Transcript Document

Chapter 6:
Microbial Growth
Microbial Growth
• Microbial growth = growth in population
─ Increase in number of cells, not cell size
• Two main categories of requirements for microbial
growth:
─ Physical requirements (environmental conditions)
◦ Temperature, pH, osmotic pressure
─ Chemical requirements
Physical Requirements for Growth:
Temperature
• Temperature
─ Minimum growth temperature
─ Optimum growth temperature
─ Maximum growth temperature
• Three main classifications
─ Psychrophiles (optimum ~120C)
─ Psychrotrophs (optimum ~230C)
─ Mesophiles (optimum ~370C)
─ Thermophiles (optimum above 500C)
Physical Requirements for Growth:
Temperature
Refrigeration
Cause majority of
food spoilage
Figure 6.1
Hansen’s Disease
(Leprosy)
• Mycobacterium leprae
• Optimal growth temperature: 30°C
─ Grows in peripheral nerves, nasal mucosa and skin cells
Figure 22.8
Physical Requirements for Growth:
pH
• pH
─ Most bacteria grow between pH 6.5 and 7.5
─ Molds and yeasts grow optimally between pH 5 and 6
─ Acidophiles grow in acidic environments (pH<5.5)
─ Alkaliphiles grow in basic environments (pH>8.5)
• Acidic foods (pickles, sauerkraut) preserved by acids
from bacterial fermentation
• Growth media used in the laboratory contain buffers
Physical Requirements for Growth:
Osmotic Pressure
• Osmotic Pressure
─ Hypertonic environments (=high osmotic pressure), increased salt
or sugar, cause plasmolysis
◦ Obligate halophiles require high osmotic pressure
◦ Facultative halophiles tolerate high osmotic pressure (>2% salt)
─ Nutrient agar has a high percentage of water to maintain low
osmotic pressure (bacterial cells are 80-90% water)
Low osmotic pressure
High osmotic pressure
Water flow
Low solute concentration/
High water concentration
High solute concentration/
Low water concentration
Physical Requirements for Growth:
Osmotic Pressure
• Plasmolysis: cell growth is inhibited when the plasma membrane
pulls away from the cell wall
─ Added salt or sugar is another method of preserving food
Isotonic solution
Hypertonic solution
(high osmotic pressure)
Figure 6.4
Chemical Requirements for Growth
• Carbon
─ Structural organic molecules, energy source
◦ Heterotrophs use organic carbon sources
◦ Autotrophs use CO2
• Nitrogen, Sulfur, Phosphorus
─ For synthesis of amino acids, nucleotides, vitamins,
phospholipids
─ Most bacteria decompose proteins to obtain N
─ Inorganic ions are sources for these elements (NH4+,
NO3-, PO43-, SO42-)
Chemical Requirements for Growth
• Trace Elements (Iron, Copper, Zinc)
─ Inorganic elements required in small amounts, usually
as enzyme cofactors
─ Often present in tap water
• Organic Growth Factors
─ Organic compounds obtained from the environment
(i.e. the organism cannot synthesize them)
─ Vitamins, amino acids
Chemical Requirements for Growth:
Oxygen
• Oxygen (O2)
Obligate
aerobes
Facultative
anaerobes
Obligate
anaerobes
Aerotolerant
anaerobes
Microaerophiles
Chemical Requirements for Growth:
Oxygen
• Aerotolerance of individual organisms depends on their
ability to handle oxygen toxicity
─ Oxygen radical species: O2-, O22-, OH .
