1. Isolation and Culturing of Bacteria
September 9, 2014

2. 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

3. Criteria for Culture Media
Must contain the necessary nutrients to promote growth
Incubated at optimal temperature
Sterility

4. Agar can be added to broth to produce solid media
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

5. Culture Media

6. 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

7. Table 6.2 A Chemically Defined Medium for Growing a Typical Chemoheterotroph, Such as Escherichia coli

8. Table 6.4 Composition of Nutrient Agar, a Complex Medium for the Growth of Heterotrophic Bacteria

9. 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)

10. Envelope containing sodium bicarbonate and sodium borohydride
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
CO2 H2
Palladium catalyst pellets
Anaerobic indicator (methylene blue)
Petri plates

11. Figure 6.7 An anaerobic chamber.
Air lock
Arm ports

12. Capnophiles Microbes that require high CO2 conditions CO2 packet
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

13. 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

14. 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

15. EMB plate
E. coli colonies on an EMB plate

16. 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

17. 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

18. 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

19. Uninoculated Staphylococcus epidermis Staphylococcus aureus
Figure 6.10 Differential and Selective medium.
Uninoculated
Staphylococcus
epidermis
Staphylococcus
aureus