Cultivation of microorganisms

Purpose of work:

Acquaintance by cultivation of microorganisms and crops technics

Materials and equipment:

Culture of microorganisms, agar-agar, test tubes, flasks, Petri dishes, bacteriological needles, loops, spatulas, racks,slide and cover glasses, dye, K₂Cr₂O₇ (6%) and concentarted H₂SO₄ (6%), Erlenmeier flasks.

A microbiological culture, or microbial culture, is a method of multiplying microbial organisms by letting them reproduce in predetermined culture media under controlled laboratory conditions. Microbial cultures are used to determine the type of organism, its abundance in the sample being tested, or both. It is one of the primary diagnostic methods of microbiology and used as a tool to determine the cause of infectious disease by letting the agent multiply in a predetermined medium. For example, a throat culture is taken by scraping the lining of tissue in the back of the throat and blotting the sample into a medium to be able to screen for harmful microorganisms, such as Streptococcus pyogenes, the causative agent of strep throat. Furthermore, the term culture is more generally used informally to refer to "selectively growing" a specific kind of microorganism in the lab.

Microbial cultures are foundational and basic diagnostic methods used extensively as a research tool in molecular biology. It is often essential to isolate a pure culture of microorganisms. A pure (or axenic) culture is a population of cells or multicellular organisms growing in the absence of other species or types. A pure culture may originate from a single cell or single organism, in which case the cells are genetic clones of one another.

For the purpose of gelling the microbial culture, the medium of agarose gel (agar) is used. Agar is a gelatinous substance derived from seaweed. A cheap substitute for agar is guar gum, which can be used for the isolation and maintenance of thermophiles.

Microbiological cultures can be grown in petri dishes of differing sizes that have a thin layer of agar-based growth medium. Once the growth medium in the petri dish is inoculated with the desired bacteria, the plates are incubated at the best temperature for the growing of the selected bacteria (for example, usually at 37 degrees Celsius for cultures from humans or animals, or lower for environmental cultures).

Another method of bacterial culture is liquid culture, in which the desired bacteria are suspended in liquid broth, a nutrient medium. These are ideal for preparation of an antimicrobial assay. The experimenter would inoculate liquid broth with bacteria and let it grow overnight (they may use a shaker for uniform growth). Then they would take aliquots of the sample to test for the antimicrobial activity of a specific drug or protein (antimicrobial peptides).

As an alternative, the microbiologist may decide to use static liquid cultures. These cultures are not shaken and they provide the microbes with an oxygen gradient.

When microorganisms are cultivated in the laboratory, a growth environment called a medium is used. The medium may be purely chemical (a chemically defined medium), or it may contain organic materials, or it may consist of living organisms such as fertilized eggs. Microorganisms growing in or on such a medium form a culture. A culture is considered a pure culture if only one type of organism is present and a mixed culture if populations of different organisms are present. When first used, the culture medium should be sterile, meaning that no form of life is present before inoculation with the microorganism.

General microbial media. For the cultivation of bacteria, a commonly used medium is nutrient broth, a liquid containing proteins, salts, and growth enhancers that will support many bacteria. To solidify the medium, an agent such as agar is added. Agar is a polysaccharide that adds no nutrients to a medium, but merely solidifies it. The medium that results is nutrient agar.

Many media for microorganisms are complex, reflecting the growth requirements of the microorganisms. For instance, most fungi require extra carbohydrate and an acidic environment for optimal growth. The medium employed for these organisms is potato dextrose agar, also known as Sabouraud dextrose agar. For protozoa, liquid media are generally required, and for rickettsiae and viruses, living tissue cells must be provided for best cultivation.

For anaerobic microorganisms, the atmosphere must be oxygen free. To eliminate the oxygen, the culture media can be placed within containers where carbon dioxide and hydrogen gas are generated and oxygen is removed from the atmosphere. Commercially available products achieve these conditions. Anaerobic chambers can also be used within closed compartments, and technicians can manipulate culture media within these chambers. To encourage carbon dioxide formation, a candle can be burned to use up oxygen and replace it with carbon dioxide.

Special microbial media. Certain microorganisms are cultivated in selective media. These media retard the growth of unwanted organisms while encouraging the growth of the organisms desired. For example, mannitol salt agar is selective for staphylococci because most other bacteria cannot grow in its high-salt environment. Another selective medium is brilliant green agar, a medium that inhibits Gram-positive bacteria while permitting Gram-negative organisms such as Salmonella species to grow.

Still other culture media are differential media. These media provide environments in which different bacteria can be distinguished from one another. For instance, violet red bile agar is used to distinguish coliform bacteria such as Escherichia coli from noncoliform organisms. The coliform bacteria appear as bright pink colonies in this media, while noncoliforms appear a light pink or clear.

Certain media are both selective and differential. For instance, MacConkey agar differentiates lactose-fermenting bacteria from nonlactose-fermenting bacteria while inhibiting the growth of Gram-positive bacteria. Since lactose-fermenting bacteria are often involved in water pollution, they can be distinguished by adding samples of water to MacConkey agar and waiting for growth to appear.

In some cases, it is necessary to formulate an enriched medium. Such a medium provides specific nutrients that encourage selected species of microorganisms to flourish in a mixed sample. When attempting to isolate Salmonella species from fecal samples, for instance, it is helpful to place a sample of the material in an enriched medium to encourage Salmonella species to multiply before the isolation techniques begin.

In order to work with microorganisms in the laboratory, it is desirable to obtain them in pure cultures. Pure cultures of bacteria can be obtained by spreading bacteria out and permitting the individual cells to form masses of growth called colonies. One can then pick a sample from the colony and be assured that it contains only one kind of bacteria. Cultivating these bacteria on a separate medium will yield a pure culture.

To preserve microbial cultures, they may be placed in the refrigerator to slow down the metabolism taking place. Two other methods are deep-freezing and freeze-drying. For deep-freezing, the microorganisms are placed in a liquid and frozen quickly at temperatures below –50°C. Freeze-drying (lyophilization) is performed in an apparatus that uses a vacuum to draw water off after the microbial suspension has been frozen. The culture resembles a powder, and the microorganisms can be preserved for long periods in this condition.

Isolation methods. To obtain separated colonies from a mixed culture, various isolation methods can be used. One is the streak plate method, in which a sample of mixed bacteria is streaked several times along one edge of a Petri dish containing a medium such as nutrient agar. A loop is flamed and then touched to the first area to retrieve a sample of bacteria. This sample is then streaked several times in the second area of the medium. The loop is then reflamed, touched to the second area, and streaked once again in the third area. The process can be repeated in a fourth and fifth area if desired. During incubation, the bacteria will multiply rapidly and form colonies (Figure 5).

A second isolation method is the pour plate method. In this method, a sample of bacteria is diluted in several tubes of melted medium such as nutrient agar. After dilution, the melted agar is poured into separate Petri dishes and allowed to harden. Since the bacteria have been diluted in the various tubes, the plates will contain various dilutions of bacteria, and where the bacteria are most diluted, they will form isolated colonies (Figure 5).

Aseptic techniques

Aseptic techniques are important to protect the worker from infection from the clinical specimen and also to prevent contamination of the material under process. Aseptic conditions can be achieved by following steps:

Open the caps and lids of the containers containing the specimen for the briefest period required.

Figure 5. Two processes for isolating bacteria from a mixed culture. (a) The streak plate technique. (b) The pour plate technique.

Control questions:

1. Methods of cultivation of microorganisms?

2. Technique of planting of microorganisms?

3. Storage of microorganisms?

4. What means pure culture?