Microbial Growth Physical Requirements of Microbes Temperature (optimal enzyme operation)...
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Microbial Growth• Physical Requirements of Microbes
• Temperature (optimal enzyme operation)
• Psychrophiles, mesophiles, thermophiles
• 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
• 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
Figure 6.2
“2-40-140”If > 2 hrs at 40-140oF, don’t eat it!
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)
• Psychrophiles, mesophiles, thermophiles
• 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
• Selective, Differential, Enrichment
• Bacterial Population Growth• Growth Curve: Lag, Log, Stationary, Death
• Quantifying Growth
• 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
The Requirements for Growth: Chemical Requirements
Microbial Growth• Physical Requirements of Microbes
• Temperature (optimal enzyme operation)
• Psychrophiles, mesophiles, thermophiles
• 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
• Selective, Differential, Enrichment
• Bacterial Population Growth• Growth Curve: Lag, Log, Stationary, Death
• Quantifying Growth
• Singlet oxygen: O2 boosted to a higher-energy state
• Handling superoxide free radicals: O2
2O2- + 2O2
- + 8H+ 4H2O2
oxygen radicals hydrogen peroxide• Superoxide Dismutase (SODS)
• Handling peroxide anion: O22
2H2O2 2H2O + O2
hydrogen peroxide water oxygen gas
Catalase (Peroxidase)
Catalase Test: Bacteria + H2O2 bubbles
How Toxic Forms of Oxygen Are Handled
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)
• Psychrophiles, mesophiles, thermophiles
• 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
• 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
Brewer or anaerobic jar CO2 packet
Candle jar
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)
• Psychrophiles, mesophiles, thermophiles
• 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
• Selective, Differential, Enrichment
• Bacterial Population Growth• Growth Curve: Lag, Log, Stationary, Death
• Quantifying Growth
• Goal: To chemically (or physically) suppress unwanted microbes and encourage desired microbes.
Selective Media
Figure 6.9b, c
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
MSA
EMB
MA
• Distinguish between different species based on a metabolic ability.
Differential Media
Figure 6.9a
Blood agar(sheep’s blood) reveals if hemolytic
Mannitol salt agar contains the pH sensitive dye phenol red (yellow when acidic)
Se Sa
• Encourages growth of desired microbe by providing special growth conditions or added growth factors
Enrichment Media
Thioglycollate
Anaerobic or Brewer Jar
Glucose Salts Agar (enriches for microbes that can growth
only on glucose and some inorganic nutrients
Lysed red blood cells provide unique nutrients in
blood/chocolate agar
• 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)
Pure Cultures Used To Study Characteristics Of A Particular Species
Microbial Growth• Physical Requirements of Microbes
• Temperature (optimal enzyme operation)
• Psychrophiles, mesophiles, thermophiles
• 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
• Selective, Differential, Enrichment
• Bacterial Population Growth• Growth Curve: Lag, Log, Stationary, Death
• Quantifying Growth
Figure 6.12b
Bacterial Growth is Exponential (Logarithmic)
Bacterial “growth” means an increase in the number of individuals, not an increase in cell size.
Figure 6.14
Growth Curve for Bacteria (Logarithmic Plot)
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
• After incubation, count colonies on plates that have 30-300 colonies (CFUs)
Plate Assays: Spread Plate or Pour Plate Methods
Figure 6.15
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
Direct Measurements of Microbial Growth
Figure 6.19
• Filtration: Good for measuring very dilute samples of bacteria
Direct Measurements of Microbial Growth
Figure 6.17a, b
• Multiple tube MPN test
• Count positive tubes and compare to statistical MPN table
• Produces a range of concentrations
Direct Measurements of Microbial Growth
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
• Turbidity
Estimating Bacterial Numbers by Indirect Methods
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)
• Psychrophiles, mesophiles, thermophiles
• 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
• Selective, Differential, Enrichment
• Bacterial Population Growth• Growth Curve: Lag, Log, Stationary, Death
• Quantifying Growth