Microbial Growth

1
Chapter 6

• Microbial Growth

2
Bacterial Cell Division

• New cells are formed by cell fission
• Cells do not grow they double their cytoplasmic
  contents and membrane
• They synthesize essential molecules needed for
  their metabolic processes

3
Binary Fission in Bacteria
4
Partitioning

• Prior to cell division, bacteria copy their DNA(
  replicate their DNA)
• They then partition the DNA by constructing a
  cell wall between the two molecules of DNA
• This insures that the new cell receives a copy of
  the chromosome
• The division or partitioning of chromosomes is
  more difficult in those organisms that have more
  than one chromosome

5
Prokaryote vs. Eukaryote

• Prokaryote cells do not go through the cell cycle
  like eukaryote cells
• They divide by fission
• In some species there is some linkage which forms
  tetrads, sarcinae, and even staphylococci

6
Growth

• Increase in cellular constituents that may result
  in
• increase in cell number
• when microorganisms reproduce by budding or
  binary fission
• increase in cell size
• coenocytic microorganisms have nuclear divisions
  that are not accompanied by cell divisions. Fungi
  have a syncytium and their nuclei are not
  separated.
• Microbiologists usually study population growth
  rather than growth of individual cells

7
The Growth Curve

• Observed when microorganisms are cultivated in
  batch culture
• culture incubated in a closed vessel with a
  single batch of medium
• Usually plotted as logarithm of cell number
  versus time
• Usually has four distinct phases

8
population growth ceases
maximal rate of division and population growth
decline in population size
no increase
Figure 6.1
9
Lag Phase

• Cell synthesizing new components
• to replenish spent materials
• to adapt to new medium or other conditions
• varies in length
• in some cases can be very short or even absent

10
Exponential Phase

• Also called log phase
• Rate of growth is constant
• Population is most uniform in terms of chemical
  and physical properties during this phase

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cells are dividing and doubling in number at
regular intervals
12
Each individual cell divides at a slightly
different time
Curve rises smoothly rather than as discrete steps
13
E. Coli Growth Curve
14
Balanced growth

• During log phase, cells exhibit balanced growth
• cellular constituents manufactured at constant
  rates relative to each other

15
Unbalanced growth

• Rates of synthesis of cell components vary
  relative to each other
• Occurs under a variety of conditions
• change in nutrient levels
• shift-up (poor medium to rich medium)
• shift-down (rich medium to poor medium)
• change in environmental conditions

16
Stationary Phase

• total number of viable cells remains constant
• may occur because metabolically active cells stop
  reproducing
• may occur because reproductive rate is balanced
  by death rate

17
Possible reasons for entry into stationary phase

• nutrient limitation
• limited oxygen availability
• toxic waste accumulation
• critical population density reached

18
Starvation responses

• morphological changes
• endospore formation
• decrease in size, protoplast shrinkage, and
  nucleoid condensation
• production of starvation proteins
• long-term survival
• increased virulence

19
Death Phase

• cells dying, usually at exponential rate
• death
• irreversible loss of ability to reproduce
• in some cases, death rate slows due to
  accumulation of resistant cells

20
The Mathematics of Growth

• Generation (doubling) time
• time required for the population to double in
  size
• Mean growth rate constant
• number of generations per unit time
• usually expressed as generations per hour

21
The Generation Time

• The generation time for most species is between
  twenty minutes and 24 hours.
• Some organisms take a longer time to go through
  the lag phase
• Some organisms due to their characteristics like
  Mycobacterium tuberculosis grow slowly due to the
  cell wall

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Synchronous Growth

• Cells doubling or dividing every 20 minutes

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Measurement of Microbial Growth

• Can measure changes in number of cells in a
  population
• Can measure changes in mass of population

26
Measurement of Cell Numbers

• Direct cell counts
• counting chambers
• electronic counters
• on membrane filters
• Viable cell counts
• plating methods
• membrane filtration methods

27
Counting chambers

• easy, inexpensive, and quick
• useful for counting both eucaryotes and
  procaryotes
• cannot distinguish living from dead cells

Figure 6.5
28
Electronic counters

• microbial suspension forced through small orifice
• movement of microbe through orifice impacts
  electric current that flows through orifice
• instances of disruption of current are counted

29
Electronic counters

• cannot distinguish living from dead cells
• quick and easy to use
• useful for large microorganisms and blood cells,
  but not procaryotes

30
Direct counts on membrane filters

• cells filtered through special membrane that
  provides dark background for observing cells
• cells are stained with fluorescent dyes
• useful for counting bacteria
• with certain dyes, can distinguish living from
  dead cells

