Lab 13-
Bacterial cultivation
Selective /differential media
Lab activities:
Demo selective and differentiation plates
Streaking bacteria on differentiation plates
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).
Growth needs
Fastidious bacteria – relatively complex growth needs
Non-fastidious bacteria- relatively basic and straightforward growth needs
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.
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
Media classification and function
Enrichment – Used to enhance growth of a particular pathogen
Supportive – support growth of most non fastidious bacteria
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.
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
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)
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
Eosine Methylene blue (EMB),
Differentiatial between lactose fermenting and non fermenting enteric bacteria
Tellurite Glycine Agar (TGA)
Selective- Tellurite glycine and lithium inhibit most bacteria
Preferential growth of Staphyloccoci coagulase positive (Staphyloccocus aureus)
Bacteria streaked in lab
Staphylococcus aureus
Staphylococcus epidermidis
Salmonella pullorum,
E.coli

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