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