Transcript Microbial culture and growth - Microbiology and Molecular Genetics

Chapter 4: Bacterial Culture, Growth,
and Development
We are only 10%
human the rest is
pure microbes
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Chapter Overview
How microbes uptake nutrients
● How microbes are cultured
● How microbes are counted
● The microbial growth cycle
● What is a biofilm?
● Cell differentiation, and how some
prokaryotes “behave” like eukaryotes
●
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Introduction
The adage “To eat well is to live well” is as
true for microbes as it is for humans.
Over eons, bacteria have evolved ingenious
strategies to find, acquire, and metabolize
a wide assortment of food sources.
- This owes to the remarkable plasticity of
microbial genomes.
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We are Carbon-based life forms on
Earth
All living organisms require:
•Proteins, which are the building blocks from which the structures of living
organisms are constructed (example: enzymes).
•Nucleic acids, which carry genetic information.
•Carbohydrates, which store energy in a form that can be used by living cells.
•Fats, which also store energy, but in a more concentrated form, and which may
be stored for extended periods in the bodies of animals.
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Elements of Life
Essential nutrients are those that must be
supplied from environment.
Macronutrients
- Major elements in cell macromolecules
- C, O, H, N, P, S
- Ions necessary for protein function
- Mg2+, Ca2+, Fe2+, K+
Micronutrients
- Trace elements necessary for enzyme function
- Co, Cu, Mn, Zn
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Microbial Nutrition
Based on its niche, an organism may have
evolved to require additional growth
factors.
- Specific nutrients not required by all cells.
- Refer to Table 4.1.
A defined minimal medium contains only the
compounds needed for an organism to grow.
- Refer to Table 4.2.
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Table 4-1 Growth factors and natural habitats of organisms associated with disease.
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Table 4-2 Composition of commonly used media.
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Some organisms have adapted so well to their natural habitat
that we still don’t know how to grow them in the lab.
- Rickettsia prowazekii grows only within the cytoplasm of
eukaryotic cells.
- Body temperature of the armadillo is low enough to favor
the growth of the leprosy-causing bacterium
Mycobacterium leprae.
Figure 1.1
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How Microbes Obtain Carbon?
All of Earth’s life-forms are
based on carbon, which they
acquire in different ways.
- Autotrophs fix CO2 and
assemble into organic
molecules (mainly sugars).
- Heterotrophs use
preformed organic
molecules.
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How Microbes Obtain Energy?
In addition to carbon, all organisms require an
energy source.
- Phototrophs obtain energy from chemical
reactions triggered by light.
- Chemotrophs obtain energy from oxidationreduction reactions.
- Lithotrophs use inorganic molecules
as a source of electrons, while…
- Organotrophs use organic molecules.
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Microbial Nutrition
In short, microbes are classified based on their
carbon and energy acquisition as follows:
- Autotrophs
- Photoautotrophs
- Chemoautotrophs (or lithotrophs)
- Heterotrophs
- Photoheterotrophs
- Chemoheterotrophs (or organotrophs)
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Figure 4.2
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The Nitrogen Cycle
N2 makes up 79% of Earth’s atmosphere but
is unavailable for use by most organisms.
Nitrogen fixers possess nitrogenase, which
converts N2 to ammonium ions (NH4+).
Nitrifiers oxidize ammonia to nitrate (NO3–).
Denitrifiers convert nitrate to N2.
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The Nitrogen Cycle
Figure 4.4
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The Nitrogen Cycle
Nitrogen-fixing bacteria
may be free-living in
soil or water, or they
may form symbiotic
associations with
plants.
- Rhizobium and
legumes
Figure 4.5
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Nutrient Uptake
Membranes are designed to separate what is
outside the cell from what is inside.
Selective permeability is achieved in three ways:
- Substrate-specific carrier proteins, or
permeases
- Dedicated nutrient-binding proteins that patrol
the periplasmic space
- Membrane-spanning protein channels or pores
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Uptake of Nutrients
Some nutrients enter by passive diffusion
Most nutrients enter by:
•
•
•
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facilitated diffusion
active transport
group translocation (form of active
transport0
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Facilitated Diffusion
Facilitated diffusion helps solutes move
across a membrane from a region of high
concentration to one of lower concentration.
