Differential, Selective Differential, Selective and Enrichment and Enrichment

MediaMedia

Microbiology SCI 204 E2Microbiology SCI 204 E2Spring 2011Spring 2011

Maria C. Carles, Ph.D. M(ASCP)Maria C. Carles, Ph.D. M(ASCP)

Differential MediaDifferential Media

Used distinguish between or among different Used distinguish between or among different microorganisms based on a difference in colony microorganisms based on a difference in colony appearance (color, shape, or growth pattern) on appearance (color, shape, or growth pattern) on the medium. the medium.

Dyes in the medium (e.g.: eosin/methylene blue Dyes in the medium (e.g.: eosin/methylene blue in EMB)in EMB)

pH indicators change the color of the medium as pH indicators change the color of the medium as sugars in the medium are fermented to produce sugars in the medium are fermented to produce acid products  acid products  

Example:Example:Lactose in EMB & MacConkey's Lactose in EMB & MacConkey's Mannitol in MSAMannitol in MSA

Examples of Differential Examples of Differential MediaMedia

Mannitol Salt AgarMannitol Salt Agar

MacConkey AgarMacConkey Agar

EMBEMB

Selective MediaSelective Media

Allows the growth of certain types of Allows the growth of certain types of organisms, while inhibiting the growth of organisms, while inhibiting the growth of other organismsother organisms

Dyes in the medium (e.g.: methylene Dyes in the medium (e.g.: methylene blue in EMB & crystal violet in blue in EMB & crystal violet in MacConkey's) MacConkey's)

High salt concentration in the medium High salt concentration in the medium (e.g.: 7% salt in MSA) inhibit the growth (e.g.: 7% salt in MSA) inhibit the growth of unwanted microorganismsof unwanted microorganisms

Why to use Selective Media?1. Microbes are found mostly as

MIXED CULTURES2. Microbes must be isolated and

cultivated as PURE CULTURES to be tested and identified

Examples of Selective Examples of Selective MediaMedia

MacConkeyMacConkey

Mannitol Salt AgarMannitol Salt Agar

Enrichment MediaEnrichment Media

Media which promotes the growth of a Media which promotes the growth of a particular organism, sometimes termed particular organism, sometimes termed fastidious organismsfastidious organisms, that require , that require specialized environments due to their specialized environments due to their complex nutritional requirementscomplex nutritional requirements

It provides essential nutrients required for It provides essential nutrients required for growth of a particular organismgrowth of a particular organism

It may contain inhibitory substances to It may contain inhibitory substances to prevent the growth of normal competitorsprevent the growth of normal competitors

Enrichment Media

• Examples of basic growth media for bacteria are: • Nutrient Broth (NB)• Nutrient Agar (NA)• Tryptic Soy Broth (TSB)• Tryptic Soy Agar (TSA)

• These preparations may be made in various forms: • Plates• Broth• Slant• Deep

S B D

Types of Nutrient media dispensed into tubes

• Nutrient agar slant (S)

• Nutrient broth (B)

• Nutrient agar deep (D)

Other Examples of Other Examples of Enrichment MediaEnrichment Media

Selenite F brothSelenite F broth

Favours growth of Favours growth of SalmonellaSalmonella

Prevents growth of normal competitors Prevents growth of normal competitors (i.e. (i.e. Eschrichia coliEschrichia coli..

Others like Blood Agar and Chocolate Others like Blood Agar and Chocolate Agar.Agar.

Selective & Differential Media

• Media designed to enhance the isolation procedure by inhibiting growth of certain organisms, while encouraging the growth of others

• Contain indicators to expose differences between organisms

Selective & Differential Media

• To isolate pathogenic bacteria• Gram positive cocci • Gram negative bacilli

• From human and environmental samples containing a mixture of microorganisms

•Selective and differential medium •Isolation of staphylococci from clinical and nonclinical materials.•Nutritive medium that contains peptones and beef extract (essential growth factors, such as nitrogen, carbon, sulfur and trace nutrients).•Contains 7.5% sodium chloride - partial or complete inhibition of bacterial organisms other than staphylococci.

