Download Dynamics of Prokaryotic Growth

Dynamics of Prokaryotic
Growth
Chapter 4
Principles of Bacterial Growth
 Prokaryotic cells divide by binary
fission


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One cell divides into two
 Two into four etc.
Cell growth is exponential
 Doubling of population with each cell
division
 Exponential growth has important
health consequences
Generation time
 Time it takes for population to double
 A.k.a doubling time
 Varies among species
Principles of Bacterial Growth
 Growth can be calculated
 Nt = N0 x 2n
 (Nt ) number of cells in population
 (N0 ) original number of cells in the population
 (n) number of divisions
 Example
 N0 = 10 cells in original population
 n = 12
 4 hours assuming 20 minute generation time
 Nt = 10 x 212
 Nt = 10 x 4,096
 Nt = 40,960
Bacterial Growth in Nature
 Conditions in nature have
profound effect on microbial
growth

Biofilm layer
Cells sense changing
environment
 Synthesize compounds
useful for growth
 Cells produce
multicellular associations
to increase survivability
 Example
 Biofilms
 Slime layers
Bacterial Growth in Nature
 Biofilm
 Formation begins with planktonic bacteria attach to
surfaces
 Other bacteria attach and grow on initial layer
 Has characteristic architecture
 Contain open channels for movement of nutrient and
waste
 Cells within biofilms can cause disease
 Treatment becomes difficult
 Architecture resist immune response and antimicrobials

Bioremediation is beneficial use of biofilm
Bacterial Growth in Nature
 Interactions of mixed microbial communities

Prokaryotes live in mixed communities

Many interactions are cooperative
 Waste of one organism nutrient for another

Some cells compete for nutrient
 Synthesize toxic substance to inhibit growth of
competitors
Obtaining Pure Culture
 Pure culture defined as population of cells derived
from single cell

All cells genetically identical
 Cells grown in pure culture to study functions of
specific species
 Pure culture obtained using special techniques

Aseptic technique
 Minimizes potential contamination
 Cells grown on culture media
 Can be broth (liquid) or solid form
Obtaining Pure Culture
 Culture media can be liquid
or solid
 Liquid is broth media

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Used for growing large
numbers of bacteria
Solid media is broth
media with addition of
agar

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
Agar marine algae
extract
Liquefies at
temperatures above
95°C
Solidifies at 45°C
 Remains solid at room
temperature and body
temperature

Bacteria grow in colonies
on solid media surface


All cells in colony
descend from single cell
Approximately 1 million
cells produce 1 visible
colony
Obtaining Pure Culture
 Streak-plate method
 Simplest and most
commonly used in
bacterial isolation
 Object is to reduce
number of cells being
spread
 Solid surface dilution
 Each successive
spread decreases
number of cells per
streak
Bacterial Growth in
Laboratory Conditions
 Cells in laboratory grown in closed or batch
system

No new input of nutrient and no release of
waste
 Population of cells increase in predictable
fashion

Follows a pattern called growth curve
Bacterial Growth in
Laboratory Conditions
 The Growth Curve
 Characterized by five
distinct stages
 Lag stage
 Exponential or log
stage
 Stationary stage
 Death stage
 Phase of prolonged
decline
Bacterial Growth in
Laboratory Conditions
 Lag phase


Number of cells does not increase
in number
Cells prepare for growth

“tooling up”
 Log phase
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Period of exponential growth
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Produce primary metabolites

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Doubling of population with each
generation
Compounds required for growth
Cells enter late log phase

Synthesize secondary
metabolites
 Used to enhance survival
 Antibiotics
Bacterial Growth in
Laboratory Conditions
 Stationary phase

Overall population remains
relatively stable

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Cells exhausted nutrients
Cell growth = cell death
 Dying cell supply metabolites
for replicating cells
 Death phase

Total number of viable cells
decreases

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Decrease at constant rate
Death is exponential

Much slower rate than growth
Bacterial Growth in
Laboratory Conditions
 Phase of prolonged decline
 Once nearly 99% of all cells
dead, remaining cells enter
prolonged decline
 Marked by very gradual
decrease in viable population
 Phase may last months or
years
 Most fit cells survive
 Each new cell more fit that
previous
Bacterial Growth in
Laboratory Conditions
 Colony growth on solid medium
 In colony, cells eventually compete for resources

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Cells grow exponentially and eventually compete for
nutrients
Position within colony determines resource
availability

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Cells on edge of colony have little competition and
significant oxygen stores
Cells in the middle of colony have high cell density
 Leads to increased competition and decreased
availability of oxygen
Bacterial Growth in
Laboratory Conditions
 Continuous culture

