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Selective and Differential Media
Media for Isolation of Microbes
• General (all purpose): contains basic nutrients for most bacteria
• Enriched: contains extra growth factors & nutrients (Fastidius
organism)
• Selective: contains ingredients that inhibit growth of some
bacteria & allow growth of others
• Differential: contain indicators that change appearance of media
in response to differential use of an ingredient
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General & Enriched Media
• General (all purpose): contains basic nutrients for most
bacteria.
• Enriched: extra growth factors & nutrients allow growth of
certain bacteria
• Organisms requiring enrichment:
• Acid Fast bacteria
• Spirochetes
• Chlamydia
• Viruses
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Selective & Differential media
• Selective and differential media are used to isolate or identify
particular organisms.
• Selective media allow certain types of organisms to grow, and
inhibit the growth of other organisms. The selectivity is
accomplished in several ways:
• For example, organisms that can utilize a given sugar are easily
screened by making that sugar the only carbon source in the
medium.
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• On the other hand, selective inhibition of some types of
microorganisms can be achieved by adding dyes,
antibiotics, salts or specific inhibitors which affect the
metabolism or enzyme systems of the organisms. For
example, media containing sodium azide will inhibit the
growth of Gram-negative bacteria.
• Media supplemented with penicillin (5-50 units/ml) or
crystal violet (2 mg/l) will inhibit the growth of Grampositive bacteria.
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Differential media
• Differential media does not necessarily inhibit bacterial growth,
but instead makes the bacteria look different
• Differential media works best with closely related organisms, and
the differential agent is what causes the bacteria look different
• Owing to the presence of certain dyes or chemicals in the media,
the organisms will produce characteristic changes or growth
patterns that are used for identification or differentiation.
• A variety of selective and differential media are used in medical,
diagnostic and water pollution laboratories, and in food and dairy
laboratories.
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• Some media are both selective and differential, that is, they are
able to select against the growth of certain organisms while the
organisms that do grow may exhibit some differential growth
characteristics.
• Three of the more common selective and differential media are
described below and will be used in the laboratory exercise.
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Mannitol Salt Agar (MSA)
• Mannitol salt agar is a selective medium used for the isolation
of pathogenic staphylococci.
• The medium contains mannitol, a phenol red indicator, and
7.5% sodium chloride.
Ferment mannitol (differential):
• Phenol red = pH indicator
• Pink : pH >7.4 (basic)
• Orange: pH = 7 (neutral)
• Yellow: pH <6.8 (acidic)
• Selective: The high salt concentration inhibits the growth of
most bacteria other than staphylococci.
Note: The high salt content does not kill Gram negative bacteria, it
just inhibits growth.
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• On MSA, pathogenic Staphylococcus aureus
produces small colonies surrounded by yellow
zones. The reason for this change in color is that
S. aureus ferments the mannitol, producing an
acid, which, in turn, changes the indicator from
red to yellow.
• Other Staphylococcus don’t ferment mannitol
don’t produce a color change from the normal
red-pink color of the medium.
• The growth of other types of bacteria is
generally inhibited.
• S. aureus = salt tolerant; ferments mannitol
(yellow)
• S. epidermidis = salt tolerant; does NOT ferment
(red)
• M. luteus = not salt tolerant; does NOT grow on
mannitol
S. epidermidis
M. luteus
S. aureus
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Eosin Methylene Blue Agar (EMB agar)
• EMB is an undefined selective/differential medium. It contains
aniline dyes (methylene blue and eosin), which inhibit the growth
of Gram-positive bacteria selecting for Gram-negative bacteria.
•
EMB also contains lactose which makes the media differential
based on an organisms ability to ferment lactose.
• Sucrose is also included in the medium because certain members
of the Enterobacteria or coliform group ferment sucrose more
readily than they ferment lactose. These sugars provide favorable
conditions for the growth of fecal coliforms.
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• Lactose and sucrose fermenters will grow as dark colonies
accompanied by a metallic green sheen (E. coli).
• Organisms that slowly ferment lactose will appear as pink
colonies (Enterobacter aerogenes are usually mucoid and much
larger than colonies of E. coli).
• Non-fermenters lactose or sucrose will remain colorless or take
on the color of the medium such as Salmonella (one of the
causative agents of food poisoning).
• Note: This media is used to confirm the presence of E. coli in
water samples contaminated with sewage or fecal material. You
will use this agar again with in the water sampling experiment to
differentiate between E. coli and Enterobacter spp]
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The
dark
colonies
produced on EMB agar
is a result of the acid
produced during lactose
or sucrose fermentation
precipitating the dyes in
the media.
K. pneumoniae produces
dark colonies on EMB
agar.
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MacConkey Agar
• Used to isolate Gram negative enteric (coliforms)
• Selective and differential medium.
• Selective - Gram positive bacteria are inhibited by the presence of bile salts
and crystal violet inhibitors in the medium. Most of gram negative bacteria will
grow.
• Differentiate- Between Gram negative bacteria by their ability to ferment
lactose.
• Pink colonies- Bacteria that ferment lactose ( These reactions are due to the
acid produced by the fermentation of lactose. The acid end-products act on bile
salts, and neutral red is absorbed by the precipitated salts. ).
• Pale colonies- Non fermenters are no colored and transparent.
• (Note: The Gram positive bacteria do not die on this media, their growth is just
inhibited)
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Blood agar
• Nutrient agar with 5% sheep blood
• Cultivation of fastidious and non fastidious bacteria.
• This media is differential because:
• Certain bacteria produce enzymes hemolysins (exotoxin) that act
on the red cells to produce either:
• Beta hemolysis: Enzymes lyse the blood cells completely,
producing a clear area around the colony.
• Alpha hemolysis: Incomplete hemolysis produces a greenish
discoloration around the colony.
• Gamma hemolysis: No effect on the red cells.
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The End
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