You can watch a video of how general growth media (complex vs. defined) and how to prepare agar plates using the link below.
Robert Koch first introduced gelatin as a solidifying agent to make solid media cultures in the late 1800s. Since then, gelatin has been replaced with agar, which is derived from red seaweed. Agar melts at 100℃ and solidifies at 42℃. It is this temperature difference which allows microbiologists to grow thermophilic microorganisms (those that grow well at high temperature) on a solid medium (plates/slants) without risk of the solidifying agent from melting.
Chemically defined growth media contains known ingredients in known concentrations. Complex growth media contains at least one unknown ingredient or concentration. For example, the addition of yeast or beef extract will make growth media complex. The precise components of the extract are not fully known.
Premade growth media can be purchased from various supply companies. Media is also available in dehydrated forms - simply add water and then sterilize the media using an autoclave. Care should be taken in reading the ingredients of growth media. Some dehydrated media may already contain agar.
Broth media can be solidified by the addition of agar. As a general rule, we add 1% agar (10 g of agar per 1000 milliliters of broth). When testing motility (as seen in our motility lab), we use 0.7% agar (7 g of agar per 1000 milliliters of broth).
Listed below are descriptions of the types of growth media this manual references.
Muller-Hinton is used for antibiotic susceptibility test. It is low in certain antibiotic inhibitors and gives consistent results. It used growing non-fastidious microorganisms (organisms that do not require special nutrients in the growth media).
Ingredients include:
- Beef Infusion: Provides nitrogen, amino acids and vitamins.
- Casamino acids: Nitrogen source from completely hydrolyzed proteins.
- Starch: Carbon source, mediates antibiotic diffusion and absorbs toxic metabolic products from
bacteria that might interfere with susceptibility testing.
Nutrient broth and agar are used for the growth of a wide variety of non-fastidious microorganisms.
Ingredients include:
- Bacto Beef Extract: Provides amino acids, carbon, nitrogen and vitamins.
- Bacto Peptone: Enzymatic digest of protein (amino acid & nitrogen sources)
- Sodium chloride: To establish proper osmotic balance for the growth of microorganisms.
Depending on the microorganism you are trying to culture, glucose can also be added to the growth media.
If you are not able to access nutrient broth, Brain Heart Infusion (BHI) or Luria-Bertani (LB) is an acceptable substitute.
BHI broth and agar are used for the growth of a wide variety of non-fastidious microorganisms,
especially streptococci.
Ingredients include:
- Calf brains: Provides amino acids, vitamins and other growth factors.
- Beef heart: Provides nitrogen, amino acids and vitamins.
- Peptone: Enzymatic digest of protein (amino acid & nitrogen sources).
- Dextrose: Carbon source
- Sodium chloride: To establish proper osmotic balance for the growth of microorganisms.
- Disodium Phosphate: Buffer that maintains pH.
LB broth and agar are used for the growth of a wide variety of non-fastidious microorganisms,
especially members of the Enterobacteriaceae family.
Ingredients include:
- Tryptone: Enzymatic digest of casein protein (amino acid & nitrogen sources).
- Yeast Extract: Provides organic compounds for growth.
- Sodium chloride: To establish proper osmotic balance for the growth of microorganisms.
Depending on the microorganism you are trying to culture, glucose can also be added to the growth
media.
These agar plates contain a grid to permit precise location and counting of bacterial cells present on environmental surfaces. These plates are also known as Contact Plates.
Estimates of bacterial numbers may be made after gross examination of incubated agar plates and counting the colonies of bacteria on the plate. The use of RODAC® plates permits the most precise counting of bacterial numbers because there is a grid on the plate surface that facilitates performing a colony count.
Ingredients include:
- Trypticase Soy Agar*: See below
- Lecithin: neutralizes disinfectants on the test surface that may inhibit microbial growth.
- Polysorbate 80 / Tween 80: neutralizes disinfectants on the test surface that may inhibit microbial growth.
*Other RODAC plates may have other growth media agar in it.
Nutrient broth and agar are used for the growth of a wide variety of fastidious (organisms that have complex nutritional requirements) and non-fastidious microorganisms. It typically is more nutrient rich than nutrient broth or LB.
Ingredients include:
- Tryptone: Enzymatic digest of casein protein (amino acid & nitrogen sources).
- Soytone: Enzymatic digest of soybean meal (amino acid & Nitrogen sources).
- Dextrose: Carbon source.
- Sodium chloride: To establish proper osmotic balance for the growth of microorganisms.
- Potassium phosphate: Buffer that maintains pH.
Source:
Difco Laboratories: Difco Manual.