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1

 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

 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

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

 Growth can be calculated
 N t
= N
0 x 2 n



(N t
) number of cells in population after certain time
(N
0
) original number of cells in the population
(n) number of divisions

Example
 N
0
= 10 cells in original population
 n = 12
 N
4hr
 N
4hr
 N
4hr
 4 hours assuming 20 minute generation time
= 10 x 2 12 n

60 min/ 1 Hr * n

12 generation s
4 Hr * 1 generation / 20 min
= 10 x 4,096
= 40,960


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
30000
25000
20000
15000
10000
5000
0
0 1 2 3 4 5 6 7 8 9 10 11 12
No. of Divisions


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

 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 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

 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 O
2
 Metabolic waste of one can serve as nutrient for other
 Interactions often competitive

Some synthesize toxic compounds to inhibit competitors

(Antibiotics)





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)

 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

 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

Microorganisms
 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

 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

Nutrients not renewed; wastes not removed
 Termed batch cultures

Yields characteristic growth curve
 Open system required to maintain continuous growth

Termed continuous culture

Nutrients added, wastes removed continuously

 Growth curve characterized by five stages
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10 10
Stationary phase
Death phase
Phase of prolonged decline
10 8
10 6
Log or exponential phase
10 4
10 2
Lag phase
10 0
Time (hr) (days/months/years)


Lag phase
 Number of cells does not increase
 Begin synthesizing enzymes required for growth

 Delay depends on conditions
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Exponential (log) phase
Stationary
 Cells divide at constant rate
 Generation time measured
 Most sensitive to antibiotics
Log
Primary metabolite
Secondary metabolite  Production of primary metabolites

Important commercially
Lag
Time (hr)
 Secondary metabolite production occurs as nutrients are depleted and wastes accumulate

 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

 Colonies and liquid cultures share similarities
 Important differences based on location
 Position of single cell determines its environment
 Edge of colony has O
2
, nutrients
 Center of colony has depleted O
2
, nutrients

Accumulation of potentially toxic wastes including acids
 Colony may range from exponential growth at edges, death phase in center

 Chemostats can maintain continuous growth
 Continually drips fresh medium into culture in chamber
 Equivalent volume removed

Contains cells, wastes, spent medium
 Nutrient content and speed of addition can be controlled

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
 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





Found in Arctic and Antarctic regions
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Psychrotroph: 20 ° to 30°C
Hyperthermophile
Mesophile
Thermophile

Important in food spoilage
Psychrotroph
Mesophile: 25 ° to 45°C

Pathogens 35 ° to 40°C
Psychrophile
Thermophiles: 45 ° to 70°C
–10
0

Common in hot springs
Hyperthermophiles: 70 ° to 110°C
10 20 30 40 50 60 70
Temperature ( °C)
80 90 100 110 120

Usually members of Archaea

Found in hydrothermal vents

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 O
2
; cool to just above solidifying temperature; innoculate; gently swirl

Growth demonstrates organism’s O
2 requirements

Oxygen Requirements
 Reactive oxygen species
 Using O
2 in aerobic respiration produces harmful reactive oxygen species (ROS) as by-products

Includes superoxide (O
2
–
) and hydrogen peroxide
 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

Inactivates superoxide by converting to O
2 and H
2
O
2
 Almost all also produce catalase


Convert H
2
O
2 to O
2 and H
2
O
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

Pump out protons if in acidic environment

Bring in protons if in alkaline environment
 Most microbes are neutrophiles

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
CO
2 and ammonia to decrease acidity of surroundings
Acidophiles grow optimally at pH below 5.5

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 )
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Halophiles: require high
Dissolved substances
(solute) salt concentrations
Cytoplasmic membrane pulls away from the cell wall (plasmolysis).

Marine bacteria ~3%
Cytoplasmic membrane

Extreme halophiles ≥ 9%
(Dead Sea, Utah’s salt flats)
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

Heterotrophs use organic carbon

Autotrophs use inorganic carbon as CO
2 fixation)
(carbon
 Nitrogen required for amino acids, nucleic acids

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 CO
2
Photoheterotrophs: energy from sunlight; carbon from organic compounds


Chemolithoautotrophs (also termed chemoautotrophs, chemolithotrophs): energy from inorganic compounds; carbon from CO
2
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% O
2
)

Broth cultures shaken to provide maximum aeration
 Many medically important bacteria (e.g., Neisseria ,
Haemophilus ) grow best with increased CO
2

Some are capnophiles, meaning require increased CO
2

One method is to incubate in candle jar
 Microaerophilic
 Require lower O
2 candle jar concentrations than achieved by


Can incubate in gas-tight container with chemical packet
Chemical reaction reduces O
2 to 5 –15%

Providing Appropriate Atmospheric Conditions
 Anaerobic: obligate anaerobes sensitive to O
2
 Anaerobic containers useful if microbe can tolerate brief
O
2 exposures; can also use semisolid culture medium containing reducing agent (e.g., sodium thioglycolate)

Reduce O
2 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
Inoculate and incubate plate
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.
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 grid
Counting chamber
Side view
0 0 0 0 2 5 8 9
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 × 10 6 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
50,000 cells/ml
1 ml to 9 ml diluent
1:10 dilution
5,000 cells/ml
1 ml to 9 ml diluent
1:100 dilution
500 cells/ml
1 ml to 9 ml diluent
1:1,000 dilution
50 cells/ml
1 ml to 9 ml diluent
1:10,000 dilution
5 cells/ml
1 ml
Too many cells produce too many colonies to count.
Too many cells produce too many colonies to count.
Too many cells produce too many colonies to count.
Between 30
–300 cells produces a countable plate.
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
Culture, diluted as needed
0.1
–0.2 ml
0.1
–1.0 ml
Solid agar
Incubate
Bacterial colonies appear only on surface.
Spread cells onto surface of pre-poured solid agar.
Pour-plate method
Melted cooled agar
Add melted cooled agar and swirl gently to mix.
Incubate
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
Combination of positives
MPN Index/
100 ml
10 ml
1 ml
0.1 ml
+
–
–
+
+
+
+
–
+
+
4
3
1
26
27
33
34
23
30
40
30
50
60
13
17
17
21
26
22
4-3-0
4-3-1
4-4-0
5-0-0
5-0-1
5-0-2
5-1-0
5-1-1
5-1-2
4-0-0
4-0-1
4-1-0
4-1-1
4-1-2
4-2-0
4-2-1
– – – – +

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.
Light detector
Bottom scale=
Optical density
(absorbance)
Light source
Dilute cell suspension (a) The cloudiness, or turbidity, of the liquid in the tube on the left is proportional to the concentration of cells.
40 50
4.
(b) A spectrophotometer is used to measure turbidity.
Concentrated cell suspension
(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
 CO
2 production
 ATP production using enzyme luciferase to produce light

4.8. Methods to Detect and Measure Microbial Growth