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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
Growth curve
Microbial metabolism
Growth of microorganisms
Nutritional factors
Required elements
Energy sources
C, H, O sources (amino acids,
lipids, nucleic acids, sugars)
Sunlight for phototrophs
N source (amino acids and
nucleic acids)
Oxidation of chemical
compounds for chemotrophs
S source (amino acids)
Nutritional diversity
(concerning the energy
source and carbon source)
Photoautotrophs
(primary producers)
Photoheterotrophs
Chemoautotrophs
Chemoheterotrophs
P source (nucleic acids,
membrane lipids, ATP)
K, Mg, Ca, Fe (enzyme
cofactors, etc.)
Growth factors
Compounds that bacteria
require but cannot synthesize
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.
Environmental factors for microbial growth
Temperature
Oxygen availability
Psychrophile (15 oC - 20 oC)
Obligate aerobe
Mesophile (30 oC - 37 oC)
Obligate anaerobe
Thermophile (50 oC - 60 oC)
Facultative anaerobe
pH
Neutrophile (pH 6 - 8)
Microaerophile (5-10% O2)
Water availability
Acidophile (pH 1-5)
Osmophile
Alkaliphile (pH 9-11)
Halophile
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.
Streak-plate
method
Pour plate method
Culture of microorganisms
Culture media
Differential media
Complex (rich) media
Substances that certain
bacteria change in a
recognizable way are added.
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
Monopotassium
phosphate
Magnesium sulfate
Water
Ammonium sulfate
Calcium chloride
Iron sulfate
Water
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
A balance between slow loss of cells through death and
the formation of new cells through growth and division.
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.
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.
Glycolysis
(the EmbdenMeyerhoff-Parnas
pathway)
Substrate-level
phosphorylation
Oxidative phosphorylation
The electron transport chain
water
ATP synthesis by proton motive force
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.
Saccharomycetes
Clostridium
Propionebacterium
E. coli
Enterobacter
Streptococcus
Lactobacillus
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
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.
Mechanisms of gene transfer
Transformation: uptake of naked exogenous DNA by
living cells.
Conjugation: mediated by self-transmissible plasmids.
Transduction: phage-mediated genetic recombination.
Demonstration of
transformation
Avery, MacLeod, and
McCarty (1944)
Transformation
Natural competence
Transformation
Artificial competence
or
plasmid
Plasmid
Electroporation
Transduction
Conjugation
Transfer of plasmid
F+ cell (donor)
F plasmid
R plasmid
F- cell (recipient)
F+ cell
F+ cell
R plasmid
R: drug resistance
RTF: transfer of R
plasmid
Obligate
aerobe
Facultative
anaerobe
Obligate
anaerobe
Microaerophile
Culture methods
Anaerobic
Anaerobic jar; anaerobic
chamber; reducing agents
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Increased CO2 (for capnophils)
Candle jar; CO2 incubator
Microaerophilic
MacConkey agar plate
Blood agar plate
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Caused by
tautomeric shift
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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
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Enrichment cultures
Isolating an organism from natural sources
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.
Methods to detect and measure bacterial growth
Direct cell count
Viable cell count
Count under a
microscope;
Plate counts;
membrane filtration;
cell-counting instrument
Measuring biomass
Turbidity;
total weight;
chemical constituents
Detecting cell products