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Mannitol Salt Agar (MSA):
Mannitol salt agar is both a selective and differential media used for the isolation of pathogenic Staphylococci from mixed cultures.
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 growth of other types of is that S. aureus have the ability to ferment the is usually inhibited. This growth mannitol, producing an acid, which, in turn, changes the indicator color from red to yellow. The bacteria differentiates S.aureus from S.epidermidis, which forms colonies with red zones or both zones.
Ingredients per liter of deionized water
a source of proteins. Neutral pH red indicator . Components: Bile salts .e. . Crystal violet dye.Inhibits certain Gram-positive bacteria thus selecting for Gram negatives.Inhibits most Gram-positive bacteria.2. MacConkey’s Agar (MAC): MacConkey’s Agar is both a selective and differential media. others cannot. Lactose. amino acids for microbial growth. Staphylococcus aureus. it is selective for Gram negative bacteria and can differentiate those bacteria that have the ability to ferment lactose.Some bacteria can ferment lactose acid-end products.Stains microbes fermenting lactose * * * tan in alkaline pH hot rose pink in in acid neutral pH pH Peptone . except Enterococcus and some species of Staphylococcus i.
Salmonella. Lac+ (Lactose positive) bacteria such as Escherichia coli. They are usually circular colonies and arranged randomly. causing the medium surrounding the colony to become hazy appearance. The bile salts in the medium precipitate in the immediate neighborhood of the colony.Figure2 : MacConkey's Agar By utilizing the available lactose in the medium. and will use peptone instead. they can also look golden to brown with dark centers. Proteus species. in some cases.8 and results in the appearance of red or pink colonies. which lowers the pH of the agar below 6. and leads to the formation of white or colorless colonies in the plate. Formula: Ingredients per liter of deionized water . which raises the pH of the agar. Pseudomonas aeruginosa and Shigella cannot utilize lactose in the medium. Non-lactose fermenting bacteria such as. This results in the formation of ammonia. But. Enterobacter and Klebsiella will produce acid in the medium.
3. differential medium used for the Components: Lactose – a disaccharide which can be fermented by some bacterial enzymes to produce acidic end products. They also react with any acidic products resulted from lactose fermentation to color the colonies. Eosin Methylene Blue (EMB) Agar (Levine): Eosin methylene blue agar (EMB) is both a selective and detection and isolation of Gram-negative intestinal pathogens. Eosin and Methylene Blue – these are dyes which inhibit the growth of most Gram positive bacteria. .
dark colonies with a green metallic sheen. Escherichia coli produce small. Salmonella and Shigella sp produces colorless colonies because it does not ferment lactose. Pseudomonas. Phenylethyl Alcohol Agar: . 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.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. Proteus. Formula: Ingredients per liter of deionized water 4.
Phenylethyl Alcohol (PEA) Agar with or without 5% sheep blood is a selective medium for the isolation of gram-positive organisms. Figure 4: Uninoculated PEA Agar Formula:Ingredients per liter of deionized water 5. particularly gram-positive cocci. 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. thus selecting for Gram positives. Component: Phenylethyl alcohol – Inhibits the growth of Gram negatives since it selectively and reversibly inhibits DNA synthesis. Components: Bile salts: Inhibits the growth of most Gram positive organisms. . from specimens of mixed gram-positive and gram-negative flora.
Ferric ammonium citrate: provides a source of iron that allows the production of hydrogen sulfide from sodium thiosulfate. Salmonella species appear as blue-green colonies with or without black centers. which provides a source of sulfur. Sodium thiosulfate: provides a source of sulfur. Shigella species develop green-colored colonies blue-green centers. allows the visualization of hydrogen sulfide production by reacting with hydrogen sulfide gas to form a black 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 into in the presence of the with bromthymol darker blue indicator. Lactose. sucrose. Formula: Ingredients per liter of deionized water . and salicin: provide fermentable carbohydrates to encourage the growth and differentiation of enterics. This also precipitate. resulting in improved recovery. Producers of H2S will form black-centered colonies in the presence of the ferric ammonium citrate indicator. Bromthymol blue and acid fuchsin dyes: have a lower toxicity than that of many other enteric media.
oxygenstable 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. beta-hemolytic streptococci are small opaque or semi translucent colonies surrounded by clear zones in a red opaque medium.6. Escherichia coli. Different streptococci produce different effects on the red blood cells in blood agar. this type of hemolysis is seen as a distinct greening of the agar in the hemolytic zone. a nonantigenic. Two types of beta lysins are produced: Streptolysin O and Streptolysin S. oxygen-labile enzyme. The blood that is incorporated into this medium is an enrichment ingredient for the cultivation of fastidious organisms such as the Streptococcus species. and thus this group of streptococci has also been referred to as the viridans group. an antigenic. Characteristically. Streptolysin O. Such substances are referred to as hemolysins. When growing on blood agar. A number of streptococcal species produce substances that destroy red blood cells. Those that produce incomplete hemolysis and only partial destruction of the cells around colonies are called alpha-hemolytic Streptococci. Species whose hemolysins cause complete destruction of red cells in the agar zones surrounding their colonies are said to be beta-hemolytic. Blood Agar: Blood agar is both differential and enriched medium. The activity of streptococcal hemolysins also known as streptolysins can be readily observed when the organisms are growing on a blood agar plate. and other bacteria also may show beta-hemolysis. . that is. and streptolysin S. Some strains of Staphylococci. they cause lysis of the red cell wall with subsequent release of hemoglobin.
no change is seen in the red blood cells around them. defibrinated blood.S. S. Mix thoroughly and then dispense into plates while a liquid. epidermidis and S. saprophyticus are almost always nonhemolytic. agar. On blood whereas S.Some species of Streptococci do not produce hemolysins. always reliable characteristic.saprophyticus either a bright yellow or white pigment. Blood agar base for use in making blood agar also can be purchased. 7. always.Chocolate Agar: . However.aureus is usually. aureus usually pigmentation but is not displays not a light a to golden yellow pigment. when their colonies grow on blood agar. On blood agar.epidermidis has a white pigment andS. S. beta-hemolytic. Therefore. 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 50°C and aseptically add 50 ml of sterile. These species are referred to as nonhemolytic or gamma hemolytic streptococci. A combination of hemoglobin and a commercial nutrient supplement can be used in place of defibrinated blood.
Fastidious organisms such as Haemophilus and Neisseria require specially enriched culture media and microaerophilic incubation conditions. so species differentiation among the members of Haemophilus must be performed in another manner. Chocolate agar may be made selective forHaemophilus species by the addition of bacitracin. however. Formula: Ingredients per liter of deionized water. 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. essential in the metabolism of some species that lack it. Figure 7 :Uninoculated Chocolate Agar Plate Two special growth factors. “Chocolate” agar is commonly used for primary isolation of Haemophilus from clinical specimens. The X factor is hemin. are required by some Haemophilus species. a heat-stable derivative of hemoglobin. Yeast extracts contain V factor and are one of the most convenient supplements of chocolate agar or other media used forHaemophilus. The red blood cells in chocolate agar have been heated until they are lysed. called X and V. producing the characteristic brown color of this medium. This medium contains hemoglobin derived from bovine red blood cells as well as other enrichment growth factors. Chocolate agar. The V factor is a heat-labile coenzyme (nicotinamide adenine dinucleotide. does not reveal hemolysis data. . or NAD).
defibrinated sheep blood to the sterile and molten agar.Note: Aseptically add 5% sterile. Heat at 80°C for 15 minutes or until a chocolate color develops. .
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