Culture Media.docx

7/29/2019 Culture Media.docx

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1. Mannitol Salt Agar (MSA):

Mannitol salt agar is both a selective and differential media used for the isolation of

pathogenic Staphylococcifrom 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 mannitolpositive 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


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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

ofStaphylococcus 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.


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Figure2 : MacConkey's Agar

By utilizing the available lactose in the medium, Lac+ (Lactose positive) bacteria such

as Escherichia coli, Enterobacterand 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:



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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.


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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 coliproduce small, dark colonies with a green metallic sheen. Pseudomonas,

Proteus, Salmonella and Shigella sp produces colorless colonies because it does not ferment

lactose.

Formula:


4. Phenylethyl Alcohol Agar:


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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:-


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.


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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-greencenters. 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:



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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 streptococciproduce 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 ofstreptococci 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 ofStaphylococci, Escherichia coli, and other bacteria also

may show beta-hemolysis.


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Some species ofStreptococcido 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 whitepigment. 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:


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 dispenseinto 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:


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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 ofHaemophilus must be performed in another manner.

Formula:

Ingredients per liter of deionized water.


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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.

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Culture Media.docx