Transcript Ch4 - Morgan Community College

Chapter 04
*Lecture Outline
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1
A Glimpse of History
 German physician Robert Koch (1843–1910)


Studied disease-causing bacteria; Nobel Prize in 1905
Developed methods of cultivating bacteria


Worked on methods of solid media to allow single bacteria
to grow and form colonies
Tried potatoes, but nutrients limiting for many bacteria
• Solidifying liquid nutrient media
with gelatin helped
• Limitations: melting temperature,
digestible
• In 1882, Fannie Hess, wife of
associate, suggested agar, then
used to harden jelly
Introduction
 Prokaryotes found growing in severe conditions


Ocean depths, volcanic vents, polar regions all
harbor thriving prokaryotic species
Many scientists believe that if life exists on other
planets, it may resemble these microbes
 Individual species have limited set of conditions

Also require appropriate nutrients
 Important to grow microbes in culture


Medical significance
Nutritional, industrial uses
4.1. Principles of Bacterial Growth
Prokaryotic cells divide by binary fission



One cell divides into two, two
into four, 48, 816, etc…
Exponential growth: population
doubles each division
Generation time is time it takes
to double



Varies among species
Environmental conditions
Exponential growth has
important consequences

10 cells of food-borne pathogen in
potato salad at picnic can become
40,000 cells in 4 hours
Cell gets longer and
DNA replicates.
DNA is moved into
each future daughter
cell and cross wall forms.
Cell divides into two cells.
Cells separate.
Daughter cells
4.1. Principles of Bacterial Growth
 Growth can be calculated
 Nt = N0 x 2n
 (Nt ) number of cells in population after certain time
 (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
n  60 min/1Hr * 4 Hr *1generation/ 20 min
 N4hr = 10 x 212
n  12generations
 N4hr = 10 x 4,096
 N4hr = 40,960
4.1. Principles of Bacterial Growth
 The power of exponential growth

Rapid generation time with optimal conditions can
yield huge populations quickly
Remember that generation time depends on
species and growth conditions
45000
40000
35000
No. of Cells

30000
25000
20000
15000
10000
5000
0
0 1 2 3 4 5 6 7 8 9 10 11 12
No. of Divisions
4.2. Prokaryotic Growth in Nature
 Microorganisms historically studied in laboratory
 But dynamic, complex conditions in nature have
profound effect on microbial growth, behavior



Cells sense changes, adjust to surroundings
Synthesize compounds useful for growth
Can live singly



Most live in polysaccharideencased communities
Termed biofilms
Cause slipperiness of rocks
in stream bed, slimy “gunk”
in sink drains, scum in toilet
bowls, dental plaque
Biofilms
 Formation of biofilm
Planktonic bacteria
move to the surface
and adhere.
Bacteria multiply
and produce
extracellular polymeric
substances (EPS).
Other bacteria may
attach to the EPS
and grow.
Cells communicate
and create channels
in the EPS that allow
nutrients and waste
products to pass.
Some cells detach
and then move to
other surfaces to
create additional
biofilms.
Biofilms
 Biofilms have characteristic architectures


Channels through which nutrients and wastes pass
Cells communicate by synthesizing chemical signals
 Biofilms have important implications




Dental plaque leads to tooth decay, gum disease
Most infections (e.g., ear infections, cystic fibrosis)
Industrial concerns: accumulations in pipes, drains
Biofilm structure shields microbes growing within


May be hundreds of times more resistant
Biofilms can also be helpful

Bioremediation, wastewater treatment
Interactions of Mixed Microbial
Communities
 Prokaryotes regularly grow in close association


Many different species
Interactions can be cooperative

Can foster growth of species otherwise unable to
survive
 Strict anaerobes can grow in mouth if others consume O2
 Metabolic waste of one can serve as nutrient for other

Interactions often competitive


Some synthesize toxic compounds to inhibit competitors
(Antibiotics)
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 (agar)
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
Growing
Microorganisms
on a Solid Medium
 Maintaining stock cultures




