Transcript Selective and Differential media

General Microbiology Laboratory
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 (TSA & TSB)
Enriched: extra growth factors & nutrients
allow growth of certain bacteria
Organisms requiring enrichment:
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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 potassium tellurite, sodium azide or thallium
acetate (at concentrations of 0.1 - 0.5 g/l) 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 Gram-positive
bacteria.
• Tellurite agar, therefore, is used to select for Gram-positive
organisms, and nutrient agar supplemented with penicillin can
be used to select for Gram negative organisms.
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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)
• Select for salt-tolerant bacteria
• Usually Gram positive bacteria
 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.
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Ferment mannitol (differential):
– Phenol red = pH indicator
• Red: pH >7.4 (basic)
• Pink: 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. epidermidis
M. luteus
1. S. aureus = salt tolerant; ferments mannitol
(yellow)
2. S. epidermidis = salt tolerant; does NOT
ferment (red)
3. M. luteus = not salt tolerant; does NOT grow
on mannitol
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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EMB agar
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MacConkey AGAR
 Used to isolate Gram negative enterics (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 noncolored and transparent.
 (Note: The Gram positive bacteria do not die on this media, their growth is just
inhibited)
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MacConkey AGAR
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MAC agar
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Differential media: 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)…hemolice-ins) 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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Procedure
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1. Cultures of: Staphylococcus aureus
Staphylococcus epidermidis
Escherichia coli
Enterobacter aerogenes
Salmonella enteritidis
2. Petri dishes of Mannitol Salt agar, EMB agar, MacConkey’s agar and nutrient
agar
Procedure:
1. Divide each plate of MSA and MacConkey’s agar into two sections. Divide
the
EMB agar plate into three sections.
2. Inoculate the MSA plate with S. aureus and S. epidermidis.
3. Inoculate MacConkey’s agar with E. coli and Salmonella enteritidis.
4. Inoculate the 3-sectional EMB agar plate with E. coli, Enterobacter
aerogenes, and Salmonella enteritidis.
5. Divide one plate of nutrient agar into two sections and one into three
sections. Inoculate the 2-sectional plate with the two species of Staphylococcus;
inoculate the 3-sectional plate with the remaining three organisms.
6. Incubate all plates at 37°C for 48 hours.
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Observe the growth and appearance of
colonies on all plates.
Notice that nutrient agar is neither a selective
nor differential medium.
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End of lecture
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