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Cultivation is the process of propagating

organisms by providing the proper environmental
conditions. Growing microorganisms are making
replicas of themselves, and they require the
elements present in their chemical composition.
Nutrients must provide these elements in
metabolically accessible form. In addition,
organisms require metabolic energy to synthesize
macromolecules and maintain essential chemical
gradients across their membranes. Factors that
must be controlled during growth include the
nutrients, pH, temperature, aeration, salt
concentration, and ionic strength of the medium
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 Culture Media
• Culture medium: Nutrients prepared for
microbial growth
• Sterile: No living microbes
• Inoculum: Introduction of microbes into medium
• Culture: Microbes growing in/on culture medium
• Synthetic media
• Defined synthetic media— it has specified amount of
macromolecules
• Complex media – the amount or what is in the
nutrient used is not known.
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 Agar
• Complex polysaccharide
• Used as solidifying agent for culture media in
Petri plates, slants, and deeps
• Generally not metabolized by microbes
• Liquefies at 100°C
• Solidifies ~40°C

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 Culture Media
• Chemically defined media: Exact chemical
composition is known
• Complex media: Extracts and digests of yeasts,
meat, or plants
– Nutrient broth
– Nutrient agar

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Culture Media

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 Selective Media
• Suppress unwanted
microbes and
encourage desired
microbes.

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 Differential Media
• Make it easy to distinguish colonies of different
microbes.

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 Enrichment Media
• Encourages growth of desired microbe
• Assume a soil sample contains a few phenol-
degrading bacteria and thousands of other
bacteria
– Inoculate phenol-containing culture medium with the soil and
incubate
– Transfer 1 ml to another flask of the phenol medium and
incubate
– Transfer 1 ml to another flask of the phenol medium and
incubate
– Only phenol-metabolizing bacteria will be growing
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• Methods of Obtaining Pure Culture
• 1) The Streak Plate Method – sterile wire loop
and streaked in different patterns on agar
• 2) Pour Plate Method- serial dilutions using
melted agar

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 Preserving Bacteria Cultures
• Deep-freezing: –50°to –95°C
• Lyophilization (freeze-drying): Frozen (–54° to –
72°C) and dehydrated in a vacuum

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 Bacteria on McConkey agar plate
Intended use of MacConkey Agar:  for the
selective isolation and identification of
Enterobacteriaceae from feces, urine,
wastewater and foods.
Composition of MacConeky Agar:
Enzymatic Digest of Gelatin, Casein and Animal
tissue: provides nitrogen, vitamins, minerals
and amino acids essential for growth.
Lactose: fermentable carbohydrate providing
carbon and energy.
Bile Salts: selective agents and inhibit Gram
positive organisms.
Crystal Violet: Use: Gram positive bacteria are
generally inhibited by crystal violet.
Sodium Chloride: supplies essential
electrolytes for transport and osmotic balance.
Neutral Red: pH indicator. which is red in color
at pH’s below 6.8.
When lactose is fermented, the pH of the
medium decreases, changing the color of
neutral red to pink
Agar : Solidifying agent
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How two strains of one bacterium combine to cause
flesh-eating infection
Date:November 11, 2019
Source:University of Maryland
Summary:A new study used genetic analysis to reveal how two different strains of a single species of flesh-eating bacteria
worked in concert to become more dangerous than either one strain alone. The work suggests that other difficult-to-treat
infections may be polymicrobial and treating only one organism in a polymicrobial infection could be the cause of many
secondary infections and chronic infections that resist treatment.

The original infection -- cultured from a patient who developed

the severe flesh-eating disease known as necrotizing fasciitis --
was diagnosed as a monomicrobial disease. Traditional
diagnostics could only determine that the infection was caused
by a single species of bacteria called Aeromonas hydrophila. But
the disease baffled clinicians when it rapidly turned deadly,
requiring a quadruple amputation to save the patient's life.
Through genetic analysis of the culture, Colwell and her team
discovered important differences among the individual bacterial
cultures that could not be detected through standard diagnostic
methods.
T6SS and exoA of flesh-eating Aeromonas hydrophila in peritonitis and necrotizing fasciitis during
mono- and polymicrobial infections. PNAS, 2019  20
Flower-like patterns in mixtures
of E. coli and A. baylyi.
Flower-like patte rns in mixtures of  E. coli and A. baylyi.

(a) The pattern after 3 days of growth on a 0.5% LB agar surface. (b) Time-lapse bright-field images of the developing
pattern. (c) Pure E. coli and pure A. baylyi colonies show no patterns. (d) Radius of the colony vs time for pure E.
coli (green), pure A. baylyi (red), and the mixture of E. coli and A. baylyi (blue). The radius is defined
as Area/π−−−−−−√A⁢r⁢e⁢a/π where AreaA⁢r⁢e⁢a is the area of the colony which is calculated after image segmentation.
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Your observations on your plates

•Sample type
•Type and number of colonies
•Impressions

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Escherichia coli: Pink colonies
Proteus mirabilis: colorless colonies
Salmonella typhimurium: colorless colonies
Staphylococcus aureus: No growth

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