Streak-plate method yields pure cultures
Can be maintained as stock culture
Often stored in refrigerator as agar slant
Cells can be frozen at –70°C for long-term storage


Requires solution that prevents ice crystal formation
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Can be freeze-dried
7 Streak final area.
8
Isolated
colonies
develop
after incubation.
© Fred Hossler/Visuals unlimited
4.4. Prokaryotic Growth in Laboratory Conditions
 Prokaryotes grown on agar plates or in tubes or
flasks of broth

Closed systems


Termed batch cultures


Nutrients not renewed; wastes not removed
Yields characteristic growth curve
Open system required to maintain continuous
growth


Termed continuous culture
Nutrients added, wastes removed continuously
The Growth Curve
 Growth curve characterized by five stages
Number of cells (logrithmic scale)
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Stationary
phase
1010
Death
phase
108
106
Log or
exponential
phase
Phase of
prolonged decline
104
102
Lag
phase
100
Time (hr)
(days/months/years)
The Growth Curve
 Lag phase
 Number of cells does not increase
 Begin synthesizing enzymes required for growth
 Delay depends on conditions
 Exponential (log) phase
 Cells divide at constant rate
 Generation time measured
 Most sensitive to antibiotics
 Production of primary
metabolites
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Log
Primary
metabolite
Secondary
metabolite
Lag
Time (hr)


Important commercially
Secondary metabolite production occurs as
nutrients are depleted and wastes accumulate
Synthesis of metabolites
Number of cells (logarithmic scale)
Stationary
The Growth Curve
 Stationary phase


Nutrient levels too low to sustain growth
Total numbers remain constant

Some die, release contents; others grow
 Death phase

Total number of viable cells decrease


Cells die at constant rate
Exponential, but usually much slower than cell growth
 Phase of prolonged decline



Some fraction may survive
Adapted to tolerate worsened conditions
Living off other decomposing cells
Colony Growth
 Colonies and liquid cultures share similarities
 Important differences based on location



Position of single cell determines its environment
Edge of colony has O2, nutrients
Center of colony has depleted O2, nutrients


Accumulation of potentially toxic wastes including acids
Colony may range from exponential growth at
edges, death phase in center
Continuous Culture
 Chemostats can maintain continuous growth


Continually drips fresh medium into culture in
chamber
Equivalent volume removed


Nutrient content and speed of addition can be
controlled


Contains cells, wastes, spent medium
Achieve constant growth rate and cell density
Produces relatively uniform population for study
4.5. Environmental Factors That Influence Microbial
Growth
 Prokaryotes inhabit nearly all environments


Some live in comfortable habitats favored by
humans
Some live in harsh environments

Termed extremophiles; most are Archaea
 Major conditions that influence growth


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Temperature
Atmosphere
pH
Water availability
4.5. Environmental Factors That Influence Microbial
Growth
Temperature Requirements
 Each species has well-defined temperature range
 Optimum growth usually close to upper end of range
 Psychrophile: –5° to 15°C

Psychrotroph: 20° to 30°C



Hyperthermophile
Psychrotroph
Psychrophile
Pathogens 35° to 40°C
–10
0
10
Common in hot springs
Hyperthermophiles: 70° to 110°C

Thermophile
Mesophile
Thermophiles: 45° to 70°C


Important in food spoilage
Mesophile: 25° to 45°C


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Growth rate

Found in Arctic and Antarctic regions
Usually members of Archaea
Found in hydrothermal vents
20
30
40
50
60
70
Temperature (°C)
80
90
100 110 120
Temperature Requirements
 Proteins of thermophiles resist denaturing
 Thermostability comes from amino acid sequence

Number and position of bonds, which determine structure
 Temperature and food preservation
 Refrigeration (~4°C) slows spoilage by limiting growth
of otherwise fast-growing mesophiles