31
Plating methods

• plate dilutions of population on suitable solid
  medium
• ?
• count number of colonies
• ?
• calculate number of cells in population

• Measure number of viable cells
• Population size is expressed as colony forming
  units (CFU)

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Spread Plate

• Samples are diluted by using 1 ml of broth
  culture and 9 ml of sterile nutrient broth
• Mix
• Then 1 ml of the 110 ( first dilution) is added
  to another 9ml of fresh nutrient broth
• Mix
• Samples are diluted by using 1ml of broth culture
  and 9 ml of sterile nutrient broth
• Mix

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Standard Dilutions
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Spread plate

• A ml of each dilution is pipetted with a plastic
  transfer pipet to the center of an agar plate
• A spreader( looks like a hockey stick) is used to
  spread the cells across the surface
• This is designed to produce an even distribution
  throughout

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Colony Counter
36
Colony Counter

• To make an exact count of the colonies you place
  the plate on a grid
• You then illuminate the plate.
• You count the colonies in the grid by going
  across a horizontal row and then vertically to
  the next row until you have covered the whole
  plate
• The final count is multiplied x the dilution
  factor. This number is the number of bacteria
  that were in 1 ml of culture
• It is assumed that each colony is equal to 1
  original cell in the broth culture

37
Applications of this technique commonly used in
the laboratory

• Determination of coliforms in the environment( E.
  coli)
• Determination of cells transformed by genetic
  engineering
• Determination of bacteria contaminating soil in
  the environment

38
Problems with colony counts using plates

• There is error in this method
• If the dilutions are homogeneous, there can be
  errors
• This may not capture all organisms in a broth
  because some may not be able to grow on the
  chosen media

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Colony Counts
40
Pour Plates

• Add 1 ml of a serial dilution to 9 ml of melted
  and slightly warm nutrient agar
• Mix
• Pour into a Petri dish and allow it to harden
• Colonies will develop both in the media and on
  the media
• Cells may be damaged by the hot agar in this
  experiment

41
Plating methods

• simple and sensitive
• widely used for viable counts of microorganisms
  in food, water, and soil
• inaccurate results obtained if cells clump
  together

42
Most Probable Number

• Most probable number is used for environmental
  samples
• Trying to determine the presence of an organism
• Use dilution factors as previously described
• Use multiple tubes for dilutions
• Check broth for cloudiness or turbidity( signs of
  bacterial growth)
• Use culture tubes containing sugars( lactose,
  sucrose, glucose) These can be checked for the
  presence of gas with a small tube on the interior
  called a Durham tube.
• See chart on page 149 for clarification

43
Membrane filtration methods
Figure 6.6
especially useful for analyzing aquatic samples
44
Measurement of Cell Mass

• dry weight
• time consuming and not very sensitive
• quantity of a particular cell constituent
• protein, DNA, ATP, or chlorophyll
• useful if amount of substance in each cell is
  constant
• turbidometric measures (light scattering)
• quick, easy, and sensitive

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more cells ? more light scattered ? less
light detected
Figure 6.8
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The Continuous Culture of Microorganisms

• growth in an open system
• continual provision of nutrients
• continual removal of wastes
• maintains cells in log phase at a constant
  biomass concentration for extended periods
• achieved using a continuous culture system

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The Chemostat

• rate of incoming medium rate of removal of
  medium from vessel
• an essential nutrient is in limiting quantities

Figure 6.9
48
Dilution rate and microbial growth
dilution rate rate at which medium
flows through vessel relative to vessel size
note cell density maintained at wide range of
dilution rates and chemostat operates best at low
dilution rate
Figure 6.10
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The Turbidostat

• regulates the flow rate of media through vessel
  to maintain a predetermined turbidity or cell
  density
• dilution rate varies
• no limiting nutrient
• turbidostat operates best at high dilution rates

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Importance of continuous culture methods

• constant supply of cells in exponential phase
  growing at a known rate
• study of microbial growth at very low nutrient
  concentrations, close to those present in natural
  environment
• study of interactions of microbes under
  conditions resembling those in aquatic
  environments
• food and industrial microbiology

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The Influence of Environmental Factors on Growth

• most organisms grow in fairly moderate
  environmental conditions
• extremophiles
• grow under harsh conditions that would kill most
  other organisms

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Solutes and Water Activity

• water activity (aw)
• amount of water available to organisms
• reduced by interaction with solute molecules
  (osmotic effect)
• higher solute ? lower aw
• reduced by adsorption to surfaces (matric effect)

