Lab 13- Bacterial cultivation

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Lab 13- Bacterial cultivation

• Selective /differential
• media

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Lab activities

• Demo selective and differentiation plates
• Streaking bacteria on differentiation plates

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Cultivation

• The process of growing microorganisms in culture
  by taking bacteria from the infection site (in
  vivo or environment) and grow them in artificial
  environment in the laboratory (in vitro).

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Growth needs

• Fastidious bacteria relatively complex growth
  needs
• Non-fastidious bacteria- relatively basic and
  straightforward growth needs

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Phases of growth media

• Broth- Growth of bacteria will change the liquid
  from clear to turbid (cloudy).
• Solid Agar plates, slants Bacterial cells
  inoculated on solid media will multiply enough to
  be seen by naked eye.

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Colony- (clone)

• Colony- A bacterial population derived from one
  bacterial cell. The cells within the colony have
  identical, genus, species, genetic and phenotypic
  characteristics.
• Pure bacteria - derived from a single colony.
• Selection of a pure colony -most important for
  bacterial identification

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Media classification and function

• Enrichment Used to enhance growth of a
  particular pathogen
• Supportive - support growth of most non
  fastidious bacteria

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Media classification and function

• Selective - Contain inhibitory agents that are
  inhibitory to all organisms except those sought
• Differential - Contain factors that allow
  bacterial species to manifest certain metabolic
  characteristics that distinguish them from other
  species.
• Media can be both selective and differential
  based on the ingredients of the medium.

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Blood agar plate (BA)

• Nutrient agar with 5 sheep blood
• Cultivation of fastidious and non fastidious
  bacteria.
• Differential Identify hemolysis - Some bacteria
  secrete enzymes that lyse red blood cells
  (hemolysins) such that a clearing around the
  colony appears.
• b hemolysis- complete clearing (white hemolysis)
• a hemolysis incomplete clearing (green
  hemolysis)
• g hemolysis- no hemolysis

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Mannitol Salt Agar (MSA)

• Both selective and differential medium.
• High salt concentration - inhibits most bacteria.
• Selective for Staphylococcus sp.
• Differentiate between Staphylococcus sp. by the
  sugar mannitol fermentation .
• Mannitol fermention produce acids that change
  the medium pH.
• Peach color- neutral- no fermentation
• Bright yellow- Acidic mannitol fermentation
  (Staph. coag. pos.- Staph. aureus)

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MacConkey Agar (MAC)

• Selective and differential medium.
• Selective - Gram positive bacteria are inhibited
  by the presence of bile salts and crystal violet
  inhibitors in the medium Most of gram negative
  bacteria will grow.
• Differentiate- Between Gram negative bacteria by
  their ability to ferment lactose.
• Pink colonies- Bacteria that ferment lactose
  (precipitation of some salts in media by acid
  production).
• Pale colonies- Non fermenters

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Eosine Methylene blue (EMB),

• Differentiatial between lactose fermenting and
  non fermenting enteric bacteria

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Tellurite Glycine Agar (TGA)

• Selective- Tellurite glycine and lithium inhibit
  most bacteria
• Preferential growth of Staphyloccoci coagulase
  positive (Staphyloccocus aureus)

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Bacteria streaked in lab

• Staphylococcus aureus
• Staphylococcus epidermidis
• Salmonella pullorum,
• E.coli

S.aureus
S.epidermidis
S.pullurum
E.coli
EMB and TGA