specific identification of bacterial growth
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Specific Identification of Bacterial Growth
Investigating Bacteria of the Cape Fear River
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Last week…
• 10 culture plates were created from Cape Fear River water sample:
– “Spread Plates”
– MacConkey Agar Gram-negative Bacteria
– Incubated at ~37°C Enteric Bacteria
• Subculture plates were created by each lab group:
– “Streak Plates”
– MacConkey Agar Lactose Fermenting Bacteria selected
– Incubated at ~ 37°C
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Bacterial Isolation
• Isolation Methods
– 1850s: Louis Pasteur developed concept of a “pure culture”
– 1880s: Robert Koch developed “streaking for isolation”
• Isolated Colonies
– Colony: A population of millions of cells that are identical and are descendent from a single founder cell
– Bacteria replicate via binary fission• Average replication rate
Photo Source: micro.cornell.edu
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Bacterial Growth Curve: Five Phases
• Lag Phase
• Log Phase
• Stationary Phase
• Death Phase
• Long-term Stationary Phase
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Bacterial Characterization
• You selected a sample of a single colony from the spread plate to create your streak plates
• Characterizing Bacterial Growth:
– MAC:
• Gram Negative Bacteria
• Lactose Fermenters
• Replication seen when incubated at temps between 35-37°C
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Bacterial Characterization
• These characteristics typically indicate the presence of Coliform Bacteria in the family Enterobacteriaceae:
– Escherichia
– Klebsiella
– Enterobacter
– Hafnia
– Citrobacter
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Coliform Bacteria
• Can be found in water, soil, and on vegetation
• Universally present (and abundant) in the feces of warm-blooded animals (including humans)
• Not typically the cause of serious illness
• Indicator bacteria
– We’ll discuss coliform bacteria further next week…
Photo by Eric Erbe, digital colorization by Christopher Pooley, both of USDA, ARS, EMU. E. coli at 10 000 x
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Specific IdentificationThrough Further Characterization
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New Media for New Clues
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Simmons Citrate Agar Tubes: Slant
+ -
• Tests ability of bacteria to utilize citrate as sole carbon source:
– Uninoculated
• pH of 6.9
• Bromthymol blue pH indicator dye is green
– Inoculated: Negative Result
• pH of 6.9
• Bromthymol blue pH indicator dye is green
– Inoculated: Positive Result
• pH of 7.5+
• Bromthymol blue pH indicator dye changes from green to royal blue
Photo Source: quizlet.com
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Christensen Urea Agar Tubes: Slant• Tests ability of bacteria to
utilize urea as sole nitrogen source:– Uninoculated
• pH of 6.8 • Pheonol red pH indicator dye is
yellow
– Inoculated: Negative Result• pH of 6.8• Pheonol red pH indicator dye is
yellow
– Inoculated: Positive Result• pH of 8.1+• Pheonol red pH indicator dye
changes from yellow to bight pink+ -
Photo Source: quizlet.com
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SIM Agar Tube: Stab
• Tests ability of bacteria to:
– Reduce sulfur (S) and produce hydrogen sulfide gas
– Produce indol (I) through the hydrolysis of tryptophan
– Be motile (M) in the agar
Photo Source: deltabiology.com
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Final Identification
Bacteria Genus Lactose Sulfur Indole Motility Citrate Urease
Escherichia + - + + - -
Klebsiella + - +/- - + +
Yersinia + +/- +/- - - +
Enterobacter + - - + + -
Citrobacter + + +/- + + + Slow
Shigella - - - - - -
Salmonella - + - + + -
Proteus - + +/- + +/- +
Edwardsiella - + + +/- - -