Bacterial culture can be maintained

Continuous exponential growth can be sustained
by use of chemostat
 Continually drips fresh nutrients in
 Releases same amount of waste product
Environmental Factors on Growth
 As group, prokaryotes inhabit nearly all environments
 Some live in “comfortable” habitats
 Some live in harsh environments
 Most of these are termed extremophiles and belong to
domain Archaea
 Major conditions that influence growth
 Temperature
 Oxygen
 pH
 Water availability
Environmental Factors on Growth
 Temperature

Each species has well
defined temperature
range
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Within range lies
optimum growth
temperature
Prokaryotes divided into
5 categories

Psychrophile
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Psychrotroph
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25°C to 45°C
 More common
 Disease causing
Thermophiles
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20°C to 30°C
 Important in food spoilage
Mesophile
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Optimum temperature -5°C to
15°C
 Found in Arctic and Antarctic
regions
45°C to 70°C
 Common in hot springs
Hyperthermophiles
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70°C to 110°C
 Usually members of Archaea
 Found in hydrothermal vents
Environmental Factors on Growth
 Oxygen
 Prokaryotes divided based on oxygen requirements
 Obligate aerobes
 Absolute requirement for oxygen
 Use for energy production
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Obligate anaerobes
 No multiplication in presence of oxygen
 May cause death

Facultative anaerobes
 Grow better with oxygen
 Use fermentation in absence of oxygen

Microaerophiles
 Require oxygen in lower concentrations
 Higher concentration inhibitory

Aerotolerant anaerobes
 Indifferent to oxygen, grow with or without
 Does not use oxygen to produce energy
Environmental Factors on Growth
 pH
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Bacteria survive within pH range
Neutrophiles
 Multiply between pH of 5 to 8
 Maintain optimum near neutral

Acidophiles
 Thrive at pH below 5.5
 Maintains neutral internal pH pumping out protons (H+)

Alkalophiles
 Grow at pH above 8.5
 Maintain neutral internal pH through sodium ion exchange
 Exchange sodium ion for external H+
Environmental Factors on Growth
 Water availability
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All microorganisms require water for growth
Water not available in all environments
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In high salt environments
 Bacteria increase internal solute concentration
 Synthesize small organic molecules
 Osmotolerant bacteria tolerate high salt
environments
 Bacteria that require high salt for cell growth termed
halophiles
Nutritional Factors on Growth
 Growth of prokaryotes depends on nutritional
factors as well as physical environment
 Main factors to be considered are:
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Required elements
Growth factors
Energy sources
Nutritional diversity
Nutritional Factors on Growth
 Required elements
 Major elements
 Carbon, oxygen, hydrogen, nitrogen, sulfur,
phosphorus, potassium, magnesium, calcium and iron
 Essential components for macromolecules

Organisms classified based on carbon usage
 Heterotrophs
 Use organism carbon as nutrient source
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Autotrophs
 Use inorganic carbon (CO2) as carbon source
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Trace elements
 Cobalt, zinc, copper, molybdenum and manganese
 Required in minute amounts
Nutritional Factors on Growth
 Growth factors
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Some bacteria cannot synthesize some cell
constituents
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These must be added to growth environment
 Referred to as growth factors
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Organisms can display wide variety of factor
requirements
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Some need very few while others require many
 These termed fastidious
Nutritional Factors on Growth
 Energy Sources

Organisms derive energy from sunlight or
chemical compounds
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Phototrophs
 Derive energy from sunlight
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Chemotrophs
 Derive energy from chemical compounds

Organisms often grouped according to energy
source
Nutritional Factors on Growth
 Nutritional Diversity
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Organisms thrive due to their ability to use diverse sources of
carbon and energy
Photoautotrouphs
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Use sunlight and atmospheric carbon (CO2) as carbon source
 Called primary producers (Plants)
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Chemolithoautotrophs
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Photoheterotrophs
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A.k.a chemoautotrophs or chemolitotrophs
Use inorganic carbon for energy and use CO2 as carbon source
Energy from sunlight, carbon from organic compounds
Chemoorganoheterotrophs
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a.k.a chemoheterotrophs or chemoorganotrophs
Use organic compounds for energy and carbon source
Most common among humans and other animals
Laboratory Cultivation
 Knowing environmental and nutritional factors
makes it possible to cultivate organisms in the
laboratory
 Organisms are grown on culture media

Media is classified as complex media or
chemically defined media
Laboratory Cultivation
 Complex media
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Contains a variety of ingredients
There is no exact chemical formula for
ingredients
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Can be highly variable
Examples include
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Nutrient broth
Blood agar
Chocolate agar
Laboratory Cultivation
 Chemically defined media
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Composed of precise amounts of pure
chemical
Generally not practically for routine laboratory
use
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Invaluable in research
 Each batch is chemically identical
 Does not introduce experimental variable
Laboratory Cultivation
 Special types of culture media
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These are used to detect or isolate particular
organisms
Are divided into selective and differential
media
Laboratory Cultivation
 Selective media
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Inhibits the growth of unwanted organisms
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Allows only sought after organism to grow
Example