- It does not use energy and cannot move a
molecule against its gradient.
Example: The aquaporin family that
transports water and small polar molecules
such as glycerol
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Passive Diffusion and Facilitated
Diffusion
No energy is spent !
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•rate of facilitated
diffusion increases
more rapidly and
at a lower
concentration
•diffusion rate
reaches a plateau
when carrier
becomes
saturated
Figure 5.3
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Active Transport Requires Energy
Coupled transport systems are those in
which energy released by moving a driving
ion down its gradient is used to move a solute
up its gradient.
- In symport, the two molecules travel in the
same direction.
- In antiport, the actively transported
molecule moves in the direction opposite to
the driving ion.
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Figure 4-7 Coupled transport.
Sodium-Calcium
exchanger
Co-transport of glucose and Na+
in to the cells
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ABC Transporters
The largest family of energy-driven transport
systems is the ATP-binding cassette
superfamily, or ABC transporters.
- They are found in all three domains of life.
Are of two main types:
- Uptake ABC transporters are critical for
transporting nutrients
- Efflux ABC transporters are generally used as
multidrug efflux pumps
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Figure 4-8 ABC transporters.
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Siderophores
Siderophores are specialized molecules
secreted to bind ferric ion (Fe3+) and transport
it into the cell.
- The iron is released into the cytoplasm and
reduced to the more useful ferrous (Fe2+) form.
Note: Neisseria gonorrhoeae employs receptors
on its surface that bind human iron complexes
and wrest the iron from them.
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Iron Uptake
ferric iron is very
insoluble so uptake is
difficult
microorganisms use
siderophores to aid
uptake
siderophore complexes
with ferric ion
complex is then
transported into cell
Figure 5.8
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Figure 4-9 Siderophores and iron transport.
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Group Translocation
Group translocation is a process that uses
energy to chemically alter the substrate
during its transport.
The phosphotransferase system (PTS) is an
example present in all bacteria.
- It uses energy from phosphoenolpyruvate
(PEP) to attach a phosphate to specific
sugars.
- The system has a modular design that
accommodates different substrates.
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Figure 4-10 Group translocation: the phosphotransferase system (PTS) of E. coli.
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Culturing Bacteria
Microbes in nature exist in complex,
multispecies communities, but for detailed
studies pure cultures are needed.
We have succeeded in culturing only 0.1%
of the microorganisms around us.
Bacteria are grown in culture media, which
is of two main types:
- Liquid or broth
- Solid (usually gelled with agar)
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Figure 4-11 Separation and growth of microbes on an agar surface.
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Culturing Bacteria
Pure colonies can be isolated via two main
techniques:
1) Dilution streaking
- Dragging a loop across the surface of
an agar plate
2) Spread plate
- Tenfold serial dilutions are performed
on a liquid culture.
- A small amount of each dilution is
then plated.
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Figure 4-14 Tenfold dilutions, plating, and viable counts.
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Types of Media
Complex media are nutrient-rich but poorly defined.
Synthetic media are precisely defined.
Enriched media are complex media to which
specific blood components are added (to grow
fastidious organisms).
Selective media favor the growth of one organism
over another. (bile salts inhibit G+ bacteria)
Differential media exploit differences between two
species that grow equally well.
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What is the difference between
enriched media and enrichment
media?
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MacConkey medium, both selective and
differential.
•Selects Gram negative organisms
•Lactose fermenting bacteria such as
E.coli, Enterobacter and Klebsiella will
produce acid.
•Non-Lactose fermenting bacteria such as
Salmonella, Pseudomonas, Proteus and
Shigella cannot utilize lactose
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Counting Bacteria
A viable bacterium is defined as being
capable of replicating and forming a colony
on a solid medium.
- Viable cells can be counted via the pour
or spread plate method.
Microorganisms can be counted indirectly via
biochemical assays of cell mass, protein
content, or metabolic rate.
- Also by measuring optical density
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Counting Bacteria
Microorganisms can be counted directly by
placing dilutions on a special microscope slide,
called a Petroff-Hausser counting chamber.
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Live-dead stain.
Fluorescent Dyes:
•Propidium Iodide – stains
dead cells
•Syto-9 – stains both dead
and live cells
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Figure 4-18 Fluorescence-activated cell sorter.
