GENERAL AND FOOD MICROBIOLOGY PART I - L. V. Krasnikova - 2016

4. CULTIVATION OF MICROORGANISMS

Objective: to provide An Overview of the various Types of nutrient media used for growing microorganisms; to familiarize students with inoculation and subculturing techniques.

The growth of microorganisms on nutrient media is referred to as cultivation (from Latin cultus - cultivation), and the resulting Microbial growth is called a culture. A distinction is made between a pure culture, which consists of the progeny of Cells of a single species; an enrichment culture, dominated by cells of a single species or a group of related microorganisms; and a mixed culture, representing a consortium of several microbial species.

The Introduction of a test material (such as a Water sample, food product, or soil sample) into a sterile nutrient medium is called inoculation. Transferring an already grown culture into a fresh sterile medium is called subculturing, and through repeated transfers, serial passage (from Latin passis - alternation).

When cultivating microorganisms, it is essential to create optimal conditions for their growth. The primary factors influencing

growth rate include Temperature, medium pH, the presence or absence of oxygen, as well as the COMPOSITION AND PROPERTIES of the medium.

Microorganisms are grown at their respective optimum temperatures using incubators or temperature-controlled rooms. This process is known as incubation (from Latin incubatio - rearing, hatching). Specifically, mesophilic microorganisms are typically grown at temperatures ranging from 25 to 30 °C, thermophiles at 40-45 °C, and coliform Bacteria usually at 37 °C.

Microorganisms are highly sensitive to the reaction of the medium. Therefore, prior to sterilization, it is necessary to adjust the medium to the required pH value, taking into account a slight drop in pH that occurs during sterilization.

Laboratory cultivation of microorganisms is carried out using glassware such as flasks, test tubes, Petri dishes, Roux bottles (flat Glass bottles), liquid media (broth), milk, or solid nutrient media.

Pure microbial cultures are usually grown in test tubes on Agar slants or in broth media. When a solid medium is required for tube cultivation, the melted medium is dispensed to fill 1/3 to 1/4 of the tube volume prior to sterilization. After sterilization, the tubes containing the molten medium are laid out in a slanted position. Tubes with solidified medium are referred to as slants.

For cultivating aerobic bacteria in liquid media, test tubes are filled with nutrient broth to 1/3 of their capacity, whereas for anaerobic bacteria, they are filled to 2/3. Typical signs of microbial growth in broth include uniform turbidity, surface pellicle formation, and the appearance of a characteristic sediment type, among others.

Test tubes filled to half their volume with a solidified solid medium are called stabs. They are used for stab inoculation.

Flasks are used exclusively for liquid nutrient media, while Petri dishes are used for solid media.

Petri dishes are generally used to cultivate aerobic and facultatively anaerobic microorganisms. They form distinct colony types On the surface of solid nutrient media.

Special devices known as anaerobic jars (Fig. 4.1) are used for cultivating anaerobic bacteria.

Class="center">Fig. 4.1. Anaerobic jar

An anaerobic jar consists of a cylinder sealed with a lid via a vacuum rubber gasket. The lid features a vacuum gauge and two Valves: one connects to a vacuum pump for air evacuation, and the other connects to gas cylinders (N₂, CO₂, H₂). After placing the Petri dishes or test tubes inside, air is evacuated from the jar using the vacuum pump, the valve is tightly closed with a specialized screw, the jar is disconnected from the pump, and it is transferred to the incubator.

4.1. Nutrient Media

Nutrient media varying in composition and purpose are used for the detection, enumeration, isolation, and characterization of microorganisms in food products. Any nutrient medium must meet the following requirements: contain all essential nutritional components in an easily assimilated form; have optimal moisture, viscosity, and pH; be isotonic; and be as transparent as possible. Each nutrient medium is sterilized using a specific method depending on its composition.

Based on their composition, nutrient media are divided into synthetic and natural media.

Synthetic media are aqueous solutions of specific, chemically pure compounds in precise concentrations, meaning their composition is fully known. However, synthetic media are used only for a few nutritionally less demanding microorganisms.

Natural, or complex, media consist of products of plant or animal origin and possess a complex, undefined chemical composition. Examples include meat extract (peptone) broth and agar, malt wort and wort agar, skimmed and hydrolyzed milk, and various vegetable decoctions.

Depending on their intended application, nutrient media are classified into basic (general-purpose), selective, and differential-diagnostic media.

General-purpose media are those used for cultivating A wide variety of bacteria. These include meat-peptone broth (MPB), as well as tryptic hydrolysates of meat, fish, and casein. Such media serve as the foundation for preparing complex growth media by adding components such as Blood, sugar, milk, and other ingredients.

Selective, or enrichment, media were introduced into microbiological practice by S.N. Winogradsky and M. Beijerinck. They are designed for the isolation and accumulation of specific microorganisms (or specific groups of microorganisms) from sources containing diverse microflora. Concomitant microorganisms either fail to grow on these media entirely or their development is heavily suppressed. The design of selective media relies on the biological traits that distinguish these target microorganisms from many others.

Differential media make it possible to rapidly identify and distinguish (differentiate) certain species or groups of microorganisms from others. Their composition is formulated to clearly reveal the most Characteristic Properties of a given species. This is frequently achieved by incorporating special indicator Dyes that stain the colonies of the target microorganisms in specific colors. In particular, differential media such as Endo, Kessler, Eijkman, Ploskirev, and Levin agars are used to detect coliform bacteria.

