isolation and culturing of bacteria september 9, 2014
TRANSCRIPT
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.
Lid with O-ring gasket
Envelope containing sodium bicarbonate and sodium borohydride
Anaerobic indicator (methylene blue)
Petri plates
Clamp with clamp screw
Palladium catalyst pellets
CO2H2
Figure 6.7 An anaerobic chamber.
Arm ports
Air lock
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
• 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 and Differential 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
Selective Media
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
Staphylococcusepidermis
Staphylococcusaureus