Psychrophiles, psychrotrophs can still grow, but slowly
Freezing preserves food; not effective at killing
microbes
 Temperature and disease
 Temperature of different parts of human body differs


Some microbes cause disease in certain parts
E.g., Hansen’s disease (leprosy) involves coolest regions
(ears, hands, feet, fingers) due to preference of M. leprae
Oxygen Requirements
 Boil nutrient agar to drive off O2; cool to just above
solidifying temperature; innoculate; gently swirl

Growth demonstrates organism’s O2 requirements
Oxygen Requirements
 Reactive oxygen species
 Using O2 in aerobic respiration produces harmful reactive
oxygen species (ROS) as by-products



Damaging to cellular components
Cells must have mechanisms to protect


Obligate anaerobes typically do not
Almost all organisms growing in presence of oxygen
produce enzyme superoxide dismutase


Includes superoxide (O2–) and hydrogen peroxide
Inactivates superoxide by converting to O2 and H2O2
Almost all also produce catalase


Convert H2O2 to O2 and H2O
Exception is aerotolerant anaerobes; makes for useful test
pH
 Bacteria survive a range of pH; have optimum

Most maintain constant internal pH, typically near neutral



Most microbes are neutrophiles

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

Range of pH 5 to 8; optimum near pH 7
Food can be preserved by increasing acidity
H. pylori grows in stomach; produces urease to split urea into
CO2 and ammonia to decrease acidity of surroundings
Acidophiles grow optimally at pH below 5.5


Pump out protons if in acidic environment
Bring in protons if in alkaline environment
Picrophilus oshimae has optimum pH of less than 1!
Alkaliphiles grow optimally at pH above 8.5
Water Availability
 All microorganisms require water for growth

Dissolved salts, sugars make water unavailable to cell





If solute concentration is higher outside of cell, water diffuses out
(osmosis)
Salt, sugar used to preserve food
Some microbes withstand or even require high salt
Halotolerant: withstand up to 10% (e.g., Staphylococcus)
Halophiles: require high
salt concentrations
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Dissolved substances
(solute)
Marine bacteria ~3%
 Extreme halophiles ≥ 9%
(Dead Sea, Utah’s salt flats)

Cytoplasmic membrane
pulls away from the
cell wall (plasmolysis).
Cytoplasmic
membrane
Cell wall
Water flows
out of cell
4.6. Nutritional Factors That Influence Microbial Growth
 Prokaryotes have remarkable metabolic diversity
 Require nutrients to synthesize cell components


Lipid membranes, cell walls, proteins, nucleic acids
Made from subunits: phospholipids, sugars, amino acids,
nucleotides

Subunits composed of chemical elements including carbon and
nitrogen
 Key considerations:




Required elements
Growth factors
Energy sources
Nutritional diversity
4.6. Nutritional Factors That Influence Microbial Growth
 Required elements


Major elements make up cell components
Carbon source distinguishes different groups



Nitrogen required for amino acids, nucleic acids




Heterotrophs use organic carbon
Autotrophs use inorganic carbon as CO2 (carbon
fixation)
Many use ammonia (some convert nitrate to ammonia)
Nitrogen fixation important
Iron, phosphorous often limiting
Trace elements usually
available (cobalt, zinc, copper
molybdenum, manganese)
4.6. Nutritional Factors That Influence Microbial Growth
 Growth factors

Some microbes cannot synthesize certain
molecules



Amino acids, vitamins, purines, pyrimidines
Only grow if these growth factors are available
Reflects biosynthetic capabilities



E. coli synthesizes all cellular components from glucose,
has wide metabolic capabilities
Neisseria unable to synthesize many, requires
numerous growth factors
Termed fastidious: have complicated nutritional
requirements
4.6. Nutritional Factors That Influence Microbial Growth
 Energy sources

Sunlight, chemical compound
 Phototrophs obtain energy from sunlight

Plants, algae, photosynthetic bacteria
 Chemotrophs extract energy from chemicals



