Download Dynamics of Prokaryotic Growth

Dynamics of Prokaryotic
Growth
Chapter 4
Principles of Bacterial Growth
• Prokaryotic cells divide by binary
fission
– One cell divides into two
• Two into four etc.
– Cell growth is exponential
• Doubling of population with each cell
division
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 right now
(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
– Cells sense changing
environment
Biofilm layer
• Synthesize compounds
useful for growth
• Cells produce multicellular
associations to increase
survivability
Big question: how applicable is growing these
things in lab if they do different things in nature.
– Example
» Biofilms
» Slime layers
Bacterial Growth in Nature
• Biofilm
– Formation begins when planktonic bacteria attach to
surfaces
• Other bacteria attach and grow on initial layer
– Has characteristic architecture
• Contains open channels for movement of nutrients and waste
– Cells within biofilms can cause disease
• Treatment becomes difficult
– Architecture resists immune response and antimicrobials
– Bioremediation is beneficial use of biofilm
• Add nutrients - biostimulation
• Add other bacteria - bioaugmentation
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
• Used for growing large
numbers of bacteria
– Solid media is broth media
with addition of agar
• 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
• Multiple techniques
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
especially in broths
• 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
– Cells prepare for growth
• “tooling up”
• Log phase
– Period of exponential growth
• Doubling of population with each
generation
– Produce primary metabolites
• Compounds required for growth
– Late log phase
• Endospores
• Synthesize secondary metabolites
– Used to enhance survival
– Antibiotics
Bacterial Growth in
Laboratory Conditions
• Stationary phase
– Overall population remains
relatively stable
• Cells exhausted nutrients
• Cell growth = cell death
– Dying cell supply metabolites for
replicating cells
• Death phase
– Total number of viable cells
decreases
• 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 than
previous
• Evolution – change in genetics
over time
World population
Bacterial Growth in
Laboratory Conditions
• Colony growth on solid medium
– In colony, cells eventually compete for
resources
– Position within colony determines resource
availability
• 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
Chemostats allow for continuous growth
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
• Within range lies optimum
growth temperature
– Prokaryotes divided into 5
categories
•
Psychrophile
– Optimum temperature -5°C to
15°C
• Found in Arctic and Antarctic
regions
•
Psychrotroph
– 20°C to 30°C
• Important in food spoilage
•
Mesophile
– 25°C to 45°C
• More common
• Disease causing
•
Thermophiles
– 45°C to 70°C
• Common in hot springs
•
Hyperthermophiles
– 70°C to 110°C
• Usually members of Archaea
• Found in hydrothermal vents
Psychro = “cold”
Environmental Factors on Growth
• Oxygen
– Prokaryotes divided based on oxygen requirements
• Obligate aerobes
– Absolute requirement for oxygen
» Use for energy production
• Facultative anaerobes
– Grow better with oxygen
» Use fermentation in absence of oxygen
• Microaerophiles
– Require oxygen in lower concentrations
» Higher concentration inhibitory
• Obligate anaerobes
– No multiplication in presence of oxygen
» May cause death
• Aerotolerant anaerobes
– Indifferent to oxygen, grow with or without
» Do not use oxygen to produce energy
Oxygen
Environmental Factors on Growth
• pH
– 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 protons
Environmental Factors on Growth
• Water availability
– All microorganisms require water for growth
– Water not available in all environments
• In high salt environments
– Bacteria increase internal solute concentration
» Synthesize small organic molecules
– Osmotolerant bacteria tolerate high salt environments
– Halophiles - bacteria that require high salt for cell growth
termed
Nutritional Factors on Growth
• Growth of prokaryotes depends on
nutritional factors as well as physical
environment
• Main factors to be considered are:
– 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 organic carbon as nutrient source
• Autotrophs
– Use inorganic carbon (CO2) as carbon source
