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)

Type: General Purpose: Cultivation of non-fastidious bacteria Interpretation: Growth indicates non-fastidious bacteria present

Examples Chocolate Agar

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

Examples

Thayer-Martin Agar

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

Examples MacConkey (lactose) Agar

Type: Selective and differential Purpose: Contains bile salts and crystal violet which selects for gram-negative enterics, differentiates lactose-fermenters from nonfermenters. 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

Examples Eosin-methylene Blue Agar (EMB)

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

Examples

Hektoen Agar

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

Examples Mannitol Salt Agar

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

Examples Triple Sugar Iron Agar (TSI)

Type: Multi-purpose, differential Purpose: Detects glucose, lactose, sucrose fermentation; gas and H 2S 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

Examples
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 () 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 hemolytic. beta () 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 hemolytic. Many other bacteria besides streptococci can be hemolytic, including Staphylococcus aureus, Pseudomonas aeruginosa, Listeria monocytogenes, etc., and hemolytic reactions can also be a useful diagnostic tool for these organisms. no () hemolysis: No apparent hemolytic activity or discoloration is produced (also called gamma hemolysis).

Sheep Blood Agar

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

Examples
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