Selective media[edit] Selective media are used for the growth of only selected microorganisms.

For example, if a microorganism is resistant to a certain antibiotic, such as ampicillin or tetracycline, then that antibiotic can be added to the medium in order to prevent other cells, which do not possess the resistance, from growing. Media lacking an amino acid such as proline in conjunction with E. coli unable to synthesize it were commonly used by geneticists before the emergence of genomics to map bacterial chromosomes. Selective growth media are also used in cell culture to ensure the survival or proliferation of cells with certain properties, such as antibiotic resistance or the ability to synthesize a certain metabolite. Normally, the presence of a specific gene or an allele of a gene confers upon the cell the ability to grow in the selective medium. In such cases, the gene is termed a marker. Selective growth media for eukaryotic cells commonly contain neomycin to select cells that have been successfully transfected with a plasmid carrying the neomycin resistance gene as a marker. Gancyclovir is an exception to the rule as it is used to specifically kill cells that carry its respective marker, the Herpes simplex virus thymidine kinase (HSV TK). Four types of agar plates demonstrating differential growth depending on bacterial metabolism. Some examples of selective media include: eosin methylene blue (EMB) that contains methylene blue toxic to Gram-positive bacteria, allowing only the growth of Gram negative bacteria YM (yeast and mold) which has a low pH, deterring bacterial growth MacConkey agar for Gram-negative bacteria Hektoen enteric agar (HE) which is selective for Gram-negative bacteria mannitol salt agar (MSA) which is selective for Gram-positive bacteria and differential for mannitol Terrific Broth (TB) is used with glycerol in cultivating recombinant strains of Escherichia coli. xylose lysine desoxyscholate (XLD), which is selective for Gram-negative bacteria buffered charcoal yeast extract agar, which is selective for certain gram-negative bacteria, especially Legionella pneumophila

Blood agar plates are often used to diagnose infection. On the right is a positive Streptococcus culture; on the left a positive Staphylococcus culture.

Blood-free, charcoal-based selective medium agar (CSM) for isolation of Campylobacter

Selective media allows the growth of certain type of organisms, while inhibiting the growth of other organisms. This selectivity is achieved in several ways. For example, organisms that have the ability to utilize a given sugar are screened easily by making that particular sugar the only carbon source in the medium for the growth of the microorganism. Like-wise, the selective inhibition of some types of microorganisms can be studied by adding certain dyes, antibiotics, salts or specific inhibitors that will affect the metabolism or enzymatic systems of the organisms. For example, media containing potassium tellurite, sodium azide or thallium acetate at different concentrations of 0.1 - 0.5 g/l will inhibit the growth of all Gram-negative bacteria. Media supplemented with the antibiotic penicillin concentration 550 units/ml or crystal violet 2 mg/l inhibit the growth of Gram-positive bacteria. Tellurite agar, is used to select for Gram-positive organisms, and nutrient agar supplemented with the antibiotic penicillin can be used to select for the growth of Gram negative organisms. Eg., Mannitol salt agar, Hektoen enteric agar (HE), Phenylethyl alcohol agar

1. Mannitol Salt Agar (MSA): Mannitol salt agar is both a selective and differential media used for the isolation of pathogenic Staphylococci from mixed cultures. Components: 7.5% NaCl selects for species of Staphylococcus. This concentration of salt is too high for most other bacteria to withstand and , therefore, inhibits their growth. Mannitol alcohol of the carbohydrate mannose. Mannitol fermentation produces acid end products which turn the medium yellow. Yellow indicates mannitol positive and no color change indicates mannitol negative. Phenol red pH indicator yellow in acid pH (The same indicator that is used in phenol red carbohydrate fermentation broths).