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Osmotolerant organisms

• grow over wide ranges of water activity
• many use compatible solutes to increase their
  internal osmotic concentration
• solutes that are compatible with metabolism and
  growth
• some have proteins and membranes that require
  high solute concentrations for stability and
  activity

55
Effects of NaCl on microbial growth

• halophiles
• grow optimally at gt0.2 M
• extreme halophiles
• require gt2 M

Figure 6.11
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pH

• negative logarithm of the hydrogen ion
  concentration

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pH

• acidophiles
• growth optimum between pH 0 and pH 5.5
• neutrophiles
• growth optimum between pH 5.5 and pH 7
• alkalophiles
• growth optimum between pH8.5 and pH 11.5

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pH

• most acidophiles and alkalophiles maintain an
  internal pH near neutrality
• some use proton/ion exchange mechanisms to do so
• some synthesize proteins that provide protection
• e.g., acid-shock proteins
• many microorganisms change pH of their habitat by
  producing acidic or basic waste products
• most media contain buffers to prevent growth
  inhibition

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Temperature

• organisms exhibit distinct cardinal growth
  temperatures
• minimal
• maximal
• optimal

Figure 6.13
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Figure 6.14
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Temperature and bacterial growth
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Adaptations of thermophiles

• protein structure stabilized by a variety of
  means
• more H bonds
• more proline
• chaperones
• histone-like proteins stabilize DNA
• membrane stabilized by variety of means
• more saturated, more branched and higher
  molecular weight lipids
• ether linkages (archaeal membranes)

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Oxygen Concentration
ignore oxygen
lt 2 10 oxygen
need oxygen
prefer oxygen
oxygen is toxic
Figure 6.15
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Basis of different oxygen sensitivities

• oxygen easily reduced to toxic products
• superoxide radical
• hydrogen peroxide
• hydroxyl radical
• aerobes produce protective enzymes
• superoxide dismutase (SOD)
• catalase

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Figure 6.14
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Pressure

• barotolerant organisms
• adversely affected by increased pressure, but not
  as severely as nontolerant organisms
• barophilic organisms
• require or grow more rapidly in the presence of
  increased pressure

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Radiation
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Radiation damage

• ionizing radiation
• x rays and gamma rays
• mutations ? death
• disrupts chemical structure of many molecules,
  including DNA
• damage may be repaired by DNA repair mechanisms

69
Radiation damage

• ultraviolet (UV) radiation
• mutations ? death
• causes formation of thymine dimers in DNA
• DNA damage can be repaired by two mechanisms
• photoreactivation dimers split in presence of
  light
• dark reactivation dimers excised and replaced
  in absence of light

70
Radiation damage

• visible light
• at high intensities generates singlet oxygen
  (1O2)
• powerful oxidizing agent
• carotenoid pigments
• protect many light-exposed microorganisms from
  photooxidation

71
Microbial Growth in Natural Environments

• microbial environments are complex, constantly
  changing, and may expose a microorganism to
  overlapping gradients of nutrients and
  environmental factors

72
Growth Limitation by Environmental Factors

• Leibigs law of the minimum
• total biomass of organism determined by nutrient
  present at lowest concentration
• Shelfords law of tolerance
• above or below certain environmental limits, a
  microorganism will not grow, regardless of the
  nutrient supply

73
Responses to low nutrient levels

• oligotrophic environments
• morphological changes to increase surface area
  and ability to absorb nutrients
• mechanisms to sequester certain nutrients

74
Counting Viable but Nonculturable Vegetative
Procaryotes

• stressed microorganisms can temporarily lose
  ability to grow using normal cultivation methods
• microscopic and isotopic methods for counting
  viable but nonculturable cells have been developed

75
Quorum Sensing and Microbial Populations

• quorum sensing
• microbial communication and cooperation
• involves secretion and detection of chemical
  signals

Figure 6.20
76
Processes sensitive to quorum sensing
gram-negative bacteria

• bioluminescence (Vibrio fischeri)
• synthesis and release of virulence factors
  (Pseudomonas aeruginosa)
• conjugation (Agrobacterium tumefaciens)
• antibiotic production (Erwinia carotovora,
  Pseudomonas aureofaciens)
• biofilm production (P. aeruginosa)

77
Quorum sensing gram-positive bacteria

• often mediated by oligopeptide pheromone
• processes impacted by quorum sensing
• mating (Enterococcus faecalis)
• transformation competence (Streptococcus
  pneumoniae)
• sporulation (Bacillus subtilis)
• production of virulence factors (Staphylococcus
  aureus)
• development of aerial mycelia (Streptomyces
  griseus)
• antibiotic production (S. griseus)