Mammalian cells, fungi, many types of prokaryotes
Sugars, amino acids, fatty acids common sources
Some prokaryotes use inorganic chemicals such as
hydrogen sulfide, hydrogen gas
4.6. Nutritional Factors That Influence Microbial Growth
 Nutritional diversity




Photoautotrophs: energy from sunlight; carbon from CO2
Photoheterotrophs: energy from sunlight; carbon from
organic compounds
Chemolithoautotrophs (also termed chemoautotrophs,
chemolithotrophs): energy from inorganic compounds;
carbon from CO2
Chemoorganoheterotrophs (also termed
chemoheterotrophs, chemoorganotrophs): energy and
carbon from organic compounds
4.7. Cultivating Prokaryotes in the Laboratory
 General categories of culture media

Complex media contains variety of ingredients



Exact composition highly variable
Often a digest of proteins
Chemically defined media composed of exact
amounts of pure chemicals


Used for specific research experiments
Usually buffered
4.7. Cultivating Prokaryotes in the Laboratory
 Hundreds of types of
media available

Regardless, some
medically important
microbes, and most
environmental ones,
have not yet been
grown in laboratory
4.7. Cultivating Prokaryotes in the Laboratory
 Special types of culture media


Useful for isolating and
identifying a specific species
Selective media inhibits growth
of certain species
• Differential media contains substance that microbes
change in identifiable way
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Colony Zone of clearing
(a)
(b)
a: © Christine Case/Visuals Unlimited; b: © L. M. Pope and D. R. Grote/Biological Photo Service
Providing Appropriate Atmospheric Conditions
 Aerobic

Most obligate aerobes and facultative anaerobes can be
incubated in air (~20% O2)


Broth cultures shaken to provide maximum aeration
Many medically important bacteria (e.g., Neisseria,
Haemophilus) grow best with increased CO2


Some are capnophiles, meaning require increased CO2
One method is to incubate in candle jar
 Microaerophilic

Require lower O2 concentrations than achieved by
candle jar


Can incubate in gas-tight container with chemical packet
Chemical reaction reduces O2 to 5–15%
Providing Appropriate Atmospheric Conditions
 Anaerobic: obligate anaerobes sensitive to O2

Anaerobic containers useful if microbe can tolerate brief
O2 exposures; can also use semisolid culture medium
containing reducing agent (e.g., sodium thioglycolate)


Reduce O2 to water
Anaerobic chamber provides more stringent approach
Enrichment Cultures
 Enrichment cultures used to isolate organism that constitutes
small fraction of mixed population
 Provide conditions promoting growth of particular species

E.g., specific carbon source
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Incubate
Medium contains nutrients
that few species other than
the one of interest can use.
Sample that contains a variety
of species, including the one of
interest, is added to the medium.
Inoculate and
incubate plate
Species of interest
multiplies, where as
others cannot.
Enriched sample is plated onto appropriate
agar medium. A pure culture is obtained by
selecting a single colony of the species
of interest.
4.8. Methods to Detect and Measure Microbial Growth
 Direct cell counts: total numbers (living plus dead)


Direct microscope count
Cell-counting instruments (Coulter counter, flow cytometer)
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Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Cover glass resting on
supporting ridges
Counting chamber
0000 25 89
Counting grid
Side view
Sample spreads over counting
grid by capillary action.
Automatic counter
Sample in
liquid
Bacterial cell
Electronic detector
Using a microscope, the cells in several large squares like the one shown
are counted and the results averaged. To determine the number of cells
per ml, that number must be multiplied by 1/volume (in ml) held in the
square. For example, if the square holds 1/1,250,000 ml, then the number
of cells must be multiplied by 1.25 × 106 ml.
4.8. Methods to Detect and Measure Microbial Growth
 Viable cell counts: cells capable of multiplying


