Objective:

 

To distinguish the growth characteristics of microorganisms in various differential, selective and enriched media.

 

Principle:

 

Much of the study of  microorganisms depends on its ability to grow  in the laboratory, and this is possible only if suitable culture media are available for the growth of microorganism. A culture medium is defined as a solid or liquid preparation used for the growth, transport, and storage of microorganisms. The effective culture medium must contain all the nutrients required for the growth of the microorganism.

Specialized media are widely employed for  the isolation and identification of microorganisms,  testing the antibiotic sensitivities, analysis of water and food, industrial microbiology, and other activities. Although all microorganisms need sources of energy, nitrogen, carbon, phosphorus, sulfur, and various minerals, the exact composition of a satisfactory medium will rely on the species one is trying to identify and cultivate because nutritional requirements vary so greatly among the microorganisms.

Knowledge of  microorganism’s normal habitat is often useful in selecting a suitable culture medium because its nutrient requirements reflect its natural surroundings.  A medium is used to select and growing specific microorganisms or to help identifying a particular species. In these cases, the function of the medium also  depends on  its composition. In addition to nutrients necessary for the growth of all bacteria, special-purpose media contain one or more chemical compounds that are essential for their functional specificity.

 

These include: Selective, Differential and Enriched Media.

 

Selective media:

 

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 5-50 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

 

Differential media:

 

Differential media are widely used for  differentiating closely related organisms or groups of organisms. Because of the  presence of certain dyes or chemicals in the media, the organisms will produce certain characteristic changes or growth patterns that are used for identification or differentiation of microorganism.

Eg., Mac Conkey (MCK) agar,  Eosin Methylene Blue (EMB) agar

 

Enriched media:

 

Enriched media are media that have been supplemented with highly nutritious materials such as blood, serum or yeast extract for the purpose of cultivating fastidious organisms.

Eg., Blood agar, Chocolate agar

 

Some of the special-purpose media are as follows:

 

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

 

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 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  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 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 and S.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 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 for Haemophilus 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 molten agar. Heat at 80°C for 15 minutes or until a chocolate color develops.