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Isolation and Culturing of
Bacteria
September 9, 2014
Culture Media
• Defined as the nutrient material prepared for
the growth of organisms in the lab
– in vivo—growth inside normal biological context
– in vitro—growth outside their biological context
• Inoculum is the microorganisms that are
introduced to the media to initiate growth
Criteria for Culture Media
1. Must contain the necessary nutrients to
promote growth
2. Incubated at optimal temperature
3. Sterility
• Broth is liquid media
• Agar can be added to broth to produce solid
media
– Complex polysaccharide derived from marine
algae
– Liquefies at 100°C and becomes solid at 40°C
– Not metabolized by microbes
– Useful in make petri dishes, slants, and deeps
Culture Media
Culture Media
• There are several different varieties of culture
media
– Chemically defined media—exact chemical
composition is known
– Chemically undefined media—exact chemical
composition is not known
• Complex media contain extracts and digests of yeasts,
meat, or plants
– Nutrient broth
– Nutrient agar
Table 6.2 A Chemically Defined Medium for Growing a Typical Chemoheterotroph, Such as Escherichia coli
Table 6.4 Composition of Nutrient Agar, a Complex Medium for the Growth of Heterotrophic Bacteria
Anaerobic Culture Methods
• Cultivation of anaerobic bacteria poses a problem
to scientists
– Must absorb all the oxygen from
medium/environment in order to grow microbes
• Reducing media
– Contain chemicals (sodium thioglycolate or oxyrase)
that combine O2 and removes all available oxygen
– Usually contained in screw cap test tubes or jars
– Media is heated before use to drive off O2 (screw cap
tube)
Figure 6.6 A jar for cultivating anaerobic bacteria on Petri plates.
Clamp with
clamp screw
Lid with
O-ring gasket
Envelope containing
sodium bicarbonate and
sodium borohydride
Anaerobic indicator
(methylene blue)
Petri plates
CO2
H2
Palladium
catalyst pellets
Figure 6.7 An anaerobic chamber.
Air
lock
Arm
ports
Capnophiles
• Microbes that require high CO2 conditions
– Camplyobacter spp.
• CO2 packet
– Chemical packets are used to generate carbon dioxide
within containers
• Candle jar
– Contains a lit candle that depletes the oxygen in an
environment and generates carbon dioxide
• Low-oxygen, high-CO2 conditions resemble the
conditions of the intestinal and respiratory tract
Selective and Differential Media
• In clinics and public health microbiology, it is
necessary to identify specific microbes to
distinguish colonies of different microbes
– Identifying the microbes that cause disease
• Clinicians use both selective and differential
media to accomplish this task
Selective Media
• Selective media contains compounds that
inhibit the growth of certain bacteria while
promoting the growth of others
– Eosin-methylene blue (EMB) plates are an
example of a selective media
• Inhibits the growth of gram positive bacteria
EMB plate
E. coli colonies on an EMB plate
Differential Media
• Differential Media grows a variety of different
organisms. Various organisms produce
different products that affects the appearance
of the microbe on the media
– Blood Agar Plate
Blood Agar Plate
• Erythrocytes are
incorporated into nutrient
agar medium
• Certain bacteria produce
products that lyse Red
Blood Cells
– Alpha-hemolytic- partial
lysis
– Beta-hemolytic- complete
lysis
– Gamma-hemolytic- no
lysis
Selective and Differential Media
• Some culture plates are both Selective AND Differential
• Mannitol Salt Agar (MSA)
– High salt concentration (7.5%) only permits
Staphylococcous spp. growth
– Mannitol is a sugar alcohol that is fermented by certain
species
– If mannitol is fermented, acidic products are formed.
Indicated by phenol red (yellow is acidic)
• Pathogenic Staph will ferment mannitol
• Non-pathogenic Staph will not ferment mannitol
Figure 6.10 Differential and Selective medium.
Uninoculated
Staphylococcus
epidermis
Staphylococcus
aureus