Basic Requirements of Microorganisms
For bacteria to successfully proliferate in vitro, any medium must meet four mandatory criteria: an aqueous base, an organic energy source, optimal pH, and suitable osmotic pressure.
For heteroorganotrophs, organic substances serve as the main carbon sources. Glucose provides cells with energy, while peptone (a product of incomplete protein digestion) supplies amino acids and peptides that the bacterium uses to build its own structures.
In addition to organics, minerals are required for the synthesis of nucleic acids and coenzymes. Phosphorus and sulfur are assimilated from inorganic salts, while nitrogen is derived from peptone and ammonium salts. Metal ions ($Ca^{2+}$, $Mg^{2+}$, $Mn^{2+}$, $Fe^{2+}$) are extremely important; they are added to the medium as phosphates and ensure the normal function of bacterial enzymes.
Physicochemical Balance
The viability of a laboratory culture is directly influenced by the parameters of the surrounding fluid:
- Acidity (pH). During metabolism, microbes release metabolic waste products that often acidify the medium. To prevent the culture from dying due to self-poisoning by acids, buffering is applied (e.g., phosphate buffer). If acid production is very intensive, a neutralizer such as calcium carbonate is added to the medium.
- Osmotic pressure. The bacterial cell wall protects them from osmotic shifts. Most species feel comfortable in an isotonic solution (0.87% NaCl). However, halophilic bacteria require a salinity above 1%. A special case involves microbes lacking a cell wall (mycoplasmas, L-forms). In standard media, they undergo osmotic lysis; therefore, hypertonic conditions are created for them by adding sucrose.
Consistency and Agar Properties
Depending on the study objectives, media can be liquid, semisolid, or solid. To give the mixture a solid consistency, agar-agar is used—a complex polysaccharide extracted from algae.
Its unique technological advantage lies in its temperature response: agar melts at 100 °C and turns into a stable gel when cooled to 45–50 °C.
The concentration of the solidifying agent depends on the medium type:
- Semisolid media: contain about 0.5% agar.
- Solid media: require 1.5–2% agar.
In addition to basic components, specific additives may be introduced: growth factors, substrates, indicators, or inhibitors of extraneous microflora.
Classification of Media by Purpose
Microbiologists use different types of media to address specific tasks:
- Simple (basal): peptone water, nutrient broth, nutrient agar.
- Complex: prepared from simple media with added nutritional components—blood, serum, or sugars (e.g., blood agar).
- Selective: create ideal conditions for a specific species while inhibiting competitors. For example, alkaline peptone water or alkaline agar (pH 9) allows the isolation of Vibrio cholerae, as the high pH is detrimental to Escherichia coli.
- Enrichment media: stimulate the proliferation of a pathogen while suppressing accompanying flora (e.g., tetrathionate or selenite broth for Salmonella).
- Differential diagnostic media: used to assess enzymatic activity (e.g., Hiss's fermentation tubes for studying saccharolytic properties).
%0A Also widely used are combination media, which combine selective and diagnostic properties. Ploskirev's agar or bismuth sulfite agar suppress normal intestinal microflora and allow visual identification of pathogenic enteric bacteria.