Mannitol Salt AgarMannitol Salt Agar

Mannitol Salt AgarMannitol Salt Agar

High salt concentration High salt concentration favors favors growth of Staphylococcigrowth of Staphylococci

Staphylococci colonies are reddish Staphylococci colonies are reddish or pink without altering the color of or pink without altering the color of the mediumthe medium

Staplylococcus aureus Staplylococcus aureus ferments ferments mannitol mannitol changing the color of changing the color of the media from red to yellowthe media from red to yellow

Mannitol Salt AgarMannitol Salt Agar

Contains the carbohydrate Contains the carbohydrate Mannitol, Mannitol, 7.5% NaCl7.5% NaCl, and a pH , and a pH indicator; Phenol Redindicator; Phenol Red

Phenol Red is:Phenol Red is:

Yellow Yellow at pH below 6.8at pH below 6.8

Red Red at pH 7.4 – 8.4 at pH 7.4 – 8.4

PinkPink at pH 8.4 and aboveat pH 8.4 and above

Growth, colonies surrounded by yellow halo: usually S. aureusGrowth, no color change around colonies: S. epidermidis Growth, large, white to orange colonies: Micrococcus sp.No growth or partial inhibition: Streptococci, Gram-negative bacteria

Mannitol Salt Agar Interpretation

Phenyl Alcohol AgarPhenyl Alcohol Agar

Selective media that allows growth Selective media that allows growth of Gram positive organisms and of Gram positive organisms and stops or inhibits growth of most stops or inhibits growth of most Gram negative organisms.Gram negative organisms.

Active ingredient is phenylethyl Active ingredient is phenylethyl alcoholalcohol

Interferes with DNA synthesis in Interferes with DNA synthesis in Gram negative organismsGram negative organisms

MacConkey agar MacConkey agar

Widely-used culture medium which is Widely-used culture medium which is both both selectiveselective AND AND differentialdifferential. .

The medium is primarily used to The medium is primarily used to differentiate between Gram negative differentiate between Gram negative bacteria while inhibiting the growth of bacteria while inhibiting the growth of most Gram positive bacteria. most Gram positive bacteria.

The medium also differentiates between The medium also differentiates between lactose-fermenting coliforms and lactose lactose-fermenting coliforms and lactose non-fermenters, which include potential non-fermenters, which include potential pathogenspathogens

MacConkey agar - MacConkey agar - ContinuationContinuation

Addition to the nutrient agar base of Addition to the nutrient agar base of bile salts and crystal violet will inhibit bile salts and crystal violet will inhibit the growth of most Gram positive the growth of most Gram positive bacteria, making MacConkey agar bacteria, making MacConkey agar selectiveselective. .

MacConkey agar - MacConkey agar - ContinuationContinuation

Lactose, a fermentable carbohydrate, and Lactose, a fermentable carbohydrate, and neutral red, a pH indicator, are added to neutral red, a pH indicator, are added to differentiatedifferentiate the lactose positive coliforms the lactose positive coliforms from the potentially pathogenic lactose from the potentially pathogenic lactose nonfermenters.nonfermenters.

When lactose is fermented, acid products When lactose is fermented, acid products lower the pH below 6.8, with the resulting lower the pH below 6.8, with the resulting colonial growth turning pinkish-red. colonial growth turning pinkish-red.

If an organism is unable to ferment lactose, If an organism is unable to ferment lactose, the colonies will be colorless. the colonies will be colorless.

Blood Agar Blood Agar

Used both as an enriched medium for Used both as an enriched medium for growing fastidious bacteria and as a growing fastidious bacteria and as a differential medium (differential medium (hemolysis)hemolysis)

Contains 5% sheep's bloodContains 5% sheep's blood

Based on the ability to break down Based on the ability to break down hemoglobin or red blood cells hemoglobin or red blood cells (hemolysins)(hemolysins)

HemolysinsHemolysins

Exotoxins called hemolysins cause lysis Exotoxins called hemolysins cause lysis of the red blood cells. of the red blood cells.

