Microbiology: Culturing Microorganisms

Importance of Media for Culturing Microbes

• The primary objective of cultivating microorganisms is to maintain viable populations of these organisms under controlled laboratory environments.
• Cultivating microorganisms is a challenging process due to their highly specific nutritional and environmental requirements and the diversity of these requirements among different species.

Nutritional Requirements of Microbes

• All-purpose medium: Tryptic Soy Broth (TSB)
• Specialized media: used in the identification of bacteria and are supplemented with dyes, pH indicators, or antibiotics
• Enriched media: contains growth factors, vitamins, and other essential nutrients to promote the growth of fastidious organisms
• Chemically defined medium: complete chemical composition of a medium is known (e.g. EZ medium)
• Complex media: contain extracts and digests of yeasts, meat, or plants, and the precise chemical composition is not known (e.g. nutrient broth, TSB, Brain Heart Infusion (BHI))

Types of Media

• Selective medium: inhibits the growth of unwanted microorganisms and supports the growth of the organism of interest (e.g. MacConkey agar)
• Enrichment medium: general purpose enrichment agar that can nourish and support the growth of gram-positive as well as gram-negative bacteria (e.g. blood agar)
• Differential medium: distinguishes between different microorganisms based on their metabolic characteristics (e.g. Eosin Methylene Blue (EMB) agar)

MacConkey Agar

• Contains bile salts and crystal violet, which interfere with the growth of many gram-positive bacteria and favor the growth of gram-negative bacteria, particularly the Enterobacteriaceae
• Lactose-fermenting organisms (e.g. E. coli) grow as pink to red colonies with or without a zone of precipitated bile
• Lactose-non-fermenting organisms (e.g. Salmonella, Shigella) form colorless or clear colonies

Blood Agar

• Enriched medium that supports the growth of fastidious bacteria and inhibits the growth of some bacteria like Neisseria and Haemophilus
• Contains 5% defibrinated mammalian blood (e.g. horse, sheep, human) added to the autoclaved basal media (TSA or Columbia Agar)

Chocolate Agar

• Enriched growth medium used for the isolation of pathogenic bacteria
• Nonselective, enriched growth medium that supports the growth of fastidious bacteria
• Contains red blood cells that have been lysed by slowly heating to 80°C, releasing intracellular nutrients

Obtaining Pure Culture

• A pure culture is a laboratory culture containing a single species of organism
• Obtained by transferring a small sample into new, sterile growth medium in such a manner as to disperse the individual cells across the medium surface or by thinning the sample many fold before inoculating the new medium
• Can be accomplished by spreading bacteria on the surface of a solid medium so that a single cell occupies an isolated portion of the agar surface

Solid vs. Semi-Solid vs. Liquid Media

• Solid media: contains a solidification agent (e.g. agar) and is used for the growth of microorganisms
• Semi-solid media (deep culture): prepared with a lower concentration of agar (0.2 to 0.5%) to observe motility of bacteria
• Liquid media (broth culture): a liquid used to grow bacteria, does not contain a solidification agent

Spread Plate Technique

• A method of isolation and enumeration of microorganisms in a mixed culture
• Distributes microorganisms evenly and makes it easier to quantify bacteria in a solution