Microorganism growth on culture media depends on a number of important factors :

• Proper nutrients must be available.
• Oxygen or other gases must be available as required.
• Moisture is necessary.
• The medium must have an appropriate pH.
• Proper temperature relations must prevail.
• The medium must be free of interfering bioburden.
• Contamination must be prevented.
  
  

A satisfactory microbiological culture medium must contain available sources of

• Carbon,
• Nitrogen,
• Inorganic phosphate and sulfur,
• Trace metals,
• Water,
• Vitamins

These were originally supplied in the form of meat infusion, Beef or yeast extracts frequently replace meat infusion in culture media. The addition of peptones, which are digests of proteins, provides readily available sources of nitrogen and carbon.

The pH of the culture medium is important for microorganism growth. Temperature is another important parameter mesophilic bacteria and fungi have optimal growth at temperatures of 25-40C; thermophilic bacteria ("heat loving") grow only at temperature greater than 45C psychrophilic ("cold loving") organisms require temperatures below 20C. Human pathogenic organisms are generally mesophiles.

Common Media Constituents

Media formulations are developed on the ability of bacteria to use media components.

CONSTITUENTS	SOURCE
Amino-Nitrogen	Peptone, protein hydrolysate, infusions and extracts
Growth Factors	Blood, serum, yeast extract or vitamins, NAD
Energy Sources	Sugar, alcohols and carbohydrates
Buffer Salts#	Phosphates, acetates and citrates
Mineral Salts and Metals	Phosphate, sulfate, magnesium, calcium, iron
Selective Agents	Chemicals, antimicrobials and dyes
Indicator Dyes	Phenol red, neutral red
Gelling agents	Agar, gelatin, alginate, silica gel

Media Ingredients

Peptone, protein hydrolysates, Infusions and extracts are the major sources of nitrogen and vitamins in culture media. Peptones are water-soluble ingredients derived from proteins by hydrolysis or digestion of the source material, e.g. meat, milk.

Carbohydrates are employed in culture media as energy sources and may be used for differentiating genera and idenifying species.

Buffers maintain the pH of culture media.

Selective Agents include Bile Salts, dyes and antimicrobial agents.

Bile Salts and desoxycholate are selective for the isolation of gram-negative microorganisms, inhibiting gram-positive cocci.

Dyes and indicators are essential in the preparation of differential and selective culture media. In these formulations, dyes act as bacteriostatic agents, inhibitors of growth or indicators of changes in acidity or alkalinity of the substrate.

Antimicrobial agents are used in media to inhibit the growth of bacteria, yeasts and fungi.

Solidifying agents, including agar, gelatin and albumin, can be added to a liquid medium in order to change the consistency to a solid or semisolid state.


Environmental Factors in Culture Media

Atmosphere

Most becteria are capable of growth under ordinary conditions of oxygen tension. Obligate aerobes require the free admission of oxygen, while anaerobes grow only in the absence of atmospheric oxygen. Between these two groups are the microaerophiles, which develop best under partial anacrobic conditions, and the facultative anaerobes, which are capable of growing in the presence or absence of oxygen. Anaerobic conditions for growth of microorganisms are obtained in a number of ways :

• Addition of small amounts of agar to liquid media;
• Addition of fresh tissue to the medium;
• Addition of a reducing substance to the medium, e.g., sodium thioglycollate, thioglycollic acid and L-cystine;
• Displacement of the air by carbon dioxide;
• Absorption of the oxygen by chemicals;
• Inoculation into the deep layers of solid media or under a layer of oil in liquid media.

Many microorganisms require an environment of 5-10% CO. Levels greater than 10% are often inhibitory due to a decrease in pH as carbonic acid forms. Culture media vary in their susceptibility to form toxic oxidation products if exposed to light and air.

Water Activity

Proper moisture conditions are necessary for continued luxuriant growth of microorganisms. Organisms require an aqueous environment and must have "free" water. "Free" water is not bound in complex structure and is necessary for transfer of nutrients and toxic waste products. Evaporation during incubation or storage results in loss of "free" water and reduction of colony size or total inhibition of organism growth.

Protective Agents and Growth Factors

Calcium carbonate, soluble starch and charcoal are examples of protective agents used in culture media to neutralize and absorb toxic metabolites produced by bacterial growth.
NAD (V factor) and hemin (X factor) are growth factors required by certain bacteria; e.g., Haemophilus species and for enhanced growth of Neisseria species.
Surfactants, including Polysorbate 80, lower the interfacial tension around bacteria suspended in the medium. This activity permits more rapid entry of desired compounds into the bacterial cell and can increase bacterial growth.