Microbial Growth
•
Physical Requirements of Microbes
•
Temperature (optimal enzyme operation)
•
•
•
•
pH (optimal enzyme operation)
•
Using buffers in media
•
Molds & yeasts versus bacteria
Chemical Requirements
•
Carbon source in medium
•
Nitrogen, sulfur, phosphorous, trace elements
•
Oxygen requirements
•
Obligate aerobes, anaerobes, facultative anaerobes
•
Free radical oxygen (O2-) and H2O2 dangers; superoxide dismutase and catalase = aerobes
Culture Media for Microbes
•
Chemically defined vs. complex media
•
Anaerobes: reducing media/Brewer jar
•
Other: animals, eggs, tissue culture, CO2
•
Media types
•
•
Psychrophiles, mesophiles, thermophiles
Selective, Differential, Enrichment
Bacterial Population Growth
•
Growth Curve: Lag, Log, Stationary, Death
•
Quantifying Growth
Characterizing Microbes By Optimal Growth Temperature
Figure 6.1
Temperature Growth Ranges and Food Safety
“2-40-140”
If > 2 hrs at 40-140oF, don’t eat it!
Figure 6.2
Alkalophiles
Neutrophiles
Acidophiles
Physical Requirements: pH
• Most bacteria grow between pH 6.5 and 7.5
• Molds and yeasts grow between pH 5 and 6
Physical Requirements: Osmotic Pressure
• Hypertonic environments, increase salt or sugar, cause plasmolysis
• Extreme or obligate halophiles require high osmotic pressure
• Facultative halophiles tolerate high osmotic pressure
Microbial Growth
•
Physical Requirements of Microbes
•
Temperature (optimal enzyme operation)
•
•
•
pH (optimal enzyme operation)
•
Using buffers in media
•
Molds & yeasts versus bacteria
Chemical Requirements
•
Carbon source in medium
•
Nitrogen, sulfur, phosphorous, trace elements
•
Oxygen requirements
•
•
•
Obligate aerobes, anaerobes, facultative anaerobes
Free radical oxygen (O2-) and H2O2 dangers; superoxide dismutase and catalase = aerobes
Culture Media for Microbes
•
Chemically defined vs. complex media
•
Anaerobes: reducing media/Brewer jar
•
Other: animals, eggs, tissue culture, CO2
•
Media types
•
•
Psychrophiles, mesophiles, thermophiles
Selective, Differential, Enrichment
Bacterial Population Growth
•
Growth Curve: Lag, Log, Stationary, Death
•
Quantifying Growth
The Requirements for Growth: Chemical Requirements
• Carbon
• Structural organic molecules, energy source
• Chemoheterotrophs use organic carbon sources
• Autotrophs use CO2
The Requirements for Growth: Chemical Requirements
• Nitrogen
• In amino acids, proteins
• Most bacteria decompose proteins
• Some bacteria use NH4+ or NO3
• A few bacteria use N2 in nitrogen fixation
• Sulfur
• In amino acids, thiamine, biotin
• Most bacteria decompose proteins
• Some bacteria use SO42 or H2S
• Phosphorus
• In DNA, RNA, ATP, and membranes
• PO43 is a source of phosphorus
• Trace Elements
• Inorganic elements required in small amounts
• Usually as enzyme cofactors
Microbial Growth
•
Physical Requirements of Microbes
•
Temperature (optimal enzyme operation)
•
•
•
•
pH (optimal enzyme operation)
•
Using buffers in media
•
Molds & yeasts versus bacteria
Chemical Requirements
•
Carbon source in medium
•
Nitrogen, sulfur, phosphorous, trace elements
•
Oxygen requirements
•
Obligate aerobes, anaerobes, facultative anaerobes
•
Free radical oxygen (O2-) and H2O2 dangers; superoxide dismutase and catalase = aerobes
Culture Media for Microbes
•
Chemically defined vs. complex media
•
Anaerobes: reducing media/Brewer jar
•
Other: animals, eggs, tissue culture, CO2
•
Media types
•
•
Psychrophiles, mesophiles, thermophiles
Selective, Differential, Enrichment
Bacterial Population Growth
•
Growth Curve: Lag, Log, Stationary, Death
•
Quantifying Growth
How Toxic Forms of Oxygen Are Handled
• Singlet oxygen: O2 boosted to a higher-energy state
• Handling superoxide free radicals: O2

2O2- + 2O2- + 8H+
oxygen radicals
•
4H2O2
hydrogen peroxide
Superoxide Dismutase (SODS)
• Handling peroxide anion: O22

2H2O2
hydrogen peroxide
2H2O
+
water
oxygen gas
Catalase (Peroxidase)
Catalase Test: Bacteria + H2O2 bubbles
O2
Obligate
aerobes
Faultative
anaerobes
Obligate
anaerobes
Aerotolerant
anaerobes
Microaerophiles
Thyoglycollate binds molecular oxygen, reducing it and removing it:
R-SH + O2  R-SO2
Microbial Growth
•
Physical Requirements of Microbes
