Culture media for bacteria

Basic needs of microorganisms

For bacteria to successfully multiply in vitro, any medium must meet four mandatory criteria: it must have an aqueous base, contain an organic energy source, have an optimal pH, and maintain appropriate osmotic pressure.

For heteroorganotrophs, organic substances serve as the main sources of carbon. Glucose supplies cells with energy, while peptone (a product of incomplete protein breakdown) provides 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, whereas nitrogen comes from peptone and ammonium salts. Metal ions ($Ca^{2+}$, $Mg^{2+}$, $Mn^{2+}$, $Fe^{2+}$) are extremely important; they are introduced into the medium as phosphates and ensure the normal function of bacterial enzymes.

Physicochemical balance

The viability of a laboratory culture is directly affected by the parameters of the surrounding liquid:

• Acidity (pH). During metabolism, microbes release metabolic 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 intense, a neutralizer—calcium carbonate—is added to the medium.
• Osmotic pressure. The bacterial cell wall protects them from osmotic fluctuations. Most species feel comfortable in an isotonic solution (0.87% NaCl). However, halophilic bacteria require a salinity above 1%. A special case is microbes lacking a cell wall (mycoplasmas, L-forms). In a regular medium, they undergo osmotic lysis, so hypertonic conditions are created for them by adding sucrose.

Consistency and properties of agar

Depending on the research objectives, media can be liquid, semi-solid, or solid. To give the mixture a thick consistency, agar-agar—a complex polysaccharide extracted from algae—is used.

Its unique technological advantage lies in its reaction to temperature: 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 type of medium:

• Semi-solid media: contain about 0.5% agar.
• Solid media: require 1.5–2% agar.

In addition to basic components, specific additives may be introduced into the composition: growth factors, substrates, indicators, or inhibitors of extraneous microflora.

Classification of media by purpose

Microbiologists use different types of media to solve specific tasks:

1. Simple (basic): peptone water, nutrient broth (NB), nutrient agar (NA).
2. Complex: prepared on the basis of simple media with the addition of nutrient components—blood, serum, or sugars (e.g., blood agar).
3. Selective: create ideal conditions for a specific species while suppressing competitors. For example, alkaline agar (pH 9) allows the isolation of the cholera vibrio, as high pH is detrimental to E. coli.
4. Enrichment media: stimulate the multiplication of the pathogen and suppress accompanying flora (medium with sodium selenite for Salmonella).
5. Differential-diagnostic: needed to assess enzymatic activity (e.g., Hiss's "differential series" to study saccharolytic properties).

Also widely used are combined media, combining selective and diagnostic properties. Ploskirev's medium or bismuth-sulfite agar suppress normal intestinal microflora and allow visual identification of pathogenic intestinal bacteria.

Briefly: answers to topical questions

Class and exam questions

What else to study on this topic

• Heteroorganotroph metabolism: glucose breakdown pathways
• Hiss media ("differential series") for assessing saccharolytic activity
• Combined culture media: Ploskirev's medium and bismuth-sulfite agar
• Halophilic bacteria and mechanisms of their resistance to high salt concentrations

Other topics in the "Microbial Physiology" section

Prepared by WhiteCoat Pro faculty in accordance with the medical school curriculum. For educational purposes only.