CULTIVATION OF MICROORGANISM

By:

Farid Ma`ruf

Ridho Muharram

INTRODUCTION

Background

Cultivation is the process of propagating organisms by providing the proper environmental conditions. Growing microorganisms are making replicas of themselves, and they require the elements present in their chemical composition. Nutrients must provide these elements in metabolically accessible form. In addition, organisms require metabolic energy to synthesize macromolecules and maintain essential chemical gradients across their membranes.

Factors that must be controlled during growth include the nutrients, pH, temperature, aeration, salt concentration, and ionic strength of the medium.

Environmental Factors Affecting Growth

A suitable growth medium must contain all the nutrients required by the organism to be cultivated, and such factors as pH, temperature, and aeration must be carefully controlled

Nutrients

In general, the following must be provided:1. hydrogen donors and acceptors: about 2 g/L;2. carbon source: about 1 g/L;3. nitrogen source: about 1 g/L;4. minerals: sulfur and phosphorus, about 50 mg/L of

each, and trace elements, 0.1–1 mg/L of each;5. growth factors: amino acids, purines, and

pyrimidines, about 50 mg/L of each, and vitamins, 0.1–1 mg/L of each.

pH

Most organisms have a fairly narrow optimal pH range. The optimal pH must be empirically determined for each species. Most organisms (neutralophiles) grow best at a pH of 6.0–8.0, although some forms (acidophils) have optima as low as pH 3.0 and others (alkaliphiles) have optima as high as pH 10.5.

Temperature

Different microbial species vary widely in their optimal temperature ranges for growth: Psychrophilic forms grow best at low temperatures (15–20°C); Mesophilic forms grow best at 30–37°C; and most Thermophilic forms grow best at 50–60°C. Most organisms are mesophilic; 30°C is optimal for many free-living forms, and the body temperature of the host is optimal for symbionts of warm-blooded animals.

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Microorganisms share with plants and animals the heat-shock response, a transient synthesis of a set of “heat-shock proteins,” when exposed to a sudden rise in temperature above the growth optimum.

Did you know…?

The relationship of growth rate to temperature for any given microorganism is seen in a typical Arrhenius plot. Arrhenius showed that the logarithm of the velocity of any chemical reaction (log k) is a linear function of the reciprocal of the temperature (1/T); since cell growth is the result of a set of chemical reactions, it might be expected to show this relationship

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Arrhenius plot

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Beyond their effects on growth rate, extremes of temperature kill microorganisms. Extreme heat is used to sterilize preparations; extreme cold also kills microbial cells, although it cannot be used safely for sterilization.

Did you know…?

Bacteria also exhibit a phenomenon called cold shock: the killing of cells by rapid—as opposed to slow—cooling. For example, the rapid cooling of Escherichia coli from 37°C to 5°C can kill 90% of the cells. A number of compounds protect cells from either freezing or cold shock; glycerol and dimethyl sulfoxide are most commonly used.

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Aeration

Many organisms are obligate aerobes, specifically requiring oxygen as hydrogen acceptor; some are facultative, able to live aerobically or anaerobically; and others are obligate anaerobes, requiring a substance other than oxygen as hydrogen acceptor and being sensitive to oxygen inhibition.

Ionic Strength and Osmotic Pressure

To a lesser extent, such factors as osmotic pressure and salt concentration may have to be controlled. For most organisms, the properties of ordinary media are satisfactory; however, for marine forms and organisms adapted to growth in strong sugar solutions, for example, these factors must be considered. Organisms requiring high salt concentrations called halophilic; those requiring high osmotic pressures are called osmophilic.

Cultivation Method

1. Growing Cells of a Given Species

Microorganisms observed microscopically to be growing in a natural environment may prove exceedingly difficult to grow in pure culture in an artificial medium. Certain parasitic forms, for example, have never been cultivated outside the host. In general, however, a suitable medium can be devised by carefully reproducing the conditions found in the organism’s natural environment. The pH, temperature, and aeration are simple to duplicate; the nutrients present the major problem. The contribution made by the living environment is important and difficult to analyze; a parasite may require an extract of the host tissue, and a free-living form may require a substance excreted by a microorganism with which it is associated in nature. Considerable experimentation may be necessary in order to determine the requirements of the organism.

2. Microbiologic Examination of Natural Materials

A given natural material may contain many different microenvironments, each providing a niche for a different species. Plating a sample of the material under one set of conditions will allow a selected group of forms to produce colonies but will cause many other types to be overlooked. For this reason, it is customary to plate out samples of the material using as many different media and conditions of incubation as is practicable. Six to eight different culture conditions are not an unreasonable number if most of the forms present are to be discovered.

3. Isolation of a Particular Type of Microorganism

A small sample of soil, if handled properly, will yield a different type of organism for every microenvironment present. For fertile soil (moist, aerated, rich in minerals and organic matter) this means that hundreds or even thousands of types can be isolated.

This is done by selecting for the desired type. One gram of soil, for example, is inoculated into a flask of liquid medium that has been made up for the purpose of favoring one type of organism, eg, aerobic nitrogen fixers (azotobacter).

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In this case, the medium contains no combined nitrogen and is incubated aerobically. If cells of azotobacter are present in the soil, they will grow well in this medium; forms unable to fix nitrogen will grow only to the extent that the soil has introduced contaminating fixed nitrogen into the medium. When the culture is fully grown, therefore, the percentage of azotobacter in the total population will have increased greatly; the method thus called “enrichment culture.”

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4. Isolation of Microorganism in Pure Culture

In order to study the properties of a given organism, it is necessary to handle it in pure culture free of all other types of organisms. To do this, a single cell must be isolated from all other cells and cultivated in such a manner that its collective progeny also remain isolated. Several methods are available… [cont]

Plating

Dilution

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Reference

Jawetz, Ernest, et al. 1996. Biologi Kedokteran (Medical Microbiology)

Edisi 20. Jakarta: Penerbit Buku Kedokteran EGC.