•
Temperature (optimal enzyme operation)
•
•
•
•
pH (optimal enzyme operation)
•
Using buffers in media
•
Molds & yeasts versus bacteria
Chemical Requirements
•
Carbon source in medium
•
Nitrogen, sulfur, phosphorous, trace elements
•
Oxygen requirements
•
Obligate aerobes, anaerobes, facultative anaerobes
•
Free radical oxygen (O2-) and H2O2 dangers; superoxide dismutase and catalase = aerobes
Culture Media for Microbes
•
Chemically defined vs. complex media
•
Anaerobes: reducing media/Brewer jar
•
Other: animals, eggs, tissue culture, CO2
•
Media types
•
•
Psychrophiles, mesophiles, thermophiles
Selective, Differential, Enrichment
Bacterial Population Growth
•
Growth Curve: Lag, Log, Stationary, Death
•
Quantifying Growth
Culture Media: Chemically Defined or Complex
Table 6.2 & 6.4
Anaerobic and Low O2 Culture Methods
Candle jar
Brewer or anaerobic jar
CO2 packet
Unusual Culture Methods
Grows only in certain cell
types: using armadillos
to culture M. leprae
Grows only inside live cells: eggs as
culture vessels for influenza virus
Grows only in certain cell types: using tissue
culture with low O2, enriched CO2 incubators
Microbial Growth
•
Physical Requirements of Microbes
•
Temperature (optimal enzyme operation)
•
•
•
•
pH (optimal enzyme operation)
•
Using buffers in media
•
Molds & yeasts versus bacteria
Chemical Requirements
•
Carbon source in medium
•
Nitrogen, sulfur, phosphorous, trace elements
•
Oxygen requirements
•
Obligate aerobes, anaerobes, facultative anaerobes
•
Free radical oxygen (O2-) and H2O2 dangers; superoxide dismutase and catalase = aerobes
Culture Media for Microbes
•
Chemically defined vs. complex media
•
Anaerobes: reducing media/Brewer jar
•
Other: animals, eggs, tissue culture, CO2
•
Media types
•
•
Psychrophiles, mesophiles, thermophiles
Selective, Differential, Enrichment
Bacterial Population Growth
•
Growth Curve: Lag, Log, Stationary, Death
•
Quantifying Growth
Selective Media
• Goal: To chemically (or
physically) suppress
unwanted microbes and
encourage desired
microbes.
MSA
Mannitol salt agar : selective for halophiles
with 7% salt (osmotic challenge) and
differential for mannitol fermenters: good for
skin bacterial cultures.
EMB Agar: kills gram positives with eosin
and methylene blue, selective for gram
negatives. Differential for lactose fermenters.
Good for growing enterics.
McConkey Agar: supresses gram positives
with crystal violet and bile salts; also
differential for
EMB
MA
Figure 6.9b, c
Differential Media
• Distinguish between different species based on a
metabolic ability.
Blood agar
(sheep’s blood)
reveals if
hemolytic
Mannitol salt agar
contains the pH sensitive
dye phenol red (yellow
when acidic)
Se
Sa
Figure 6.9a
Enrichment Media
• Encourages growth of desired microbe by providing
special growth conditions or added growth factors
Thioglycollate
Anaerobic or
Brewer Jar
Lysed red blood cells provide
unique nutrients in
blood/chocolate agar
Glucose Salts Agar (enriches
for microbes that can growth
only on glucose and some
inorganic nutrients
Pure Cultures Used To Study Characteristics Of A Particular Species
• 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 cells
• A colony is often called a colony-forming unit (CFU)
Microbial Growth
•
Physical Requirements of Microbes
•
Temperature (optimal enzyme operation)
•
•
•
•
pH (optimal enzyme operation)
•
Using buffers in media
•
Molds & yeasts versus bacteria
Chemical Requirements
•
Carbon source in medium
•
Nitrogen, sulfur, phosphorous, trace elements
•
Oxygen requirements
•
Obligate aerobes, anaerobes, facultative anaerobes
•
Free radical oxygen (O2-) and H2O2 dangers; superoxide dismutase and catalase = aerobes
Culture Media for Microbes
•
Chemically defined vs. complex media
•
Anaerobes: reducing media/Brewer jar
•
Other: animals, eggs, tissue culture, CO2
•
Media types
•
•
Psychrophiles, mesophiles, thermophiles
Selective, Differential, Enrichment
Bacterial Population Growth
•
Growth Curve: Lag, Log, Stationary, Death
•
Quantifying Growth
Bacterial Growth is Exponential (Logarithmic)
Bacterial “growth” means an increase in the number
of individuals, not an increase in cell size.