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The Lux Gene in Vibrio Fischeri
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Requirements for Nitrogen

• Nitrogen is required for the synthesis of amino
  acids that compose the structure of proteins,
  purines and pyrimidines the bases of both DNA and
  RNA, and for other derivative molecules such as
  glucosamine.
• Many microorganisms can use the nitrogen directly
  from amino acids. The amino group ( NH2) is
  derived from ammonia through the action of
  enzymes such as glutamate dehydrogenase.
• Most photoautotrophs and many nonphotosynthetic
  microorganisms reduce nitrate to ammonia and
  assimilate nitrogen through nitrate reduction. A
  variety of bacteria are involved in the nitrogen
  cycle such as Rhizobium which is able to use
  atmospheric nitrogen and convert it to ammonia. (
  Found on the roots of legumes like soy beans and
  clover) These compounds are vital for the
  Nitrogen cycle and the incorporation of nitrogen
  into plants to make nitrogen comounds.

80
Phosphorous

• Phosphorous is present in phospholipids(
  membranes), Nucleic acids( DNA and RNA),
  coenzymes, ATP, some proteins, and other key
  cellular components.
• Inorganic phosphorous is derived from the
  environment in the form of phosphates. Some
  microbes such as E. coli can use organophosphates
  such as hexose 6-phosphates .

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Mixotrophy

• Chemical energy source organic
• Inorganic H/e- donor
• Organic carbon source

82
Requirements for Nitrogen, Phosphorus, and Sulfur

• Needed for synthesis of important molecules
  (e.g., amino acids, nucleic acids)
• Nitrogen supplied in numerous ways
• Phosphorus usually supplied as inorganic
  phosphate
• Sulfur usually supplied as sulfate via
  assimilatory sulfate reduction

83
Sources of nitrogen

• organic molecules
• ammonia
• nitrate via assimilatory nitrate reduction
• nitrogen gas via nitrogen fixation

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Growth Factors

• organic compounds
• essential cell components (or their precursors)
  that the cell cannot synthesize
• must be supplied by environment if cell is to
  survive and reproduce

85
Classes of growth factors

• amino acids
• needed for protein synthesis
• purines and pyrimidines
• needed for nucleic acid synthesis
• vitamins
• function as enzyme cofactors

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Amino acids
Proteins
87

• Bases of nucleic acids
• Adenine and guanine are purines
• Cytosine, thymine, and uracil are pyrimidines
• Also found in energy triphosphates( ATP and GTP)

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Practical importance of growth factors

• development of quantitative growth-response
  assays for measuring concentrations of growth
  factors in a preparation
• industrial production of growth factors by
  microorganisms

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Uptake of Nutrients by the Cell

• Some nutrients enter by passive diffusion
• Most nutrients enter by
• facilitated diffusion
• active transport
• group translocation

92
Passive Diffusion

• molecules move from region of higher
  concentration to one of lower concentration
  because of random thermal agitation
• H2O, O2 and CO2 often move across membranes this
  way

93
Active Transport

• energy-dependent process
• ATP or proton motive force used
• moves molecules against the gradient
• concentrates molecules inside cell
• involves carrier proteins (permeases)
• carrier saturation effect is observed

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Transporters

• Molecular Properties of Bacterial Multidrug
  Transporters Monique Putnam, Hendrik van Veen,
  and Wil Konings PubMed Central. Full Text
  available .
• Microbiol Mol Biol Review. 2000 December 64 (4)
  672693

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ABC transporters

• ATP-binding cassette transporters
• observed in bacteria, archaea, and eucaryotes

Figure 5.3
96
antiport
symport
Figure 5.4
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Group Translocation

• molecules are modified as they are transported
  across the membrane
• energy-dependent process

Figure 5.5
98
Fe uptake in pathogens

• The ability of pathogens to obtain iron from
  transferrins, ferritin, hemoglobin, and other
  iron-containing proteins of their host is central
  to whether they live or die
• Some invading bacteria respond by producing
  specific iron chelators - siderophores that
  remove the iron from the host sources. Other
  bacteria rely on direct contact with host iron
  proteins, either abstracting the iron at their
  surface or, as with heme, taking it up into the
  cytoplasm