Thayer-Martin agar
 For isolation of Neisseria gonorrhoeae
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MacConkey agar
 For isolation of Gram-negative bacteria
Laboratory Cultivation
 Differential media
 Contains substance
that bacteria change in
recognizable way
 Example
 Blood agar
 Certain bacteria
produce hemolysin
to break down RBC
 Hemolysis

MacConkey agar
 Contains pH
indicator to identify
bacteria the
produce acid
Laboratory Cultivation
 Providing appropriate atmospheric conditions
 Bacteria can be grouped by oxygen
requirement
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Capnophile
Microaerophile
Anaerobe
Laboratory Cultivation
 Capnophile
 Require increased CO2
 Achieve higher CO2 concentrations via
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Candle jar
CO2 incubator
 Microaerophile
 Require higher CO2 than capnophile
 Incubated in gastight jar
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Chemical packet generates hydrogen and CO2
Laboratory Cultivation
 Anaerobe
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Die in the presence of oxygen
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Incubated in anaerobe jar
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Chemical reaction converts
atmospheric oxygen to water
Organisms must be able to
tolerate oxygen for brief
period
Reducing agents in media
achieve anaerobic environment
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Even if exposed for short
periods of time
Agents react with oxygen to
eliminate it
Sodium thioglycolate
Anaerobic chamber also used
for cultivation
Detecting Bacterial Growth
 Variety of techniques to determine growth
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Number of cells
Total mass
Detection of cellular products
Detecting Bacterial Growth
 Direct cell count
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Useful in determining total number of cells
Does not distinguish between living and dead
cells
Methods include
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Direct microscopic count
Use of cell counting instruments
Detecting Bacterial Growth
 Direct microscopic count
 One of the most rapid methods
 Number is measured in a know
volume
 Liquid dispensed in specialized
slide
 Counting chamber
 Viewed under microscope
 Cells counted
 Limitation
 Must have at least 10 million
cells per ml to gain accurate
estimate
Detecting Bacterial Growth
 Cell counting instruments
 Counts cells in suspension
 Cells pass counter in
single file
 Instrument measure
changes in environment
 Coulter counter
 Detects changes in
electrical resistance

Flow cytometer
 Measures laser light
Detecting Bacterial Growth
 Viable cell count

Used to quantify living cells
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Cells able to multiply
 Valuable in monitoring bacterial growth
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Often used when cell counts are too low for other
methods
Methods include
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Plate counts
Membrane filtration
Most probable numbers
Detecting Bacterial Growth
 Plate counts

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Measures viable cells
growing on solid culture
media
Count based on
assumption the one cell
gives rise to one colony
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Ideal number to count
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Number of colonies =
number of cells in
sample
Between 30 and 300
colonies
Sample normally diluted in
10-fold increments
Plate count methods


pour-plates
Spread-plates methods
Detecting Bacterial Growth
 Membrane filtration
 Used with relatively low numbers
 Known volume of liquid passed
through membrane filter
 Filter pore size retains organism
 Filter is placed on appropriate
growth medium and incubated
 Cells are counted
Detecting Bacterial Growth
 Most probable numbers (MPN)

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Statistical assay
Series of dilution sets created

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Each set inoculated with
10-fold less sample than
previous set
Sets incubated and results
noted

Results compared to MPN
table
 Table gives statistical
estimation of cell
concentration
Detecting Bacterial Growth
 Biomass measurement

Cell mass can be determined via



Turbidity
Total weight
Amounts of cellular chemical constituents
Detecting Bacterial Growth
 Turbidity

Measures with spectrophotometer

Measures light transmitted through sample
 Measurement is inversely proportional to cell concentration
 Must be used in conjunction with other test once to determine cell
numbers

Limitation

Must have high number of cells
Detecting Bacterial Growth
 Total Weight
 Tedious and time consuming
 Not routinely used
 Useful in measuring filamentous organisms
 Wet weight
 Cells centrifuged down and liquid growth medium
removed
 Packed cells weighed
 Dry weight
 Packed cells allowed to dry at 100°C 8 to 12 hours
 Cells weighed
Detecting Bacterial Growth
 Detecting cell products
 Acid production


pH indicator detects acids that result from sugar
breakdown
Gas production

Gas production monitored using Durham tube
 Tube traps gas produced by bacteria

ATP

Presence of ATP detected by use of luciferase
 Enzyme catalyzes ATP dependent reaction
 If reaction occurs ATP present  bacteria
present