Figure1 : Mannitol Salt Agar On MSA, only pathogenic Staphylococcus aureus produces small colonies surrounded by yellow zones. The reason for this color change is that S. aureus have the ability to ferment the mannitol, producing an acid, which, in turn, changes the indicator color from red to yellow. The growth of other types of bacteria is usually inhibited. This growth differentiates S.aureus from S.epidermidis, which forms colonies with red zones or both zones. Formula: Ingredients per liter of deionized water

Beef extract Peptone

Sodium chloride D-mannitol Agar Phenol red

1.0g 10.0g
75.0g 10.0g 15.0g 0.025g

2. MacConkeys Agar (MAC): MacConkeys Agar is both a selective and differential media; it is selective for Gram negative bacteria and can differentiate those bacteria that have the ability to ferment lactose. Components: Bile salts - Inhibits most Gram-positive bacteria, except Enterococcus and some species of Staphylococcus i.e. Staphylococcus aureus. Crystal violet dye- Inhibits certain Gram-positive bacteria thus selecting for Gram negatives. Lactose- Some bacteria can ferment lactose acid-end products, others cannot. Neutral pH red indicator - Stains microbes fermenting lactose * hot pink in acid pH * rose in neutral pH * tan in alkaline pH Peptone - a source of proteins, amino acids for microbial growth.

Figure2 : MacConkey's Agar By utilizing the available lactose in the medium, Lac+ (Lactose positive) bacteria such as Escherichia coli, Enterobacter and Klebsiella will produce acid in the medium, which lowers the pH of the agar below 6.8 and results in the appearance of red or pink colonies. The bile salts in the medium precipitate in the immediate neighborhood of the colony, causing the medium surrounding the colony to become hazy appearance. Non-lactose fermenting bacteria such as, Proteus species,Salmonella, Pseudomonas aeruginosa and Shigella cannot utilize lactose in the medium, and will use peptone instead. This results in the formation of ammonia, which raises the pH of the agar, and leads to the formation of white or colorless colonies in the plate. But, in some cases, they can also look golden to brown with dark centers. They are usually circular colonies and arranged randomly. Formula: Ingredients per liter of deionized water

3. Eosin Methylene Blue (EMB) Agar (Levine): Eosin methylene blue agar (EMB) is both a selective and intestinal pathogens. Components: Lactose a disaccharide which can be fermented by some bacterial enzymes to produce acidic end products. Eosin and Methylene Blue these are dyes which inhibit the growth of most Gram positive bacteria. They also react with any acidic products resulted from lactose fermentation to color the colonies. Figure 3: Uninoculated EMB agar plate Acid production from lactose fermentation causes precipitation of the dyes on the surface of the colony resulting in different colors. Large amounts of acid green metallic sheen Small amounts of acid pink No fermentation colorless Enterobacter aerogenes produces large colonies which are pink-to-buff around dark centers. Escherichia coli produce small, dark colonies with a green metallic sheen. Pseudomonas, Proteus, Salmonella and Shigella sp produces colorless colonies because it does not ferment lactose. Formula: Ingredients per liter of deionized water differential medium used for the detection and isolation of Gram-negative

4. Phenylethyl Alcohol Agar: Phenylethyl Alcohol (PEA) Agar with or without 5% sheep blood is a selective medium for the isolation of gram-positive organisms, particularly gram-positive cocci, from specimens of mixed gram-positive and gram-negative flora. Component: Phenylethyl alcohol Inhibits the growth of Gram negatives since it selectively and reversibly inhibits DNA synthesis, thus selecting for Gram positives. Figure 4: Uninoculated PEA Agar Formula:Ingredients per liter of deionized water

5. Hektoen Enteric (HE) Agar: Hektoen Enteric (HE) Agar is a moderately selective medium used in qualitative procedures for the isolation and cultivation of gram-negative enteric microorganisms, especially Shigella and Salmonella from a variety of clinical and nonclinical specimens. Components: Bile salts: Inhibits the growth of most Gram positive organisms. Bromthymol blue and acid fuchsin dyes: have a lower toxicity than that of many other enteric media, resulting in improved recovery. Lactose, sucrose, and salicin: provide fermentable carbohydrates to encourage the growth and differentiation of enterics.

Sodium thiosulfate: provides a source of sulfur.