99
Iron and signalling

• Iron is also used by pathogenic bacteria as a
  signal molecule for the regulation of virulence
  gene expression. This sensory system is based on
  the marked differences in free iron
  concentrations between the environment and
  intestinal lumen (high) and host tissues (low)
• Listeria Pathogenesis and Molecular Virulence
  Determinants
• José A. Vázquez-Boland,1,2 Michael Kuhn,3
  Patrick Berche,4 Trinad Chakraborty,5 Gustavo
  Domínguez-Bernal,1 Werner Goebel,3 Bruno
  González-Zorn,1 Jürgen Wehland,6 and Jürgen Kreft3

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Pathogens and Iron uptake

• Burkholderia cepacia
• Campylobacter jejuni
• Pseudomonas aeruginosa
• E. coli
• Listeria monocytogenes

101
Iron Uptake

• ferric iron is very insoluble so uptake is
  difficult
• microorganisms use siderophores to aid uptake
• siderophore complexes with ferric ion
• complex is then transported into cell

Figure 5.6
102
Listeriosis

• One involves the direct transport of ferric
  citrate to the bacterial cell
• Another system involves an extracellular ferric
  iron reductase, which uses siderophores
• The third system may involve a bacterial cell
  surface-located transferrin-binding protein

103
Iron bacteria in the environment

• There are several non-disease producing bacteria
  which grow and multiply in water and use
  dissolved iron as part of their metabolism. They
  oxidize iron into its insoluble ferric state and
  deposit it in the slimy gelatinous material which
  surrounds their cells.
• These filamentous bacteria grow in stringy clumps
  and are found in most iron-bearing surface
  waters. They have been known to proliferate in
  waters containing iron as low as 0.1 mg/l.

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Culture Media

• preparations devised to support the growth
  (reproduction) of microorganisms
• can be liquid or solid
• solid media are usually solidified with agar
• important to study of microorganisms

105
Synthetic or Defined Media

• all components and their concentrations are known

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Complex Media

• contain some ingredients of unknown composition
  and/or concentration

107
Some media components

• peptones
• protein hydrolysates prepared by partial
  digestion of various protein sources
• extracts
• aqueous extracts, usually of beef or yeast
• agar
• sulfated polysaccharide used to solidify liquid
  media

108
Types of Media

• general purpose media
• support the growth of many microorganisms
• e.g., tryptic soy agar
• enriched media
• general purpose media supplemented by blood or
  other special nutrients
• e.g., blood agar

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Types of media

• Selective media
• Favor the growth of some microorganisms and
  inhibit growth of others
• MacConkey agar
• selects for gram-negative bacteria
• Inhibits the growth of gram-positive bacteria

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Beta Hemolysis
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Types of media

• Differential media
• Distinguish between different groups of
  microorganisms based on their biological
  characteristics
• Blood agar
• hemolytic versus nonhemolytic bacteria
• MacConkey agar
• lactose fermenters versus nonfermenters

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Selective and differential media
Selects for Gram Differentiates between
bacteria based upon fermentation of lactose(
color change)
114
Organism Salt Tolerance Mannitol Fermentation
 1. S. aureus Positive - growth Positive
(yellow)  2. S. epidermidis Positive-
growth Negative( color does not change) no
fermentation of mannitol with production of
acid  3. M. luteus Negative N/A http//www.austi
n.cc.tx.us/microbugz/20msa.html  
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Web References on Media   http//www.jlindquist.ne
t/generalmicro/102diff.html - General
Reference http//medic.med.uth.tmc.edu/path/maccon
k.htm - MacConkey Agar http//www.indstate.edu/thc
me/micro/hemolys.html - Blood Agar  
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Spread-plate technique
1. dispense cells onto medium in petri dish
4. spread cells across surface
2. - 3. sterilize spreader
Figure 5.7
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Streak plate technique
inoculating loop
Figure 5.8
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Isolation of Pure Cultures

• Pure culture
• population of cells arising from a single cell
• Spread plate, streak plate, and pour plate are
  techniques used to isolate pure cultures

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Colony Morphology and Growth

• individual species form characteristic colonies

Figure 5.10b
121
Terms1. Colony shape and size round, irregular,
punctiform (tiny)2. Margin (edge) entire
(smooth), undulate (wavy), lobate (lobed)3.
Elevation convex, umbonate, flat, raised4.
Color color or pigment, plus opaque,
translucent, shiny or dull5. Texture moist,
mucoid, dry (or rough).
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Figure 5.10a
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Colony growth

• Most rapid at edge of colony
• oxygen and nutrients are more available at edge
• Slowest at center of colony
• In nature, many microorganisms form biofilms on
  surfaces