n Basic medium n Enrichment medium n Selective medium n Differentiation medium n Anaerobic medium Department of Microbiology, Harbin Medical University
Cultivation Medium: Based on physical properties Liquid medium Solid medium Semi-Solid medium Department of Microbiology, Harbin Medical University
Medium Applications n n n Liquid Medium u Pure Culture u Amplifying culture Semi-solid medium u Motion: flagellum Solid Medium u Pure culture u Isolation culture F colony Department of Microbiology, Harbin Medical University
Colony Attributes Department of Microbiology, Harbin Medical University
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