Can use selective, differential media for particular species
Plate counts: single cell gives rise to colony

Plate out dilution series: 30–300 colonies ideal
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Adding 1 ml of culture to 9 ml of diluent results in a 1:10 dilution.
Original bacterial
culture
to 9 ml diluent
1:10 dilution
to 9 ml diluent
1:100 dilution
50,000
cells/ml
5,000
cells/ml
500
cells/ml
1 ml
Too many cells
produce too
many colonies
to count.
1 ml
Too many cells
produce too
many colonies
to count.
1 ml
Too many cells
produce too
many colonies
to count.
to 9 ml diluent
1:1,000 dilution
50
cells/ml
1 ml
Between 30–300
cells produces a
countable plate.
to 9 ml diluent
1:10,000 dilution
5
cells/ml
1 ml
Does not produce
enough colonies
for a valid count.
4.8. Methods to Detect and Measure Microbial Growth

Plate counts determine colony-forming units (CFUs)
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Spread-plate method
Solid agar
Incubate
Culture, diluted
as needed
0.1–0.2 ml
Bacterial colonies
appear only on surface.
Spread cells onto surface
of pre-poured solid agar.
Pour-plate method
0.1–1.0 ml
Melted cooled agar
Incubate
Add melted cooled agar
and swirl gently to mix.
Some colonies appear on
surface; many are below surface.
4.8. Methods to Detect and Measure Microbial Growth

Membrane filtration


Concentrates microbes by filtration
Filter is incubated on appropriate agar medium
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Liquid
sample
Membrane
filter
A known volume of liquid is passed
through a sterile membrane filter; the
filter retains bacterial cells.
The membrane filter is placed on
an appropriate agar medium and
incubated.
The number of colonies that grow on the
filter indicates the number of bacterial cells
in the volume filtered.
(left): © Dennis Kunkel/Phototake; (right): © Kathy Talaro/Visuals Unlimited
4.8. Methods to Detect and Measure Microbial Growth

Most Probable Number (MPN)


Estimates cell concentration using dilution series
Sets of tubes are incubated; results are recorded and
compared to table to give statistical determination
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Volume of
inoculum
Observation after incubation
(gas production noted)
Number of positive
tubes in set of five
10 ml
Combination
of positives
MPN Index/
100 ml
4-0-0
13
4-0-1
17
4-1-0
17
4-1-1
21
4-1-2
26
4-2-0
22
4-2-1
26
4-3-0
27
4-3-1
33
4-4-0
34
5-0-0
23
5-0-1
30
5-0-2
40
5-1-0
30
5-1-1
50
5-1-2
60
4
+
–
+
+
+
1 ml
3
–
+
+
–
+
0.1 ml
1
–
–
–
+
–
4.8. Methods to Detect and Measure Microbial Growth
 Measuring biomass


Turbidity is proportional to concentration of cells
Measured with spectrophotometer
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Top scale=Percentage of
light that passes through
40
50
4.
Bottom scale=
Optical density
(absorbance)
Light
detector
Light source
(a) The cloudiness, or turbidity, of the liquid in the tube on the left
is proportional to the concentration of cells.
Dilute cell
suspension
40
50
4.
Concentrated cell
suspension
(b) A spectrophotometer is used to measure turbidity.
(c) The percentage of light that reaches the detector of the spectrophotometer
is inversely proportional to the optical density.
a: © Richard Megna/Fundamental Photographs
4.8. Methods to Detect and Measure Microbial Growth
 Measuring biomass

Total weight can be measured




Tedious and time-consuming
Typically only used for filamentous organisms that do not
readily separate into individual cells for valid plate counts
Cells in liquid culture centrifuged; pellet is weighed
Dry weight can be determined by heating pellet in oven
 Detecting cell products




pH indicators
Durham tubes (inverted tubes) to trap gas
CO2 production
ATP production using enzyme luciferase to produce
light
4.8. Methods to Detect and Measure Microbial Growth