Transcript Resolution (continued)

Microbial Growth
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Factors that influence growth:
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Physical/Environmental
Chemical/Nutritional
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Microbial adaptations are remarkable
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Extremeophiles
May adapt to a point of no return
Temperature
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Microbes within different ranges (-200C – 1200C)
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Min and max typically ~ 30°C apart
Optimum temp closer to max than min
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Psychrophiles:
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Psychrotrophs (moderate psychrophiles)
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Optimum temperature ~15°C
Optimum temperature ~25°C
Mesophiles:
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Optimum temperature ~37°C
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Thermophiles:
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Optimum temperature ~60°C
Hyperthermophiles:
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Optimum temperature above 70°C
Usually Archaea
pH
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Most microbes are neutrophiles (6.5 – 7.5)
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Some bacteria are considered acid tolerant
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Optimum pH of most bacteria is 7
Helicobacter pylori
Typically fungi grow over a wider pH range
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Usually responsible for spoilage of acidic foods
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Acidophiles optimum is lower (below 5.5)
 Sulfolobus
- Archaea from acidic hot springs
 Lactobacillus – Bacteria produces lactic acid
 Thiobacillus - Bacteria produces sulfuric
acid
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Alkalophiles have optimum above 8.0
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Bacillus alcalophilus ~ 10.5
Vibrio cholerae prefers pH of 9.0 outside host
Water Activity
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Osmotic Pressure
Adaptations
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inclusion bodies, compatible solutes, stretch receptors
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Facultative halophiles
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Obligate halophiles
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Osmotolerant/Halotolerant
Staphylococcus aureus
Fungi tend to be more tolerant than other microbes
Most marine microbes
Extreme halophiles
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Salt flats of Utah and Dead Sea
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All organisms need:
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Macroelements
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CHNOPS
Microelements
K, Ca, Cl, Na
 Trace elements
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Growth factors
Carbon
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One of the most important growth requirements
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½ the dry weight of a bacteria cell is carbon
Carbon skeleton base of organic compounds
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Hydrocarbons
Metabolic Diversity
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Organisms grouped by energy, carbon and electron source
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Energy Source
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Carbon Source
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Phototroph or Chemotroph
Autotroph or Heterotroph
Electron Source
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Lithotroph or Organotroph
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Photolithoautotroph
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Chemoorganoheterotroph
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CO2, inorganic chemicals and inorganic e- donor
Photoorganoheterotroph
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Organic carbon, organic chemicals and organic e- donor
Chemolithoautotroph
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CO2, Light and inorganic e- donor
Organic carbon, light and organic e- donor
Chemolithoheterotroph
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Organic carbon, inorganic chemicals and inorganic e- donor
Oxygen Requirements
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Oxygen has many toxic forms
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Organisms require enzyme systems to protect them
Superoxide (O2-) radical
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Neutralized by superoxide dismutase (SOD)
2 O2- + 2 H+ → H2O2 + O2
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Peroxide
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Detoxified by catalase or peroxidase
Catalase: H2O2 → H2O + O2
Peroxidase: H2O2 + 2 H+ → 2 H2O
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Obligate (Strict) Aerobes
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only aerobic metabolism
Have SOD and catalase or peroxidase
Obligate (Strict) Anaerobes
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Destroyed by oxygen
Do not have SOD, catalase or peroxidase
only anaerobic metabolism
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Facultative Anaerobes
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Have SOD and catalase or peroxidase
Grow with or without oxygen
aerobic or anaerobic metabolism
Grow faster in the presence of O2
Aerotolerant Anaerobes
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Have SOD
Grow with or without oxygen
only anaerobic metabolism
Grow faster in the absence of O2
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Microaerophiles
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Grow only in low levels O2
small amounts of SOD and catalase
Produce toxic levels of superoxide free radicals and
peroxide at high levels of O2
only aerobic metabolism
Growth in liquid media
Obligate
Aerobes
Facultative
Anaerobes
Obligate
Anaerobes
Aerotolerant
Anaerobes
Microaerophiles
Nitrogen
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Needed for amino acids, nucleic acids, and ATP
Amino acids from protein degradation
Nitrogen reduction
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Reduce nitrate to ammonia then utilize the ammonia
Nitrogen fixation
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assimilate gaseous nitrogen ( N2)
Sulfur
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Needed for building some amino acids (cysteine
and methionine), vitamins (thiamine and biotin)
and some carbohydrates
Sulfur containing amino acids from protein
degradation
Reduce sulfates (SO42-) or sulfides (H2S)
Phosphorus
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Tends to be a limiting growth requirement
