1. Tools of the Laboratory: Methods for Culturing of Microorganisms
Ch 2.1
Tools of the Laboratory:
Methods for Culturing of Microorganisms

2. SLOs for Culturing of Microorganisms
Explain what the Five I’s are and what each step entails.
Discuss three physical states of media and when each is used.
Compare and contrast selective and differential media, and give an example of each.
Provide brief definitions for defined media and complex media.

3. How to Culture Microorganisms: The Five I’s
Inoculation
Incubation
Isolation
Inspection
Identification

4. 1) Inoculation Culturing: Growing microorganisms
Medium (plural, media): Provides nutrients for the growth of microbes
Inoculum: Small sample of microbes that is __________
Inoculation: the introduction of an inoculum into media to culture microbes
Clinical specimens are obtained from body fluids, discharge, diseased tissue, etc.

5. 2) Incubation
Incubator: Chamber for ideal growth temperature and gas composition.
Minimum, optimum, and maximum incubation temperatures?
Incubation period?
Goal: Visible growth in media.

6. Fig 2.2

7. Culture Media Contained in:
Test tubes
Flasks
Petri dishes = agar plates
Inoculated with loops, needles, pipettes, or swabs
Use sterile (aseptic) technique!!
3 physical state:
Liquid
Semisolid
solid
Agar:
Complex polysaccharide from red algae.
Liquefies at 100C
Solidifies at 42C
Not digested by most microbes.

8. Chemical Content of Media
Chemically defined or synthetic media: Exact chemical composition is known (for research purposes only)
Complex media: Extracts and digests of yeasts, meat, or plants, e.g.:
Nutrient broth
Nutrient agar
TSA and TSB
Blood agar
Compare to Table 2.2A
Nutrient Agar

9. Media for Different Purposes
General purpose media
Enriched media for fastidious organisms
Selective media suppress unwanted and encourage desired microbes – e.g. EMB and mannitol salt agar etc.
Important in the 1 isolation of a specific microbe from a sample containing many different species
Differential media are changed in recognizable manner by some bacteria
 Make it easy to distinguish colonies of different microbes.
MacConkey agar
EMB
Mannitol salt agar
 and  hemolysis on blood agar

10. EMB
MacConkey

11. MSA
Compare to Fig 6.10

12. Fig 2.6
Can a medium be both?

13. Miscellaneous Media Reducing medium:
Contains a substance that absorbs oxygen or slows the penetration of oxygen
For culturing anaerobic bacteria
Carbohydrate fermentation media:
Contains sugars that can be fermented with pH indicator to show this reaction
Transport media:
Used to maintain and preserve specimens that have to be held for a period of time before clinical analysis

14. 3) Isolation
Goal is to separate one species from another and gain a pure culture, containing only one species or strain.
Most patient specimens and environmental samples contain several different kinds of microbes
Streak-plate method most commonly used.
Spread-plate method
Colony formation: A population of cells arising from a single cell or spore or from a group of attached cells (also referred to as CFU).
Only ~1% of all bacteria can be successfully cultured
Aseptic technique critical!

15. Fig. 2.9

16. 4 & 5) Inspection and Identification
Microscopic appearance
Biochemical testing
Characterization of cellular metabolism through determining nutrient requirements, products given off during growth, presence of enzymes, and mechanisms for deriving energy
Genetic and immunologic testing
The end