Ferric ammonium citrate: provides a source of iron that allows the production of hydrogen sulfide from sodium thiosulfate, which provides a source of sulfur. This also allows the visualization of hydrogen sulfide production by reacting with hydrogen sulfide gas to form a black precipitate. Figure 5:Uninoculated Hektoen Enteric Agar plate Coliforms capable of overcoming the moderately inhibitory qualities of the media will develop into orange or salmon-pink colonies in the presence of the bromthymol blue indicator. Shigella species develop into green-colored colonies with darker blue-green centers. Salmonella species appear as blue-green colonies with or without black centers. Producers of H2S will form black-centered colonies in the presence of the ferric ammonium citrate indicator. Formula: Ingredients per liter of deionized water

6. Blood Agar: Blood agar is both differential and enriched medium. The blood that is incorporated into this medium is an enrichment ingredient for the cultivation of fastidious organisms such as the Streptococcus species. A number of streptococcal species produce substances that destroy red blood cells; that is, they cause lysis of the red cell wall with subsequent release of hemoglobin. Such substances are referred to as hemolysins. The activity of streptococcal hemolysins also known as streptolysins can be readily observed when the organisms are growing on a blood agar plate.

Different streptococci produce different effects on the red blood cells in blood agar. Those that produce incomplete hemolysis and only partial destruction of the cells around colonies are called alpha-hemolytic Streptococci. Characteristically, this type of hemolysis is seen as a distinct greening of the agar in the hemolytic zone, and thus this group of streptococci has also been referred to as the viridans group.
Species whose hemolysins cause complete destruction of red cells in the agar zones surrounding their colonies are said to be beta-hemolytic. When growing on blood agar, beta-hemolytic streptococci are small opaque or semi translucent colonies surrounded by clear zones in a red opaque medium. Two types of beta lysins are produced: Streptolysin O and Streptolysin S. Streptolysin O, an antigenic, oxygen-labile enzyme, and streptolysin S, a nonantigenic, oxygen-stable lysin. The hemolytic reaction is enhanced when blood agar plates are streaked and simultaneously stabbed to show subsurface hemolysis by Streptolysin O in an environment with reduced oxygen tension. Some strains of Staphylococci, Escherichia coli, and other bacteria also may show beta-hemolysis. Some species of Streptococci do not produce hemolysins. Therefore, when their colonies grow on blood agar, no change is seen in the red blood cells around them. These species are referred to as nonhemolytic or gamma hemolytic streptococci. On blood agar, S. aureus usually displays a light to golden yellow pigment, whereas S.epidermidis has a white pigment andS.saprophyticus either a bright yellow or white pigment. However, pigmentation is not always a reliable characteristic. On blood agar,S.aureus is usually, but not always, beta-hemolytic; S. epidermidis and S. saprophyticus are almost always nonhemolytic. Figure 6:-Blood Agar

Formula:
Ingredients per liter of deionized water

Note: Dissolve the above ingredients and autoclave. Cool the sterile blood agar base to 45 to 50C and aseptically add 50 ml of sterile, defibrinated blood. Mix thoroughly and then dispense into plates while a liquid. Blood agar base for use in making blood agar also can be purchased. A combination of hemoglobin and a commercial nutrient supplement can be used in place of defibrinated blood.

7.Chocolate Agar: Fastidious organisms such as Haemophilus and Neisseria require specially enriched culture media and microaerophilic incubation conditions. Chocolate agar is commonly used for primary isolation of Haemophilus from clinical specimens. This medium contains hemoglobin derived from bovine red blood cells as well as other enrichment growth factors. Chocolate agar may be made selective forHaemophilus species by the addition of bacitracin. Figure 7 :Uninoculated Chocolate Agar Plate Two special growth factors, called X and V, are required by some Haemophilus species. The X factor is hemin, a heat-stable derivative of hemoglobin. The red blood cells in chocolate agar have been heated until they are lysed, producing the characteristic brown color of this medium. Lysing the blood with heat releases the X factor that otherwise isn't available in regular blood agar plates. This is why chocolate agar is the media of choice for culturing Haemophilus influenzae. The V factor is a heat-labile coenzyme (nicotinamide adenine dinucleotide, or NAD), essential in the metabolism of some species that lack it. Yeast extracts contain V factor and are one of the most convenient supplements of chocolate agar or other media used forHaemophilus. Chocolate agar, however, does not reveal hemolysis data, so species differentiation among the members of Haemophilus must be performed in another manner. Formula: Ingredients per liter of deionized water.

Note: Aseptically add 5% sterile, defibrinated sheep blood to the sterile and molten agar. Heat at 80C for 15 minutes or until a chocolate color develops.