Phosphorus is needed for building nucleic acids,
phospholipids, and ATP
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Phosphate ion (PO43-)is an important source
Microelements
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Several are essential for proper cell function
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Signal molecules, membrane potential, enzyme
cofactors
Trace Elements
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Minerals required in very small amounts
Iron, copper and zinc
Growth Factors
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Organic compounds essential growth
Cannot be synthesized by microorganisms
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Vitamins
Amino acids
Nucleic acid bases
Fastidious
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Neisseria
Cultivation Of Microorganisms
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culture medium
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inoculation
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nutrient material prepared for growing
microorganisms
introduction of a microorganism into medium
culture
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growth of a microorganism observed on/in a medium
Types Of Culture Media
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Chemically defined media:
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Exact composition known
Complex media:
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Exact composition varies
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Selective media:
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Favors the growth of desired microorganisms
Inhibits the growth of unwanted ones
Differential media:
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Distinguishes between groups of microorganisms
MacConkey’s Agar
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Selective medium:
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Inhibits Gram-positive bacteria growth
Encourages Gram-negative bacteria growth
Differential medium:
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Lactose fermenters produce acid and form pink
colonies
Non-lactose fermenters form colorless colonies
MacConkey’s Agar
Escherichia
Salmonella
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Differential media
 Blood
agar
Alpha Hemolysis
 Beta Hemolysis
 Gamma Hemolysis
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Microbial Growth
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Refers to increase in number of cells not size of
individual cells
Bacteria typically reproduce by binary fission
Generation time
 time required for a bacterial population to double
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Typically 1-3 hours
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Generation number is expressed as a power of 2
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Original cell is 20, 2nd generation (after one cell division)
would be 21
20=
21=
22=
23=
1
2
4
8
cell
cells
cells
cells
Phases of Bacterial Growth Curve
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In closed system or
batch culture
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Lag phase
Log phase
Stationary phase
Decline phase
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Phases of the growth curve can be observed in
liquid media
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Solid media, different colonies in different phases
Continuous cultures
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Open system
Measuring Bacterial Growth
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Direct Methods
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Viable plate count, Membrane filtration,
Microscopic count, Most Probable Number (MPN),
Electronic Counters
Indirect Methods
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Turbidity, Metabolic activity, Weight
Direct Methods
Viable Plate Count
1.
Important to limit colonies to a countable number
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30-300 colonies (CFUs)
Serial dilutions ensure colony counts within range
Advantage: only living cells
Disadvantage: incubation time, growth
requirements, may underestimate count
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Plate count methods
 pour-plates
 Spread-plates methods
2. Membrane Filtration
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Sample (liquid) passed through filter
Filter placed on surface of solid medium
Organisms retained on filter will grow
Advantage: only living cells, can be used to count low
cell concentrations
Disadvantage: must have at least 100 ml of media,
requires incubation time, may underestimate count
3. Microscopic Count
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Known volume of sample placed in counting chamber
Viewed under microscope
Cells counted
Advantage: no incubation time is required
Disadvantage: dead cells may be counted, tedious,
requires a high concentration of cell (10 million per ml)
4. Most probable numbers (MPN)
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Multi-tube statistical assay
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Advantage: measures only living cells, useful for
culturing cells that wont grow on solid media
Disadvantage: incubation time, expensive & time
consuming
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Series of dilution sets
Each set inoculated with 10X less sample than previous set
 Incubated and results compared to MPN table
 gives statistical estimation of cell concentration
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5. Electronic Counter
 Coulter Counter – electrical current
 Flow Cytometry – light transmission
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Advantage: No incubation time
Disadvantage: dead cells may be counted, not very
sensitive due to clumping and debris in media
Indirect methods
1. Turbidity
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Uses spectrophotometer
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Advantage: no incubation time
Disadvantage: must have high concentration of
cells, may count dead cells
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Measures light transmitted through sample
 Measurement is inversely proportional to cell
concentration
2. Weight
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Wet weight
 Cells centrifuged and packed cells weighed
Dry weight
 Packed cells dried at 100°C for 8 to 12 hours then
weighed
Disadvantage: Tedious and time consuming
Advantage: Useful in measuring filamentous
organisms
3. Metabolic Activity
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Based on enzyme activity
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Advantage: once metabolic rate is established
provides reliable estimate of cell number
Disadvantage: requires incubation time, requires
metabolic rate be established in advance
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