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The Bacterial Growth Curve
Exponential growth never lasts indefinitely.
The simplest way to model the effects of a changing
environment is to culture bacteria in a batch
culture.
- A liquid medium within a closed system
The changing conditions in this system greatly affect
bacterial physiology and growth.
- This illustrates the remarkable ability of bacteria
to adapt to their environment.
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The Growth Curve
Observed when microorganisms are
cultivated in batch culture culture
incubated in a closed vessel with a single
batch of medium
Usually has four distinct phases
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lag phase
no increase
log phase
maximal rate of division
and population growth
stationary phase
population growth ceases
death phase decline in
population size
Figure 6.6
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The Mathematics of Growth
Generation (doubling) time
time required for the population to double in
size
varies depending on species of
microorganism and environmental
conditions
range is from 10 minutes to several days for
some microorganisms
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Exponential Growth
Simple binary fission is not the only kind of
division that generates an exponential curve.
- e.g.: Plasmodium falciparum invades an
RBC, releasing about 20 progeny per
generation.
Figure 4.20
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Figure 4-21 Bacterial growth curves.
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The Mathematics of Growth
Generation time is the time it takes for a
population to double.
For cells undergoing binary fission,
Nt = No x 2n
where Nt is the final cell number
No is the original cell number
n is the number of generations
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Calculation of number of cells, generation
times, and growth rates
No = initial population number
Nt = population at time t
n = number of generations at time t
g = generation time
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If in 8 h an exponentially growing
cell population increases from 5 ×
106 cells/ml to 5 × 108 cells/ml,
calculate g and n.
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Continuous Culture
In a continuous culture, all cells in a population
achieve a steady state, which allows detailed
study of bacterial physiology.
The chemostat ensures logarithmic growth by
constantly adding and removing equal amounts of
culture media.
Note that the human gastrointestinal tract is
engineered much like a chemostat.
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Figure 4-23 Relationships between dilution rate, cell mass, and generation time.
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Figure 4-22 Chemostats and continuous culture.
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Cell Differentiation
Bacteria faced with environmental stress
undergo complex molecular reprogramming
that includes changes in cell structure.
Examples include:
- Endospores of Gram-positive bacteria
- Heterocysts of cyanobacteria
- Fruiting bodies of Myxococcus xanthus
- Aerial hyphae and arthrospores of
Streptomyces
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Figure 4-24 Biofilms.
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Figure 4-25 Biofilm development.
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Bacterial Endospores
Clostridium and Bacillus species can produce
dormant spores that are heat-resistant.
Starvation initiates an elaborate 8-hour genetic
program that involves:
- An asymmetrical cell division process that
produces a forespore and ultimately an
endospore
Sporulation can be divided into discrete stages
based primarily on morphological
appearance.
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Figure 4.26
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Cyanobacterial Heterocysts
Anabaena differentiates
into specialized cells
called heterocysts.
- Allow it to fix nitrogen
anaerobically while
maintaining oxygenic
photosynthesis
Figure 4.27
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Fruiting Bodies
Myxococcus xanthus uses gliding motility.
- Starvation triggers the aggregation of
100,000 cells, which form a fruiting body.
Figure 4.28
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Eukaryotic-like Structures
Streptomyces bacteria form mycelia and
sporangia analogous to those of fungi.
Figure 4.29
As nutrients decline, aerial hyphae divide into
arthrospores that are resistant to drying.
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Figure 4.30
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Chapter Summary
Microbes require certain essential macronutrients
and micronutrients to grow.
● Microbes are classified on the basis of their carbon
and energy acquisition.
● Transport systems can be divided into 2 main
types: - Passive transport does not require energy.
- Simple and facilitated diffusion
- Active transport requires energy.
- Coupled transport
- ABC transporters
- Group translocation
●
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Chapter Summary
Bacteria can be cultured on solid or liquid media.
● Microorganisms in culture may be counted directly
or indirectly.
● The growth cycle of organisms grown in liquid batch
culture consists of four phases:
- Lag, logarithmic, stationary, and death
● Biofilms are complex, multicellular, surfaceattached microbial communities.
● Many bacteria can undergo cell differentiation.
- Examples: Endospores, heterocysts, fruiting
bodies, and aerial hyphae
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