– Trace elements
• Cobalt, zinc, copper, molybdenum and manganese
– Required in minute amounts
Nutritional Factors on Growth
• Growth factors
– Some bacteria cannot synthesize some cell
constituents
• These must be added to growth environment
– Organisms can display wide variety of factor
requirements
• 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
• Phototrophs
– Derive energy from sunlight
• Chemotrophs
– Derive energy from chemical compounds
– Organisms often grouped according to energy source
Nutritional Factors on Growth
• Nutritional Diversity
– Organisms thrive due to their ability to use diverse sources of
carbon and energy
– Photoautotrophs
• Use sunlight and atmospheric carbon (CO2) as carbon source
– Called primary producers (Plants)
– Chemoautotrophs
• a.k.a chemolithoautotrophs or chemolitotrophs
• Use inorganic compounds for energy and use CO2 as carbon
source
– Photoheterotrophs
• Energy from sunlight, carbon from organic compounds
– Chemoheterotrophs
• a.k.a chemoorganoheterotrophs or chemoorganotrophs
• Use organic compounds for energy and carbon source
Laboratory Cultivation
• Knowing environmental and nutritional
factors makes it possible to cultivate
organisms in the laboratory
• Organisms are grown on culture media
– complex media
– chemically defined media
Laboratory Cultivation
• Complex media
– Contains a variety of ingredients
– There is no exact chemical formula for
ingredients
• Can be highly variable
– Examples include
• Nutrient broth
• Blood agar
• Chocolate agar
Laboratory Cultivation
• Chemically defined media
– Composed of precise amounts of pure
chemical
– Generally not practical for routine laboratory
use
• Invaluable in research
– Each batch is chemically identical
» Does not introduce experimental variable
Laboratory Cultivation
• Special types of culture media
– Used to detect or isolate particular organisms
– Are divided into selective and differential
media
Laboratory Cultivation
• Selective media
– Inhibit the growth of unwanted organisms
• Allow only sought after organisms to grow
– Example
• Thayer-Martin agar - antibiotics
– For isolation of Neisseria gonorrhoeae
• MacConkey agar – crystal violet and bile salts
– For isolation of Gram negative bacteria of intestine
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
that produce acid
Laboratory Cultivation
• Providing appropriate atmospheric
conditions
• Bacteria can be grouped by oxygen
requirement
– Capnophile
– Microaerophile
– Anaerobe
Laboratory Cultivation
• Capnophile
– Require increased CO2
– Achieve higher CO2 concentrations
via
• Candle jar
• CO2 incubator
• Microaerophile
– Require higher CO2 than
capnophile
– Incubated in gastight jar
• Chemical packet generates hydrogen
and CO2
Laboratory Cultivation
• Anaerobe
– Die in the presence of oxygen
• Even if exposed for short period of
time
– Incubated in anaerobe jar
• Chemical reaction converts
atmospheric oxygen to water
• Organisms must be able to
tolerate oxygen for brief period
– Reducing agents in media achieve
anaerobic environment
• Agents react with oxygen to
eliminate it
• Sodium thyoglycolate
– Anaerobic chamber also used for
cultivation
Detecting Bacterial Growth
• Variety of techniques to determine growth
– Numbers of cells
– Total mass
– Detection of cellular products
Detecting Bacterial Growth
• Direct cell count
– Useful in determining total number of cells
– Does not distinguish between living and dead
cells
– Methods include
• 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
known 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
– Count cells in suspension
– Cells pass counter in
single file
– 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
• Cells able to multiply
– Valuable in monitoring bacterial growth
• Often used when cell counts are too low for other
methods
– Methods include
• Plate counts
• Membrane filtration
• Most probable numbers
Detecting Bacterial Growth
• Plate counts
– Measures viable cells
growing on solid culture
media
– Count based on
assumption that one cell
gives rise to one colony
• Number of colonies =
number of cells in sample
– Ideal number to count
• 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)
– Statistical assay
– Series of dilution sets created
• Each set inoculated with 10fold 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