Differential, Selective and
Enrichment Media
Microbiology SCI 204 E2
Spring 2011
Maria C. Carles, Ph.D. M(ASCP)
Differential Media
Used distinguish between or among different
microorganisms based on a difference in colony
appearance (color, shape, or growth pattern) on the
medium.
Dyes in the medium (e.g.: eosin/methylene blue in EMB)
pH indicators change the color of the medium as sugars in
the medium are fermented to produce acid products
Example:
Lactose in EMB & MacConkey's
Mannitol in MSA
Examples of Differential Media
Mannitol Salt Agar
MacConkey Agar
EMB
Selective Media
Allows the growth of certain types of organisms,
while inhibiting the growth of other organisms
Dyes in the medium (e.g.: methylene blue in EMB
& crystal violet in MacConkey's)
High salt concentration in the medium (e.g.: 7%
salt in MSA) inhibit the growth of unwanted
microorganisms
Why to use Selective Media?
1. Microbes are found mostly as MIXED
CULTURES
2. Microbes must be isolated and cultivated
as PURE CULTURES to be tested and
identified
Examples of Selective Media
MacConkey
Mannitol Salt Agar
Enrichment Media
Media which promotes the growth of a particular
organism, sometimes termed fastidious organisms,
that require specialized environments due to their
complex nutritional requirements
It provides essential nutrients required for growth of a
particular organism
It may contain inhibitory substances to prevent 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
Types of Nutrient media
dispensed into tubes
• Nutrient agar slant (S)
• Nutrient broth (B)
• Nutrient agar deep (D)
S B D
Other Examples of Enrichment
Media
Selenite F broth
Favours growth of Salmonella
Prevents growth of normal competitors (i.e.
Eschrichia coli.
Others like Blood Agar and Chocolate 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
Mannitol Salt Agar
•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 Agar
High salt concentration favors growth of
Staphylococci
Staphylococci colonies are reddish or pink
without altering the color of the medium
Staplylococcus aureus ferments mannitol
changing the color of the media from red to
yellow
Mannitol Salt Agar
Contains the carbohydrate Mannitol, 7.5%
NaCl, and a pH indicator; Phenol Red
Phenol Red is:
Yellow at pH below 6.8
Red at pH 7.4 – 8.4
Pink at pH 8.4 and above
Mannitol Salt Agar Interpretation
Growth, colonies surrounded by yellow
halo: usually S. aureus
Growth, no color change around colonies:
S. epidermidis
Growth, large, white to orange colonies:
Micrococcus sp.
No growth or partial inhibition:
Streptococci, Gram-negative bacteria
Phenyl Alcohol Agar
Selective media that allows growth of Gram
positive organisms and stops or inhibits
growth of most Gram negative organisms.
Active ingredient is phenylethyl alcohol
Interferes with DNA synthesis in Gram
negative organisms
MacConkey agar
Widely-used culture medium which is both
selective AND differential.
The medium is primarily used to differentiate
between Gram negative bacteria while inhibiting
the growth of most Gram positive bacteria.
The medium also differentiates between lactose-
fermenting coliforms and lactose non-fermenters,
which include potential pathogens
MacConkey agar - Continuation
Addition to the nutrient agar base of bile salts and
crystal violet will inhibit the growth of most Gram
positive bacteria, making MacConkey agar
selective.
MacConkey agar - Continuation
Lactose, a fermentable carbohydrate, and neutral red,
a pH indicator, are added to differentiate the lactose
positive coliforms from the potentially pathogenic
lactose nonfermenters.
When lactose is fermented, acid products lower the
pH below 6.8, with the resulting colonial growth
turning pinkish-red.
If an organism is unable to ferment lactose, the
colonies will be colorless.
Blood Agar
Used both as an enriched medium for growing
fastidious bacteria and as a differential medium
(hemolysis)
Contains 5% sheep's blood
Based on the ability to break down hemoglobin or
red blood cells (hemolysins)
Hemolysins
Exotoxins called hemolysins cause lysis of the red
blood cells.
The degree of the hemolysis is an especially useful
tool for identification of many of the Gram
positive cocci.
Particularly useful for identification of clinical
specimens of streptococci.
Hemolysins are grouped in three
categories
Alpha-hemolysins:
produces a greenish discoloration of the blood agar around
the colonies
Partial break downof RBCs and hemoglobin leaving a
green pigment (biliverdin)
Beta-hemolysins: Production of "beta-hemolysin"
(streptolysin O or S)
Completely lyses the red blood cells and hemoglobin;
Complete clearing around colonies.
Gamma-hemolysins (no hemolysis): no hemolysis is
observed
Bacteria do not produce a hemolysins
Eosin Methylene Blue Agar
(EMB)
Selective for gram-negative bacteria
Contains methylene blue and eosin dyes to inhibit the growth of
Gram-positive bacteria (e.g.: Staphylococcus aureus).
Differential for lactose fermentation
Differentiates between: lactose-fermenting coliforms and nonlactose-
fermenting coliforms
Contains lactose. Small amounts of acid production result in a pink
colored growth, while large amounts of acid cause the acid to
precipitate on the colony, resulting in a characteristic greenish,
metallic sheen.
Eosin Methylene Blue Agar
(EMB) - Continuation
The differential basis of this medium involves two
indicator dyes Eosin and methylene blue
That distinguish between lactose fermenting and
non-lactose fermenting organisms.
Lactose fermenters form colonies with dark
centers and clear borders
Non-lactose fermenters form completely
coloroless colonies.
Eosin Methylene Blue Agar
(EMB) - Continuation
This medium has been widely used in the past to screen
for coliforms in the water.
Gram-negative Enterobacteria Escherichia coli and
Enterobacter aerogenes ferment lactose
E. coli produces colonies with a characteristic green
metallic sheen on EMB agar
E. aerogenes produces pink colonies often with a central
dark purple dot (fish eye colonies) on EMB agar
Gram-negative bacteria Proteus vulgaris and Salmonella
typhimurium grow on EMB agar, but do not ferment lactose