Figure 6.12b
Growth Curve for Bacteria (Logarithmic Plot)
Figure 6.14
Estimating Bacterial Numbers by Indirect methods
• Direct Measures
• Plate counts of viable bacterial forming colonies
• Counting low viable bacterial numbers by filtration
• Counting viable bacteria with Most Probable
Number
• Counting bacteria per ml in direct microscopy
• Indirect Measures
• Turbidity/Absorbance with a spectrophotometer
• Metabolic activity tracking conversion of colored
molecules
• Dry weight by weighing a set volume and knowing
weight of one cell
Plate Assays: Spread Plate or Pour Plate Methods
• After incubation, count colonies on plates that have 30300 colonies (CFUs)
The dilution in a
particular tube =
ml of fluid added
to tube/total
volume after
addition; e.g.
1ml/(9ml + 1ml) =
1/10 = 10-2
Figure 6.15
Direct Measurements of Microbial Growth
Figure 6.19
Direct Measurements of Microbial Growth
• Filtration: Good for measuring very dilute samples of bacteria
Figure 6.17a, b
Direct Measurements of Microbial Growth
• Multiple tube
MPN test
• Count positive
tubes and
compare to
statistical MPN
table
• Produces a
range of
concentrations
Figure 6.18b
Estimating Bacterial Numbers by Indirect methods
• Direct Measures
• Plate counts of viable bacterial forming colonies
• Counting low viable bacterial numbers by filtration
• Counting viable bacteria with Most Probable
Number
• Counting bacteria per ml in direct microscopy
• Indirect Measures
• Turbidity/Absorbance with a spectrophotometer
• Metabolic activity tracking conversion of colored
molecules/enyzme assay
• Dry weight by weighing a set volume and knowing
weight of one cell
Estimating Bacterial Numbers by Indirect Methods
• Turbidity
Figure 620
Metabolic Conversion/Enzyme Assay
• 1 bacterium produces 4.6 x 1012 NADH/sec/cell under
idea growth conditions.
• In a 1 ml sample of growing cells, 5.2 x 1023
NADH/sec/ml are produced per second (as revealed by
a color-based assay of NADH on the sample)
• Therefore, (4.6 x 1012 NADH/sec/ml) x (5.2 x 1023
NADH/sec/cell) = 2.3 x 1024 cells/ml
Determining dry mass of a fixed volume
An E. coli cell has a dry mass of
about 7.0 x 10-19 mg.
A 1 ml sample with a dry mass of
2 mg therefore has:
2 mg/ml x 1 cell/7 x 10-19 mg
= 2.8 x 1020 cells/ml
Microbial Growth
•
Physical Requirements of Microbes
•
Temperature (optimal enzyme operation)
•
•
•
•
pH (optimal enzyme operation)
•
Using buffers in media
•
Molds & yeasts versus bacteria
Chemical Requirements
•
Carbon source in medium
•
Nitrogen, sulfur, phosphorous, trace elements
•
Oxygen requirements
•
Obligate aerobes, anaerobes, facultative anaerobes
•
Free radical oxygen (O2-) and H2O2 dangers; superoxide dismutase and catalase = aerobes
Culture Media for Microbes
•
Chemically defined vs. complex media
•
Anaerobes: reducing media/Brewer jar
•
Other: animals, eggs, tissue culture, CO2
•
Media types
•
•
Psychrophiles, mesophiles, thermophiles
Selective, Differential, Enrichment
Bacterial Population Growth
•
Growth Curve: Lag, Log, Stationary, Death
•
Quantifying Growth