The degree of the hemolysis is an The degree of the hemolysis is an especially useful tool for identification of especially useful tool for identification of many of the Gram positive cocci.many of the Gram positive cocci.

PPaarticularly useful for identification of rticularly useful for identification of clinical specimens of streptococci.clinical specimens of streptococci.

Hemolysins are grouped in Hemolysins are grouped in three categoriesthree categories

AAlpha-hemolysinslpha-hemolysins::produces a produces a greenisgreenish discoloration of the blood h discoloration of the blood agar around the coloniesagar around the coloniesPartial break downof RBCs and hemoglobin Partial break downof RBCs and hemoglobin leaving a green pigment (biliverdin)leaving a green pigment (biliverdin)

BBeta-hemolysinseta-hemolysins: Production of "beta-: Production of "beta-hemolysin" (streptolysin O or S)hemolysin" (streptolysin O or S)Completely lyses the red blood cells and Completely lyses the red blood cells and hemoglobin;hemoglobin;Complete Complete clearingclearing around colonies. around colonies.

GGamma-hemolysins (no hemolysis)amma-hemolysins (no hemolysis): no : no hemolysis is observedhemolysis is observed

Bacteria do not produce a hemolysinsBacteria do not produce a hemolysins

Eosin Methylene Blue Agar Eosin Methylene Blue Agar (EMB)(EMB)

Selective forSelective for gram-negative bacteriagram-negative bacteria

Contains methylene blue and eosin dyes to inhibit the Contains methylene blue and eosin dyes to inhibit the growth of Gram-positive bacteria (e.g.: growth of Gram-positive bacteria (e.g.: Staphylococcus aureusStaphylococcus aureus). ).

DDifferential forifferential for lactose fermentationlactose fermentation

Differentiates betweenDifferentiates between: lactose-fermenting coliforms : lactose-fermenting coliforms and nonlactose-fermenting coliforms and nonlactose-fermenting coliforms

Contains lactose. Small amounts of acid production Contains lactose. Small amounts of acid production result in a pink colored growth, while large amounts of result in a pink colored growth, while large amounts of acid cause the acid to precipitate on the colony, acid cause the acid to precipitate on the colony, resulting in a characteristic greenish, metallic sheen. resulting in a characteristic greenish, metallic sheen.

Eosin Methylene Blue Agar Eosin Methylene Blue Agar (EMB) - Continuation(EMB) - Continuation

The differential basis of this medium The differential basis of this medium involves two indicator dyes Eosin and involves two indicator dyes Eosin and methylene bluemethylene blue

That distinguish between lactose That distinguish between lactose fermenting and non-lactose fermenting fermenting and non-lactose fermenting organisms. organisms.

Lactose fermenters form colonies with dark Lactose fermenters form colonies with dark centers and clear borders centers and clear borders

Non-lactose fermenters form completely Non-lactose fermenters form completely coloroless colonies. coloroless colonies.

Eosin Methylene Blue Agar Eosin Methylene Blue Agar (EMB) - Continuation(EMB) - Continuation

This medium has been widely used in the This medium has been widely used in the past to screen for coliforms in the water.past to screen for coliforms in the water.

Gram-negative Enterobacteria Gram-negative Enterobacteria Escherichia coliEscherichia coli and and Enterobacter aerogenesEnterobacter aerogenes ferment lactose ferment lactoseE. coliE. coli produces colonies with a characteristic produces colonies with a characteristic green metallic sheen on EMB agargreen metallic sheen on EMB agarE. aerogenesE. aerogenes produces pink colonies often with produces pink colonies often with a central dark purple dot (fish eye colonies) on a central dark purple dot (fish eye colonies) on EMB agar  EMB agar  

Gram-negative bacteria Gram-negative bacteria Proteus vulgarisProteus vulgaris and and Salmonella typhimuriumSalmonella typhimurium grow on EMB agar, but grow on EMB agar, but do not ferment lactose do not ferment lactose