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