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 lactosefermenting 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

1) Tryptic Soy Agar (TSA)

Tryptic Soy Agar - uninoculated Type: General Purpose: Cultivation of non-fastidious bacteria Interpretation: Growth indicates non-fastidious bacteria present Examples S aureus, P aeruginosa, E. Coli

S. aureus

Tryptic Soy Agar - Staphylococcus aureus Note the carotenoid pigment typical of S. aureus. E. coli

Tryptic Soy Agar - Escherichia coli P. aeruginosa

Note the blue-green color due to pyocin production by the bacteria.

2. 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 Examples S aureus, N gonorrheae, E. Coli S. aureus

Chocolate Agar - Staphylococcus aureus Note: luxuriant growth with yellow pigmented colonies. N. gonorrhoeae

Chocolate Agar - Neisseria gonorrhoeae Note: small colonies that appear transparent on close examination. E. coli- Note: luxuriant growth of gray-white colonies

3. Thayer-Martin 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 Examples S aureus, N gonorrheae, E. Coli

S. aureus

Thayer-Martin Agar - Staphylococcus aureus Note: vancomycin in the medium inhibits the growth of staphylococci. N. gonorrhoeae

Thayer-Martin Agar - Neisseria gonorrhoeae Note: luxuriant growth of this fastidious bacterium. E. coli- Note: colistin in the media inhibits the growth of enterics.

4. MacConkey (lactose) 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 Examples S aureus, N gonorrheae, E. Coli, S enteridis

E. coli

MacConkey Agar - Escherichia coli Note: Red colonies and red precipitate due to acid production as a result of lactose fermentation. N. gonorrhoeae

MacConkey Agar - Neisseria gonorrhoeae Note: Neisseria does not grow on MacConkey. S. enteritidis

MacConkey Agar - Salmonella enteritidis Note: Growth, but no fermentation of lactose. Colorless colonies, medium is slightly yellow due to the increased pH resulting from bacterial digestion of peptone in the medium. S. aureus- Note: Gram-positives do not grow on MacConkey.

5. Eosin-methylene Blue Agar (EMB)

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 Examples S enteridis, E Coli, K pneumoniae S. enteritidis

Eosin Methylene Blue Agar - Salmonella enteritidis Note: pink colonies indicative of non-lactose fermentation. E. coli

Eosin Methylene Blue Agar - Escherichia coli Note: Green metallic sheen indicative of dye precipitation due to lactose fermentation. K. pneumonia- Mucoid colonies with dark centers due to capsule production and lactose fermentation
respectively.

6. 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 Examples E Coli, S enteridis

E. coli

Hektoen - Escherichia coli Note: Orange color indicates acid production as a result of lactose fermentation. S. enteritidis- Clear colonies indicates a non-lactose fermentor & black precipitate in center of colonies
is due to H2S production.

7. 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 Examples S epidermis, E coli, S. aureus S. epidermidis

Mannitol Salt Agar - Staphylococcus epidermidis Note: growth, but no fermentation of mannitol, medium color unchanged

S. aureus

Mannitol Salt Agar - Staphylococcus aureus Note: yellow color due to acid produced by fermentation of mannitol E coli- Streptococci, Enterics, and other organisms do not grow on Mannitol Salt Agar

8. Triple Sugar Iron Agar (TSI)

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 Examples E Coli, Salmonella, Shigella, P aeruginosa E. coli

Triple Sugar Iron Agar - Escherichia coli

Salmonella

Triple Sugar Iron Agar - Salmonella Shigella

Triple Sugar Iron Agar - Shigella P. aeruginosa

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

Sheep Blood Agar - uninoculated Type: Differential and enriched Purpose: Determine types of hemolysis (i.e., , , ) Interpretation: : partial clearing, green or brownish ring; : wide zone of clearing; : nonhemolytic Examples Alpha hemolysis, Beta hemolysis, Gamma Hemolysis, No growth

Alpha hemolysis

Sheep Blood Agar - Streptococcus pneumoniae, alpha hemolytic Beta hemolysis

Sheep Blood Agar - Streptococcus pyogenes, beta hemolytic Gamma hemolysis

Sheep Blood Agar - E. coli, gamma (non) hemolytic

No growth

Sheep Blood Agar - Neisseria gonorrhoeae Note: Neisseria are fastidious and do not grow on Sheep Blood Agar.