Microbiological Media Terms and Concepts

Forms of culture media 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 - 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.

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 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 - Deep 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 – 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)

Synthetic vs. 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

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

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

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

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 +/-)

Provision of Primary Macronutrients Element (Macronutrient) Cell need Form provided Hydrogen H2O, Organics Oxygen O2, H2O, Organics Nitrogen Amino acids, NH4Cl Carbon Glucose, acetate, amino acids

Provision of Secondary Macronutrients Element (Macronutrient) Cell need Form provided Phosphorous KH2PO4 Sulfur Na2SO4, Cysteine Potassium, Magnesium, Sodium, Calcium KCl, MgCl2, MgSO4, NaCl, CaCl2 Iron FeCl3, FeSO4 Chelators - EDTA

Micronutrients and Growth Factors 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

Example of Defined Medium Recipe Component Function or value K2HPO4, KH2PO4 (NH4)2SO4 MgSO4 CaCl2 Glucose Trace elements

Example of Complex Medium Recipe Component Function or value KH2PO4 Yeast Extract Peptone Glucose