1. Mannitol Salt Agar (MSA): Mannitol salt agar is both a selective and differential media used for the isolation of pathogenic Staphylococci from Staphylococci from mixed cultures.
Components:
7.5% NaCl – selects for species of Staphylococcus S taphylococcus.. This concentration of salt is too high for most other bacteria 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 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 bacteria
is
usually
inhibited.
This
growth
differentiates S.aureus from S.epidermidis, S.epidermidis, which forms colonies with red zones or both zones.
Formula: Ingredients per liter of deionized water
2. MacConkey’s Agar (MAC): MacConkey’s 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 rose
in in
* tan in alkaline pH
Peptone - a source of proteins, amino acids for microbial growth.
acid neutral
pH pH
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
differential medium used for the
detection and isolation of Gram-negative 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
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
indicator. Shigella species
colonies
develop
into
in
the
presence
green-colored
of
colonies
the with
bromthymol darker
blue
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 streptoc occi 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, 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. 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 and S.saprophyticus either a bright yellow or white pigment.
However,
agar,S.aureus is
pigmentation
usually,
but
is not
not
always
always,
a
reliable
characteristic.
beta-hemolytic; S.
On
blood
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 50°C 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 for Haemophilus. 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 m olten agar. Heat at 80°C for 15 minutes or until a chocolate color develops.