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Microbiological Media

Microbiological Media. Terms and Concepts. All examples here allow air to enter without dust getting in (“tortuous path”) Agar is the solidifying agent (often used interchangeably with the name of the medium) It’s not a culture until it is inoculated. Forms of culture media.

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Microbiological Media

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  1. Microbiological Media Terms and Concepts

  2. All examples here allow air to enter without dust getting in (“tortuous path”) Agar is the solidifying agent (often used interchangeably with the name of the medium) It’s not a culture until it is inoculated Forms of culture media

  3. Forms of culture media - Broth • Liquid form of medium – no agar present • Very “spillable” • Allows movement of bacteria – not good for obtaining pure cultures • TSB, NB, APT broth, LB Broth, etc.

  4. Contains solidifying agent called agar – 1.5% to 2% Tubes with molten agar from the autoclave are laid in racks at a an angle and allowed to solidify to form slant Handy, non-”spillable”, form of culture that takes up little room Not enough surface area to be useful for pure culture Forms of culture media - Slant

  5. Allowed to soldify standing up YETS motility medium: 0.2% - 0.5% Agar (semisolid or sloppy agar) Often use needle to inoculate Forms of culture media - Deep

  6. Forms of culture media – Petri plate • Most significant form • Allows for dispersal of cells leading to pure culture – individual colonies • Plate often used interchangeably with media type • Always store and incubate upside down (right image)

  7. Syntheticvs. Complex • Synthetic (defined) • All reagent grade chemicals. Exact composition is known. • Used to assess nutritional requirements of organisms • In genetics – prototroph vs. auxotroph • M9 Minimal Media

  8. Synthetic vs.Complex • Complex (undefined) • Contains “raw ingredients” whose composition is not known precisely (e.g. milk, yeast extract, casein, soy digest, etc.) • Used to culture a wide variety of organisms • Most widely used in the laboratory • Tryptic Soy Agar, Nutrient Agar, Luria- Bertrani Agar

  9. Selective, Enrichment, and Differential Media • Selective (counterselective) • Contains some poison or inhibitor that prevents the growth of certain organisms and allows for growth of the rest • APT with Azide – Azide is a potent inhibitor of respiration. Aerobes and facultatives die, while fermentative bacteria flourish • MSA – 7.5% salt inhibits gram negative bacteria and allows gram positive bacteria to grow

  10. Selective, Enrichment, and Differential Media • Enrichment (positive selection) • Contains nutrients that favor the growth of certain organisms and fails to support for growth of the rest • Sulfate deeps – contains lactate and sulfate in deep form – favors sulfate reducing bacteria • No glucose or other fermentable sugar • Deep form prevents oxygen from getting to bottom of tube • Sulfate only available electron acceptor

  11. Selective, Enrichment, and Differential Media • Allows you to distinguish one phenotype from another (all organisms still grow, but they look different) • Usual combination is some sugar and a pH indicator that will turn color if fermentation acids are produced • MSA – Mannitol fermentation is indicated by phenol red turning yellow (Man +/-) • EMB – Methylene Blue turns dark purple when lactose is fermented to acid endproducts (Lac +/-)

  12. Provision of Primary Macronutrients

  13. Provision of Secondary Macronutrients

  14. Trace Elements (metals) Cr, Co, Cu, Mn, Mo, Ni, Se, W, V, Zn Trace minerals solution Often prosthetic groups at active site of enzyme Vitamins (organic compounds) PABA, Biotin, B12, Lipoic acid, Niacin, Pantothene, Riboflavin, B1, B6, K and Q10 Vitamins solution or raw ingredients Cofactors for enzymes Micronutrients and Growth Factors

  15. Example of Defined Medium Recipe

  16. Example of Complex Medium Recipe

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