Cultivation Media for Bacteria

Isolation of bacteria is accomplished by growing ("culturing") them on the surface of solid nutrient media. Such a medium normally consists of a mixture of protein digests (peptone, tryptone) and inorganic salts, hardened by the addition of 1.5% agar. Examples of standard general purpose media that will support the growth of a wide variety of bacteria include nutrient agar, tryptic soy agar, and brain heart infusion agar. A medium may be enriched, by the addition of blood or serum. Examples of enriched media include sheep blood agar and chocolate (heated blood) agar.

Selective media contain ingredients that inhibit the growth of some organisms but allow others to grow. For example, mannitol salt agar contains a high concentration of sodium chloride that inhibits the growth of most organisms but permits staphylococci to grow.

Differential media contain compounds that allow groups of microorganisms to be visually distinguished by the appearance of the colony or the surrounding media, usually on the basis of some biochemical difference between the two groups. Blood agar is one type of differential medium, allowing bacteria to be distinguished by the type of hemolysis produced. Some differential media are also selective, for example, standard enteric agars such as MacConkey and EMB agars, which are selective for gram-negative coliforms and can differentiate lactose-fermenting and non-lactose-fermenting bacteria.

Several examples of commonly used bacteriological media, as well as examples with one or more types of bacteria cultured on them are shown below. Carefully examine the plates and observe the colony morphology, colors, and patterns of growth (or no growth) that occurs. This information can be valuable in the preliminary identification of pathogens in case studies.

Common Bacteriologic Media

Tryptic Soy Agar (TSA)
Tryptic Soy Agar
Tryptic Soy Agar - uninoculated
Type: General
Purpose: Cultivation of non-fastidious bacteria
Interpretation: Growth indicates non-fastidious bacteria present
Chocolate Agar
Chocolate Agar
Chocolate Agar - uninoculated
Type: Enriched
Purpose: Cultivation of fastidious organisms such as Neisseria or Haemophilus sp.
Interpretation: Some organisms grow on Chocolate that do not grow on standard media
Thayer-Martin Agar
Chocolate Agar
Thayer-Martin Agar - uninoculated
Type: Enriched and selective; contains antibiotics colistin (kills gram-negative coliforms), vancomycin (kills gram-positives), nystatin (kills fungi)
Purpose: To select for fastidious organisms, such as N. gonorrhoeae, in patient samples containing large numbers of normal flora, such as in the female genital tract
MacConkey (lactose) Agar
MacConkey Agar
MacConkey Agar - uninoculated
Type: Selective and differential
Purpose: Contains bile salts and crystal violet which selects for gram-negative enterics, differentiates lactose-fermenters from non-fermenters. Can include sugars other than lactose for further differentiation (for example, to differentiate enterohemorrhagic E. coli (EHEC), which does not ferment sorbitol, from other E. coli types which do.)
Interpretation: Selects for non-fastidious gram-negatives; red colonies indicate fermentation of lactose, white indicates no fermentation of lactose
Eosin-methylene Blue Agar (EMB)
Eosin Methylene Blue
Eosin Methylene Blue Agar - uninoculated
Type: Differential (lactose) and selective (dye inhibition and precipitation at acid pH)
Purpose: Differentiates lactose fermenters (E. coli) from non-fermenters (Salmonella, Shigella)
Interpretation: Lactose fermenters blue/black; non-fermenters colorless or light purple
Hektoen Agar
Hektoen - uninoculated
Type: Selective and differential
Purpose: Detects lactose fermentation, H2S production, inhibits non-enterics
Interpretation: Lactose fermenters yellow or salmon, non-fermenters colorless; H2S production produces black precipitate
Mannitol Salt Agar
Mannitol Salt Agar
Mannitol Salt Agar - uninoculated
Type: Selective and differential
Purpose: Selects for Staphylococci, which grow at high salt concentrations; differentiates Staphylococcus aureus from other Staphylococci
Interpretation: Staphylococcus aureus is yellow (ferments mannitol), other staphylococci are white
Triple Sugar Iron Agar (TSI)
Triple Sugar Iron Agar
Triple Sugar Iron Agar - uninoculated
Type: Multi-purpose, differential
Purpose: Detects glucose, lactose, sucrose fermentation; gas and H2S production. (E. coli → A/AG; Salmonella → K/A+G; Shigella → K/A; Ps. aeruginosa → K/K)
Interpretation: Yellow butt, red slant (K/A) = ferments glucose only; yellow butt and slant (A/A) = ferments glucose + lactose and/or sucrose; red but and slant (K/K) = non-fermenter of all 3 sugars; black (+) = H2S; bubbles (G) = gas production

Hemolytic Reactions Observed on Blood Agar

Observation of the hemolytic reactions on sheep blood agar is a very useful tool in the preliminary identification of bacteria, particularly streptococci. The types of hemolysis are defined as follows:

alpha (α) hemolysis: An indistinct zone of partial destruction of red blood cells (RBCs) appears around the colony, often accompanied by a greenish to brownish discoloration of the medium. Streptococcus pneumoniae and many oral streptococci are α hemolytic.

beta (β) hemolysis: A clear, colorless zone appears around the colonies, in which the RBCs have undergone complete lysis. Streptococcus pyogenes, S. agalactiae, and several other species of streptococci are β hemolytic. Many other bacteria besides streptococci can be β hemolytic, including Staphylococcus aureus, Pseudomonas aeruginosa, Listeria monocytogenes, etc., and hemolytic reactions can also be a useful diagnostic tool for these organisms.

no (γ) hemolysis: No apparent hemolytic activity or discoloration is produced (also called gamma hemolysis).

Sheep Blood Agar
Sheep Blood Agar
Sheep Blood Agar - uninoculated
Type: Differential and enriched
Purpose: Determine types of hemolysis (i.e., α, β, γ)
Interpretation: α: partial clearing, green or brownish ring; β: wide zone of clearing; γ: non-hemolytic


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