─ Presence/lack of enzymes that neutralize toxic oxygen
species
◦ SOD (Superoxide dismutase)
◦ Catalase/peroxidase
Chemical Requirements for Growth:
Oxygen
• Oxygen (O2)
Obligate
aerobes
Facultative
anaerobes
Express SOD and
catalase
Obligate
anaerobes
Don’t express
SOD/catalase
Aerotolerant
anaerobes
Tolerate oxygen
(express
SOD/catalase) but
incapable of using it
for growth
Microaerophiles
Require oxygen, but
at lower levels than in
the air
Culture Media
• Culture Medium: Nutrients prepared for microbial
growth
─ Source of energy, carbon, nitrogen, sulfur,
phosphorus, trace elements and organic growth
factors
• Sterile: No living microbes
• Inoculum: Introduction of microbes into medium to
initiate growth
• Culture: Microbes growing in/on culture medium
Culture Media:
Agar
• Complex polysaccharide
• Used as solidifying agent for culture media in Petri
plates, slants, and deeps
• Generally not metabolized by microbes
─ Agar is not a nutrient
• Liquefies above 100°C
─ Can incubate at a wide range of temperatures
Culture Media
Anaerobic Culture Media:
Broth cultures
• Reducing broth media
─ Contain chemicals (thioglycollate) that combine with
dissolved O2 to deplete it from the media
Anaerobic Culture Methods:
Agar Cultures
• Anaerobic jar
─ Oxygen and H2
combine to
form water
Figure 6.5
Culture Media:
Selective and Differential Media
• Selective media: suppress growth of unwanted
microbes and encourage growth of desired microbes
• Differential media: make it easy to distinguish
colonies of different microbes
Enterobacter aerogenes on EMB
E. coli on EMB
Figure 6.9b, c
Obtaining Pure Cultures
• A pure culture contains only one species or strain
• A colony is a population of cells arising from a single
cell or spore or from a group of attached (identical)
cells
─ One colony arises from one colony-forming unit (CFU)
• Specimens (pus, sputum, food) typically contain many
different microorganisms
─ Common way to isolate a single species from a
mixture of microorganisms: Streak plate method
Streak Plate Method for Isolation of
a Pure Species
• Use loop to pick colony
• Inoculate broth
• Pure culture
Figure 6.10a, b
Microbial Growth in Hosts:
Biofilms
• Microbial communities
─ 3-dimensional “slime”
─ i.e. dental plaque,
soap scum
• Share nutrients
• Sheltered from harmful
factors
• Cell-to-cell
communication:
quorum sensing
Figure 6.5
Bacterial biofilm growing on a micro-fibrous material
Microbial Growth in Hosts:
Biofilms & Quorum Sensing
• Quorum sensing allows a form of bacterial communication
─ Individual cells can sense the accumulation of signaling
molecules (autoinducers)
◦ Informs individual cells about surrounding cell density
◦ May change the behavior (gene expression) of individual cells
− Results in a coordinated response by the whole population
http://biofilmbook.hypertextbookshop.com/public_version/
Prokaryotic Reproduction:
Binary Fission
Figure 6.11
Reproduction in Prokaryotes:
Generation Time
Generation time: the time required for one population
doubling
─ Varies with species and environmental conditions
Reproduction in Prokaryotes:
Generation Number
• Generation number: the number of times a cell population has
doubled in a given time under given conditions
Figure 6.12b
Reproduction in Prokaryotes:
Growth Plot
Logarithmic
Arithmetic
Figure 6.13
Bacterial Growth Curve
• Lag: little/no cell division
─ Adapting to new medium
─ *Metabolically active*
• Log: exponential growth
─ Most metabolically active
─ Gen. time at constant minimum
• Stationary: equilibrium phase
─ Growth rate = death rate
─ Nutrients exhausted, waste
accumulation, pH changes
• Death: logarithmic decline
Figure 6.14
Measuring Microbial Growth
• To determine the size of a bacterial population in a
specimen, cell counting techniques are used
─ Often there are too many cells per ml or gram of
specimen…
◦ A small proportion of the specimen (a dilution) is
counted
◦ The number of cells in the original specimen can be
calculated based on the count in the small dilution
Direct Measurements of Microbial Growth:
Viable Cell Count
• Plate Counts: Perform serial dilutions of a sample
• How many cells are in 1 mL of original culture?
DF=1
DF:
10-1
10-2
10-3
10-4
10-5
Figure 6.15, top portion
Direct Measurements of Microbial Growth:
Viable Count
• Inoculate one
agar plate with
each serial dilution
Figure 6.16
Direct Measurements of Microbial Growth:
Viable Count
• After incubation, count colonies on plates that have 30300 colonies (CFUs)
Figure 6.15
Direct Measurements of Microbial
Growth
• Filtration
─ Ideal when microbial density is low in a sample
Figure 6.17a, b
Direct Measurements of Microbial
Growth
Disadvantages:
-Likely to count dead cells
-Motile cells can be difficult to count
Figure 6.19