Specific Identification of Bacterial Growth

Investigating Bacteria of the Cape Fear River

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

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

Bacterial Growth Curve: Five Phases

• Lag Phase

• Log Phase

• Stationary Phase

• Death Phase

• Long-term Stationary Phase

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

Bacterial Characterization

• These characteristics typically indicate the presence of Coliform Bacteria in the family Enterobacteriaceae:

– Escherichia

– Klebsiella

– Enterobacter

– Hafnia

– Citrobacter

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

Specific IdentificationThrough Further Characterization

New Media for New Clues

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

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

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

Final Identification

Bacteria Genus Lactose Sulfur Indole Motility Citrate Urease

Escherichia + - + + - -

Klebsiella + - +/- - + +

Yersinia + +/- +/- - - +

Enterobacter + - - + + -

Citrobacter + + +/- + + + Slow

Shigella - - - - - -

Salmonella - + - + + -

Proteus - + +/- + +/- +

Edwardsiella - + + +/- - -