Post on 29-Apr-2018
MICROBIAL GROWTH
Dr. Hala Al-Daghistani
Microbial growth: Increase in cell number, not cell size!
Physical Requirements for Growth: Temperature Minimum growth temperature
Optimum growth temperature
Maximum growth temperature
Five groups based on optimum growth temperature
1. Psychrophiles
2. Psychrotrophs
3. Mesophiles
4. Thermophiles
5. Hyperthermophiles
Microbial Growth
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Physical Requirements for Growth:
pH and Osmotic Pressure
Most bacteria grow best between pH 6.5 and 7.5:
Neutrophils
Some bacteria are very tolerant of acidity or thrive
in it: Acidophiles (preferred pH range 1 to 5)
Molds and yeasts grow best between pH 5 and 6
Hypertonic environments (increased salt or sugar)
cause plasmolysis
Obligate halophiles vs.
facultative halophiles
WATER ACTIVITY Quantifies water availability in an environment; decreases with increasing solute concentration. PLASMOLYSIS: Hypertonic solutions; cytoplasm water loss; compatible solutes. OSMOTOLERANT: Grows over a wide range of water activity; fungi > bacteria. HALOPHILE: “Salt-loving”; requires > 0.2M sodium chloride.
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Chemical Requirements for Growth: Carbon, N, S, P, etc.
Carbon
Half of dry weight
heterotrophs use organic carbon sources
Autotrophs use inorganic carbon sources
Nitrogen, Sulfur, Phosphorus
N, S Needed for protein sythesis
N, P used for NA and ATP synthesis
S in thiamine and biotin
Phosphate ions (PO43–)
Also needed K, Mg, Ca, trace elements (as cofactors),
and organic growth factors
Nutrient Concentration Effects in Batch Cultures
• Total growth will increase until limiting nutrients are exhausted (included oxygen for aerobes) or metabolic by products accumulate that change environmental conditions to inhibit growth (toxicity).
• Growth rate will also increase with increasing nutrient concentration up to a some maximum value, beyond which there is no effect (transporters are
saturated with there substrate.
Oxygen Requirement Types
2 to 10% atm O2
Super Oxide Dismutase (SOD): Superoxide radicals (O 2 •-), or superoxide anions (OH2¯² . Superoxide radicals go to hydrogen peroxide & O2.
Catalase: hydrogen peroxide go to water & O2.
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Toxic Forms of Oxygen
Singlet oxygen: O2 boosted to a higher-energy state
Superoxide free radicals: O2–
Peroxide anion: O22–
Hydroxyl radical (OH)
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Fig 6.5
Biofilms
Microbial communities form
slime or hydrogels
Starts via attachment of
planctonic bacterium to surface structure.
Bacteria communicate by chemicals via quorum sensing
Sheltered from harmful factors (disinfectants etc.)
Cause of most nosocomial infections
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Advantages of biofilms 1. Cell-to-cell chemical communication 2. Quorum sensing, allows bacteria to coordinate their activity and group together into communities that provide benefits not unlike those of Multicellular organisms. 3. Within a biofilm community, the bacteria are able to share nutrients and are sheltered from harmful factors in the environment, such as desiccation, antibiotics, and the body's immune system. 4. Facilitating the transfer of genetic information's (conjugation).
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Culture Media
Culture medium: Nutrients prepared for microbial
growth. Have to be sterile (not contain living microbes)
Inoculum: Microbes introduced into medium
Culture: Microbes growing in/on culture medium
Chemically defined media: Exact chemical composition
is known (for research purposes only)
Complex media: Extracts and digests of yeasts,
meat, or plants, e.g.: Nutrient broth
Nutrient agar
Blood agar
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Agar
Complex polysaccharide
Used as solidifying agent for
culture media in Petri plates, and
slants
Generally not metabolized
by microbes
Liquefies at 100°C
Solidifies ~40°C
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Anaerobic Culture Methods
Use reducing media, containing chemicals (e.g. thioglycollate) that combine with O2
Are heated shortly before use to drive off O2
Use anaerobic jar
Novel method in clinical labs:
Add oxyrase to growth media
OxyPlate (no need for anaerobic jar)
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Capnophiles: Aerobic Bacteria Requiring High CO2
Low oxygen, high CO2
conditions resemble those
found in
intestinal tract
respiratory tract and
other body tissues where
pathogens grow
E.g: Campylobacter jejuni
Use candle jar, CO2-
generator packets, or CO2
incubators
Candle jar
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Selective Media and Differential Media
Selective medium: Additives suppress unwanted and encourage desired microbes – e.g. EMB, mannitol salt agar etc.
