Download Lecture 3 - Bacterial Growth Chpt. 4 S11 2 slides per page

1/21/2011
Dynamics of Prokaryotic
Growth
Chapter 4
1
1/21/2011
Principles of Bacterial Growth
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
• Exponential growth has important
h lth consequences
health
– Generation time
• Time it takes for population to double
• a.k.a. doubling time
• Varies among species
2
1/21/2011
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
Generation Times Vary for Each
Organism
• E. coli
• Mycobacterium tuberculosis
3
1/21/2011
Bacterial Growth in
Laboratory Conditions
• Cells in laboratory grown in closed or
b t h system
batch
t
– 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
4
1/21/2011
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
• Stationary
St ti
phase
h
– Overall population remains
relatively stable
• Cells exhausted nutrients
• Cell growth = cell death
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
5
1/21/2011
Environmental Factors on Growth
• Temperature
•
– Optimum temperature -5°C to
15°C
– Each species has wellwell
defined temperature range
• Within range lies optimum
growth temperature
– Prokaryotes divided into 5
categories
Psychrophile
• 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
Environmental Factors on Growth
Decreasing
O2
6
1/21/2011
Environmental Factors on Growth
• pH
– Bacteria survive within various 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
– Osmotolerant bacteria tolerate high salt environments
– Bacteria that require high salt for cell growth termed
h l hil
halophiles
7
1/21/2011
Nutritional Factors on Growth
• Growth of prokaryotes depends on
nutritional
t iti
l ffactors
t
as wellll as physical
h i l
environment
• Main factors to be considered are:
– Required elements
– Growth factors
– Energy sources
– Nutritional diversity
Nutritional Factors on Growth
To live/reproduce, all living things need:
an energy source
a source of carbon and other required elements
Energy sources:
Chemicals (“chemo”)
Organic (“organo”) (ex. glucose)
Inorganic (“litho”) (ex. H2S)
Sunlight (“photo”)
Carbon sources:
CO2 (“auto”) (carbon fixation)
Organic cmpds (“hetero”)
troph = nourish
8
1/21/2011
Nutritional Factors on Growth
Required
Elements:
Nutritional Factors on Growth
Carbon Sources:
•CO2 (Carbon fixation)
•Organic Compounds
Nitrogen Sources:
•Organic Compounds
•Inorganic Compound
•N2 (Nitrogen fixation)
9
1/21/2011
Nutritional Factors on Growth
Hartford Courant, March 6, 2009
Early Vegetables A Rewarding Way To Welcome Spring
Peas are not fussy, but it does pay to soak the seed for about a half hour before
planting, then cover it with an inoculant.
You can buy an inexpensive inoculant at nurseries; it's a black powder that "inoculates"
the soil with nitrogen-fixing bacteria, prompting the peas, which are legumes, to start
producing nitrogen on their own.
10
1/21/2011
Nutritional Factors on Growth
Carbon Sources:
•CO2 (Carbon fixation)
•Organic Compounds
Nitrogen Sources:
•Organic Compounds
•Inorganic Compound
•N2 (Nitrogen fixation)
g Nutrients
Limiting
Limiting Nutrients
Nutritional Factors on Growth
1 pkg chocolate chips
3 c sugar
11
1/21/2011
Nutritional Factors that Influence Microbial Growth
1 pkg chocolate chips
10 c sugar
Seattle Times, Linda Mapes, 3/18/08
Algae-prone Green Lake is staying clean
Good news for Green Lake: It's clean and staying that way.
Efforts in 2004 to reduce algae
g g
growth in the lake by
y adding
g aluminum
sulfate, or alum, are still paying dividends. The alum binds with phosphorus
in the water, tying up a key nutrient algae need to thrive.
….the addition of nitrogen and phosphorus from fertilizers and other
pollutants, combined with warm, sunny days, can spell excessive growth.
And cyanobacteria, while quite beautiful, are toxic to small animals and
dogs,…..
dogs
12
1/21/2011
Bacterial Growth in Nature
• Conditions in nature
have profound effect on
microbial growth
– Cells sense changing
environment
• Synthesize compounds
useful for growth
• Cells produce multicellular
associations to increase
survivability
– Example
» Biofilms
» Slime layers
Biofilm layer
Bacterial Growth in Nature
• Interactions of mixed microbial
communities
iti
– 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
13
1/21/2011
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
– 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
14
1/21/2011
Laboratory Cultivation
• Chemically defined media
– Composed of precise amounts of pure chemical
– Generally not practical for routine laboratory use
Laboratory Cultivation
• Special types of culture media
– Used to detect or isolate particular organisms
– Divided into selective and differential media
15
1/21/2011
Laboratory Cultivation
• Selective media
– Inhibit the growth of unwanted organisms
• Allow only sought after organisms to grow
– Examples
• Mannitol salts agar
– For isolation of salt tolerant bacteria
• MacConkey
M C k 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
• MacConkey agar
– Contains pH indicator
to identify bacteria
that produce acid
16
1/21/2011
Laboratory Cultivation
• Providing appropriate atmospheric
conditions
diti
• Bacteria can be grouped by oxygen
requirement
– Capnophile
– Microaerophile
– Anaerobe
– Aerobe
Laboratory Cultivation
17
1/21/2011
Obtaining a Pure Culture
Streak Plate
Detecting Bacterial Growth
• Variety of techniques to determine growth
– Numbers of cells
– Total mass
– Detection of cellular products
18
1/21/2011
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
Direct Counts
Counting chamber
• Counting Chamber
Cell counting instruments
19
1/21/2011
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
Viable Cell Counts
• Plate method
• Membrane filtration
20
1/21/2011
Viable Cell Counts:
The Most Probable Number (MPN)
Method
Measure Biomass: Turbidity
gives a rough estimate
21
1/21/2011
Summary
• General principles
– binary fission, exponential growth, generation time
• Factors that influence growth
– environmental factors; temperature, O2 availability, pH, aw, nutrients
– nutritional factors; required elements, growth factors, energy source
• Bacterial growth in laboratory conditions
– closed system; the growth curve
• Bacterial growth in nature
– open system; dynamic conditions, mixed microbial communities,
biofilms
• Cultivating bacteria in the laboratory
– characteristics of media
• Methods to detect and measure bacterial growth
– direct, viable, measuring biomass
22