1. Microbiological Media
Terms and Concepts

2. 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

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. 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

5. 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

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. 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

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

13. 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

14. 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

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

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