Differential medium: changed in recognizable manner by some bacteria Make it easy to distinguish colonies of different microbes – e.g. and hemolysis on blood agar; MacConkey agar, EMB, mannitol salt agar etc.
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Enrichment Media/Culture
Encourages growth of desired microbe
Example: Assume soil sample contains a few phenol-degrading bacteria and thousands of other bacteria
Inoculate phenol-containing culture medium with the
soil and incubate
Transfer 1 ml to another flask of the phenol medium
and incubate
Transfer 1 ml to another flask of the phenol medium
and incubate
Only phenol-metabolizing bacteria will be growing
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Pure Cultures
Contain only one species or strain.
Most patient specimens and
environmental samples contain
several different kinds of bacteria
Streak-plate method is commonly used
Colony formation: A population of cells arising from
a single cell or spore or from a group of attached
cells (also referred to as CFU).
Only ~1% of all bacteria can be successfully cultured
Aseptic technique critical!
3 or 4
quadrant
methods
Streak Plate Method
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Preserving Bacterial Cultures
Deep-freezing: Rapid cooling of pure culture in
suspension liquid to –50°to –95°C. Good for several
years.
Lyophilization (freeze-drying): Frozen (–54° to –72°C)
and dehydrated in a vacuum. Good for many years.
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The Growth of Bacterial Cultures
Binary fission – exponential growth
Budding
Generation time – time required for cell to divide
(also known as doubling time)
Ranges from 20 min (E. coli) to > 24h (M. tuberculosis)
Consider reproductive potential of E. coli
Figure 6.12b
Fig 6.13
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Bacterial Growth Curve
Phases of growth
Lag phase
Exponential or
logarithmic (log) phase
Stationary phase
Death phase (decline phase)
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The lag phase - This period of little or no cell division. During this time, however, the cells are not dormant. The microbial population is undergoing a period of intense metabolic activity involving, synthesis of enzymes and various molecules The log phase (exponential phase) - The cells begin to divide and enter a period of growth, or logarithmic increase. Cellular reproduction is most active during this period, The stationary phase - The growth rate slows, the number of microbial deaths balances the number of new cells, and the population stabilizes. (period of equilibrium)
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What causes exponential growth to stop is not always clear. 1.The exhaustion of nutrients 2.The accumulation of waste products, and harmful changes in ph may all play a role. The death phase - The number of deaths eventually exceeds the number of new cells formed, and the population enters the death phase, or logarithmic decline phase.
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Direct Measurements of Microbial Growth
Viable cell counts: Plate counts: Serial
dilutions put on plates CFUs form colonies
Figure 6.15, step 1
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Additional Direct Measurements
Direct microscopic count: Counting chambers
(slides) for microscope
A specially designed slide called a petroff-hausser cell counter
is used in direct microscopic counts.
-motile bacteria are difficult to count by this method
-Dead cells are bout as likely to be counted as live ones.
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FILTRATION
Membrane filters for fluids. Pore size for bacteria: 0.2 – 0.4 m
Pore size for viruses: 0.01 m
At least 100 ml of water are passed
through a thin membrane filter whose
pores are too small to allow bacteria to
pass.
Filtration is the method of choice for low counts M.O.
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Estimating Bacterial Numbers by Indirect Methods
Spectrophotometry to measure turbidity
OD is function of cell number
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The most probable number (MPN) method - Another method for determining the number of bacteria in a sample is the most probable number (MPN) method - This statistical estimating technique is based on the fact that the greater the number of bacteria in a sample, the more dilution is needed to reduce the density to the point at which no bacteria are left to grow in the tubes in a dilution series. - It is useful when the growth of bacteria in a liquid differential medium is used to identify the microbes (such as coliform bacteria, which selectively ferment lactose to acid, in water testing).
Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings
Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings
Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings
Metabolic activity - indirect way to estimate bacterial numbers is to measure a population's metabolic activity ( acid or CO2, is in direct proportion to the number of bacteria present).
Dry weight -For filamentous bacteria and molds, the fungus is removed from the growth medium, filtered, and dried in a desiccator. It is then weighed
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Measuring Microbial Growth - Overview
Direct Methods
Plate counts
MPN
Direct microscopic
count
Filtration
Indirect Methods
Turbidity
Metabolic activity
Dry weight