FUNDAMENTALS OF MICROBIOLOGY - V. M. Samygin - 2015

CHAPTER 10. BASIC PRINCIPLES OF BACTERIAL CULTIVATION

The cultivation (growth) of microorganisms is essential for obtaining biomass or various microbial metabolites (metabolic products). Microbes are cultivated in the laboratory using artificial nutrient media, tissue cultures, and experimental animals. Cultural CHARACTERISTICS OF MICROORGANISMS are determined by their growth patterns on nutrient media. These features are of great diagnostic importance and remain constant for each microbial species.

When A large number of microbial Cells are inoculated onto solid nutrient media, continuous confluent growth occurs, forming a lawn culture. When a limited number of cells are inoculated, colonies of various SHAPES AND SIZES develop. These are visible clusters of individuals of the same microbial species formed As a result of the multiplication of a single Cell. Colonies can be flat, convex, dome-shaped, or indented; their surfaces may be smooth (S-form), rough (R-form), striated, or bumpy; and their edges can be even, serrated, fibrous, or fringed. Colony shapes vary widely, including circular, rosette-like, stellate, and arborescent. By size, colonies are categorized as large (4–5 mm in diameter), medium (2–4 mm), small (1–2 mm), and dwarf (less than 1 mm). Colonies also differ in consistency, density, and color. They can be transparent or opaque, pigmented or colorless, moist, dry, or mucoid (Figs. 10 and 11).

Class="center">Fig. 10. Main Types of microbial colonies

Fig. 11. Morphological and structural diversity of colonies: 1 - elevation profiles of colonies above the nutrient medium surface; 2 - colony outlines; 3 - edge characteristics of colonies; 4 - Internal Structure of colonies

In liquid nutrient media, microbes grow by forming diffuse turbidity, surface pellicles, or sediments visible to the naked eye. Wall growth (adherent growth) of Bacteria is also sometimes observed.

In laboratory settings, bacteria are cultivated in test tubes, Petri dishes, flasks, and bacteriologic matrices. In industrial settings, microorganism cultivation is carried out in various fermenters (fermenters, bioreactors) using specially developed technologies.

To assess growth efficiency, researchers typically measure biomass or the number of microbial cells. A direct method for measuring cell mass is determining the dry weight of cells contained in a specific volume of culture by separating them from the medium, drying them, and subsequently weighing them. However, standard balances struggle to accurately measure masses under 1 mg, yet such a small amount of dry matter can contain up to 5 billion bacteria.

A more convenient METHOD FOR DETERMINING the mass of unicellular microorganisms is the optical method—measuring The amount of light scattered by a cell suspension. It is based on the principle that Light Scattering by small particles is proportional to their concentration within certain limits, and the fraction of transmitted light serves as a measure of the suspension's optical density. Measurements are performed using a spectrophotometer or a photoelectric colorimeter. These instruments display optical density units, which represent the logarithm of The ratio of the light intensity incident on the suspension to the intensity of the transmitted light. The lower limit of sensitivity for this method is approximately 10 million cells per 1 ml.

To determine the number of cells, one can use microscopic Methods employing specialized Glass slides (counting chambers), direct cell counting using an electronic device (Coulter counter), etc. However, the Total Cell Count in a suspension is most often determined using a bacterial turbidity standard, as well as by plating microorganisms onto a solid nutrient medium in Petri dishes.

The turbidity standard consists of glass particles ranging from 0.5 to 3.5 µm in diameter suspended in distilled Water. When the sediment is shaken, this glass suspension corresponds to a specific cell concentration. The standard consists of sealed reference tubes whose turbidity levels are equivalent to 5 and 10 international turbidity units (5 • 10⁸ and 10⁹ cells/ml, respectively). A font chart containing a set of various type sizes is supplied with the set of bacterial standards. The culture suspension under investigation is placed in a standard tube, the wall thickness and glass color of which match the reference tubes. By diluting the culture with measured amounts of saline, the turbidity of the test tube is adjusted to match the standard, and the initial concentration of microbial cells is then calculated. The calculation is performed either based on the culture dilution factor or from the total cell count in the bacterial suspension.

The number of unicellular microorganisms can also be counted after plating them onto a solid nutrient medium in Petri dishes. Because viable cells that are spatially separated from one another On the surface of a solid medium form distinct, naked-eye-visible colonies during growth, preparing appropriate dilutions of the bacterial population and using them to inoculate the medium allows one to determine the Number of viable cells in the original suspension. This is done by counting the colonies that grow after incubation and multiplying that number by the dilution factor. This method is called viable cell concentration determination, as it accounts only for those microorganisms capable of growing on the nutrient medium. Determining the viable cell count is undoubtedly the most sensitive method for quantitative bacterial enumeration, as it makes it possible to detect even a single viable cell in a suspension. To establish the proportion of viable cells within a population, methods for separately determining the total cell count (using a turbidity standard) and the viable cell count can be combined.