Based on their physical state, media are classified into liquid, semi-solid, solid, and dry.

Liquid media (broths) are used to accumulate microbial biomass or their metabolic products, as well as to study the PHYSIOLOGICAL AND BIOCHEMICAL CHARACTERISTICS OF MICROORGANISMS.

Semi-solid media contain from 0.08 to 0.7 % agar.

Solid media are prepared from liquid media by adding gelling agents—either gelatin or agar (1.5 — 2.0 %). When dissolved in hot water, both substances form a colloidal solution that sets into a firm gel upon cooling. These gel-like media can be re-melted by heating. Solid media are used to isolate pure cultures of microorganisms, for diagnostic purposes (describing colony Morphology), for quantitative enumeration of microorganisms, and for determining antagonistic or proteolytic activity, among other Applications.

Dry nutrient media, manufactured by specialized enterprises, are prepared for microbiological use by adding water followed by sterilization.

4.2. Techniques for Inoculating Microorganisms onto Nutrient Media

WORKING WITH MICROORGANISMS requires specialized tools such as bacteriological loops, needles, spreaders, and pipettes. Inoculations must always be performed within the zone of a burner flame. When handling a pure culture, one should avoid sudden movements, walking, coughing, or similar actions, as air currents increase the risk of contaminating the culture tube with foreign microorganisms. Therefore, it is recommended to perform inoculations and subculturing inside a laminar flow cabinet or inoculation box. A box is a small, isolated room divided by a partition into two sections. The main workspace is entered through an ante-room, where lab coats designated exclusively for work inside the box are kept. All equipment, walls, and floors of the box are regularly cleaned and wiped down with disinfectants. Before use, the box is sterilized using bactericidal lamps for 1.0 — 1.5 hours.

Inoculation into liquid nutrient media. Inoculation is carried out using a loop or a graduated pipette. The inoculum is carefully introduced into the test tube with a bacteriological loop and gently agitated in the upper layer of the medium or rubbed against the inner wall to wash it off with the liquid.

A sterile pipette is submerged into the culture tube, a specific volume of material is drawn up, and it is transferred into a tube of fresh nutrient medium by releasing the liquid along the inner wall, or by immersing the pipette deeper into the medium before discharging its contents.

Streak inoculation onto an agar slant. The tube containing the culture and the tube containing the nutrient agar slant are held in the left hand in an inclined position. A bacteriological needle is taken in the RIGHT HAND AND heated in a Bunsen burner flame until red-hot, after which the needle holder is also passed through the flame. Using the little finger of the right hand, the cotton plugs are removed from both tubes, and the tube rims are flamed. The loop is inserted into the culture tube, cooled against the inner walls, and a small amount of the microbial culture is gently picked up. The loop with the inoculum is quickly transferred to the tube with the sterile medium and lowered almost to the bottom, where a small amount of Condensation water accumulates. Touching the agar lightly, a zigzag line is drawn along the surface without lifting the loop from the medium. After inoculation, the loop is withdrawn and flamed along with any remaining inoculum. The rims of the tubes and the inner ends of the plugs are then flamed before plugging the tubes securely. The date of inoculation and the name of the culture are marked on the outside of the tube.

Stub (stab) inoculation. The test culture is picked up using a flamed bacteriological needle. The tube containing a Column of nutrient medium is held in the right hand, inverted, and its plug is removed while being held against the palm with the little finger. Raising the tube to eye level, the solid medium is punctured vertically in the center of the column from bottom to top to its full depth using the needle. The needle is then withdrawn, the tube is plugged, labeled, and placed in an incubator for growth.

Surface inoculation of a Petri dish. Molten agar is poured into Petri dishes, allowed to solidify, and dried in an incubator. Inoculation is performed using a bacteriological loop or a Drigalski spatula (a glass rod bent in the shape of a triangle). When using a loop, inoculation is carried out by streaking. The lid of the Petri dish is opened only slightly—just enough to allow the loop to pass through the gap. The loop is laid flat on the nutrient medium to avoid scratching it, and the microbial culture is applied in zigzag strokes across the entire agar surface without lifting the loop, which allows for the isolation of individual colonies.

Spread-plate inoculation ("lawn culture") is performed using a spreader. To do this, the lid of the Petri dish is slightly opened with the left hand, and the inoculum is applied to The surface of the nutrient agar using a loop or pipette. The spreader is then passed through the burner flame, cooled against the inner side of the dish lid, and used to distribute the material evenly across the entire surface of the medium. After incubation, a uniform lawn of grown culture appears on the dish.

Pour-plate (deep) inoculation. A specific volume of the prepared test material (1.0 or 0.1 cm³) is pipetted into an empty sterile Petri dish. The cotton plug is removed from a test tube or flask containing nutrient medium melted and cooled to 45 °C, the rims are flamed, and, with the lid of the Petri dish slightly ajar, the medium is poured into the bottom of the dish.

The inoculated tubes and Petri dishes are placed in an incubator set to the optimal temperature for the specific microorganism. As a rule, mesophilic bacteria are cultivated at 37±1 °C, thermophilic bacteria at 40–55 °C, and Yeasts and Molds at 30±1 °C.

Review Questions

1. What are pure, enrichment, and mixed cultures?

2. How are nutrient media classified according to their component composition and intended use?

3. Which media allow for the preferential growth of certain microbes while simultaneously suppressing the growth of others?

4. What Methods are used to inoculate microorganisms onto nutrient media?

5. How is the cultivation of aerobic and anaerobic bacteria carried out?