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

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.

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

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.

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

Fig 2.2

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.

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

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

EMB MacConkey

MSA Compare to Fig 6.10

Fig 2.6 Can a medium be both?

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

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!

Fig. 2.9

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