Culture Media Types

General Categories of Culture Media

• Microorganism growth is influenced by environmental and nutritional factors.
• Providing appropriate conditions involves a suitable growth medium and atmosphere.
• Hundreds of media types exist, but some medically important and most environmental organisms haven't been grown in labs yet.

Complex Media

• This type of medium contains various undefined ingredients like meat juices and digested proteins that make a "tasty soup" for microbes.
• Easy to make and used routinely, ingredients' chemical compositions are variable.
• Peptone, a mix of amino acids and short peptides from digested proteins, is a common ingredient.
• Extracts made from components like lean beef helps provide vitamins and minerals.
• Nutrient broth, a common complex recipe, consists of peptone and beef extract in distilled water; adding agar creates nutrient agar.
• Fastidious, medically important bacteria need media richer than nutrient agar.
• Clinical labs use blood agar including red blood cells, a source of nutrients like hemin.
• Chocolate agar, named for its brownish appearance, are used for even-more fastidious bacteria.

Chemically Defined Media

• It is composed of specific amounts of pure chemicals, with a known, exact chemical composition.
• Typically used for research experiments requiring precise nutrient control.
• Glucose-salts broth, supports E. coli growth, containing only chemicals listed in Table 4.7, resulting in slower growth because cells must synthesize all components from glucose.
• It takes 46 ingredients to make a chemically defined medium that supports the growth of Neisseria gonorrhoeae which is a fastidious bacterium.
• Buffers are important in these media to keep pH neutral, especially since some bacteria create acids as a by-product that can hinder their growth.

Special Types of Culture Media

• To isolate a species from a mixed population, scientists use selective and differential media.

Selective Media

• This type of media contains an ingredient that inhibits growth of certain species but allows growth of the species of interest.
• Thayer-Martin agar, a chocolate agar with added antimicrobials, is used to isolate Neisseria gonorrhoeae, which are clinical specimens.
• Antimicrobials in Thayer-Martin agar inhibit fungi, Gram-positive bacteria, and Gram-negative rods, excluding most N. gonorrhoeae strains.
• MacConkey agar isolates Gram-negative rods using crystal violet (which inhibits Gram-positive bacteria) and bile salts (which inhibit non-intestinal bacteria).

Differential Media

• Contain a substance that microbes change recognizably.
• Blood agar is differential; bacteria that produce hemolysin (a protein that causes red blood cells to burst), which creates a visible zone of clearing called hemolysis.
• Some pathogens are distinguished by hemolysis type, which makes this observable characteristic important medically
• Beta hemolysis, a clear zone, helps identify Streptococcus pyogenes, which causes strep throat.
• Streptococcus species exhibit alpha hemolysis, a zone of greenish partial clearing.
• MacConkey agar contains lactose and a pH indicator and is both differential and selective.
• Bacteria can ferment lactose and produce acid, turning the pH indicator to pink-red.

Providing Appropriate Atmospheric Conditions

• Achieving appropriate atmospheric conditions is crucial for growing microorganisms on culture media in the lab.

Aerobic Conditions

• Most obligate aerobes and facultative anaerobes, when incubated on agar media, do not require special atmospheric conditions and can be incubated in air (20% O₂).
• Shaking broth cultures increases aeration, leading to optimal growth.
• Medically important bacteria like Neisseria and Haemophilus grow best in aerobic conditions with additional CO₂ and some are capnophiles.
• A candle jar builds up CO₂ levels; a candle consumes some O₂ and generates CO₂ and H₂O, with the flame extinguishing from insufficient oxygen .
• 17% O₂ remains, enough to support obligate aerobes and prevent obligate anaerobes.
• Special incubators can also maintain CO₂.

Microaerophilic Conditions

• They require O₂ concentrations lower than those in a candle jar.
• Often the microbes are incubated in gastight containers with special packets that lower O₂ concentrations to approximately 5-15%.

Anaerobic Conditions

• Obligate anaerobes are hard to grow with the sensitivity to O₂ and containing environments.
• Those that tolerate brief O₂ exposures can be incubated in an anaerobe container with disposable packets that create an anaerobic environment.
• Sodium thioglycolate, a reducing agent in a semisolid culture medium, reacts with O₂ to make water and can remove dissolved O₂ by being heated.

Enrichment Cultures

• These are used to isolate an organism only present in only a small amount of a mixed population.
• Conditions are provided that enhance the growth of the species in broth.
• Selective agents like bile salts can be added.
• If the target microbe grows using atmospheric nitrogen, that element isn't left out of the element.
• If it cannot grow on an unusual carbon source like phenol, phenol can be added as the only carbon source.
• The cultures are incubated in conditions that promote the growth of the desired organism.
• As the microbes multiply, the relative concentration of the target organism can increase.

Direct Cell Counts

• They are useful for determining total microorganism numbers in a sample, even ones that won't grow in culture.
• The methods generally do not distinguish between living and dead cells

Direct Microscopic Count

• It is one of the fastest method of determining cell concentration
• A liquid specimen is added to a hemocytometer, a special glass slide designed for counting cells.
• Each slide has a chamber that holds the liquid (known volume for a microscopic grid).
• The chamber's content can be viewed with a light microscope allowing for easy view of the cells in a given volume.
• At least 10 million bacteria (10^7) per milliliter (mL) are needed for enough cells to be seen in the scope field.

Cell-Counting Instruments

• A Coulter counter is an electronic instrument that counts cells in suspension single file.
• The suspending liquid must is an electrically conducting fluid because the machine counts changes in resistance when non-conducting particles like bacteria pass through.