2 Forms of culture mediaAll 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
3 Forms of culture media - Broth Liquid form of medium – no agar presentVery “spillable”Allows movement of bacteria – not good for obtaining pure culturesTSB, NB, APT broth, LB Broth, etc.
4 Forms of culture media - Slant 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 slantHandy, non-”spillable”, form of culture that takes up little roomNot enough surface area to be useful for pure culture
5 Forms of culture media - Deep Allowed to soldify standing upYETS motility medium: 0.2% - 0.5% Agar (semisolid or sloppy agar)Often use needle to inoculate
6 Forms of culture media – Petri plate Most significant formAllows for dispersal of cells leading to pure culture – individual coloniesPlate often used interchangeably with media typeAlways store and incubate upside down (right image)
7 Synthetic vs. Complex Synthetic (defined) All reagent grade chemicals. Exact composition is known.Used to assess nutritional requirements of organismsIn genetics – prototroph vs. auxotrophM9 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 organismsMost widely used in the laboratoryTryptic 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 restAPT with Azide – Azide is a potent inhibitor of respiration. Aerobes and facultatives die, while fermentative bacteria flourishMSA – 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 restSulfate deeps – contains lactate and sulfate in deep form – favors sulfate reducing bacteriaNo glucose or other fermentable sugarDeep form prevents oxygen from getting to bottom of tubeSulfate 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 producedMSA – 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 Element (Macronutrient)Cell needForm providedHydrogenH2O, OrganicsOxygenO2, H2O, OrganicsNitrogenAmino acids, NH4ClCarbonGlucose, acetate, amino acids
13 Provision of Secondary Macronutrients Element (Macronutrient)Cell needForm providedPhosphorousKH2PO4SulfurNa2SO4, CysteinePotassium, Magnesium,Sodium, CalciumKCl, MgCl2, MgSO4, NaCl, CaCl2IronFeCl3, FeSO4Chelators - EDTA
14 Micronutrients and Growth Factors Trace Elements (metals)Cr, Co, Cu, Mn, Mo, Ni, Se, W, V, ZnTrace minerals solutionOften prosthetic groups at active site of enzymeVitamins (organic compounds)PABA, Biotin, B12, Lipoic acid, Niacin, Pantothene, Riboflavin, B1, B6, K and Q10Vitamins solution or raw ingredientsCofactors for enzymes
15 Example of Defined Medium Recipe ComponentFunction or valueK2HPO4, KH2PO4(NH4)2SO4MgSO4CaCl2GlucoseTrace elements
16 Example of Complex Medium Recipe ComponentFunction or valueKH2PO4Yeast ExtractPeptoneGlucose
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