Introduction

An initial aim of all microbiologists is the reproducible growth of their microbial cultures, no matter whether the microorganisms are of natural origin or have been genetically engineered by man. Reproducible growth requires defined environmental conditions with respect to energy source, temperature, pH and nutrients (see chapter Microbial Growth Requirements). With this in mind Fluka supplies a range of products and services (see list of culture collections, comparison of media etc.) designed to meet the needs of general microbiologists and specialists alike.

Microorganisms

In the group of organisms classified as microorganisms, there are simple unicellular forms (cocci, bacilli, virio and spirillae) as well as multicellular forms (filaments and sheaths). The group includes the blue green algae (cyanobacteria), fungi, protozoans and bacteria.

In order to survive and grow, microorganisms require a source of energy and nourishment. Bacteria are the most primitive forms of microorganisms but are composed of a great variety of simple and complex molecules and are able to carry out a wide range of chemical transformations. Depending on their requirements and the source of energy used they are classified into different nutritional groups.

The size of microorganisms varies from a fraction of a μm for viruses, which can only be seen in the electron microscope, to several cm for filamentous algae or fungi, for example:

Microbial Growth Requirements

Microbial growth requires suitable environmental conditions, a source of energy, and nourishment. These requirements can be divided into two categories, physical and chemical.

Chemical Factors

Table of the elements required for microbial growth as found in nature compared to the chemical forms supplied to microbiological media.

*also act as buffers
1)To facilitate the solubilisation or retention of iron in solution, complexing agents such as EDTA or citrate may be added to the medium.

Physical / Environmental Factors

Temperature

Most microorganisms grow well at the normal temperatures favoured by man, higher plants and animals. However, certain bacteria grow at temperatures (extreme heat or cold) at which few higher organisms can survive. Depending on their preferred temperature range, bacteria are divided into three groups: Psychrophiles (cold-loving microorganisms) found mostly in the depths of the oceans, in ice and snow and in the arctic regions, have an optimum growth temperature between 0°C and 15°C and a maximum growth temperature of not more than 20°C. Mesophiles (moderate-temperature-loving bacteria) found in water, soil and in higher organisms, are the most common type of microbe studied. Their optimum growth temperature ranges between 25°C and 40°C. The optimum temperature for many pathogenic bacteria is 37°C, thus the mesophiles constitute most of our common spoilage and disease microbes. Thermophiles (heat-loving microbes) are capable of growth at high temperatures with an optimum above 60°C. Some organisms grow at temperatures near the boiling point of water and even above 100°C when under pressure. Most thermophiles cannot grow below 45°C.

pH

Most bacteria grow best in an environment with a narrow pH range near neutrality between pH 6.5 and 7.5. Those that grow at extremes of pH are classed as acidophiles (acid-loving) or alkalinophiles (base-loving). Acidophiles grow at pH values below 4 with some bacteria still active at a pH of 1. Alkalinophilic bacteria prefer pH values of 9-10 and most cannot grow in solutions with a pH at or below neutral. Often during bacterial growth, organic acids are released into the medium, which lower its pH and so interfere with or totally inhibit further growth. Although common media ingredients such as peptones and amino acids have a small buffering effect, an external buffer is needed in most bacteriological media to neutralise the acids and maintain the correct pH. Phosphate salts are the most commonly used buffers because they buffer in the growth range of most bacteria, are non-toxic and provide a source of phosphorus, an essential nutrient element. High phosphate concentration has the disadvantage, however, that it can result in a severe nutrient limitation caused by the precipitation of insoluble metal phosphates (such as iron) in the medium.

Osmotic Pressure

Microbes contain approximately 80-90% water and if placed in a solution with a higher solute concentration will lose water which causes shrinkage of the cell (plasmolysis). However, some bacteria have adapted so well to high salt concentrations that they actually require them for growth. These bacteria are called halophiles (salt-loving) and are found in salterns or in areas such as the Dead Sea.

Oxygen

Microbes that use oxygen for energy-yielding purposes are called aerobes, if they require oxygen for their metabolism they are called obligate aerobes. Obligate aerobes are at a disadvantage because oxygen is poorly soluble in water and much of the environment is lacking in this necessary element. Often, aerobic bacteria have retained the ability to grow without oxygen; these are called facultative anaerobes. Those bacteria that are unable to use oxygen and in fact may be harmed by it are known as obligate anaerobes. Further groups include: the microaerophiles which are aerobic microbes that tolerate only a narrow band of oxygen concentrations usually lower than that of the atmosphere and are therefore often difficult to cultivate in the laboratory, and aerotolerant bacteria that grow in the presence of oxygen but do not require it.Water

In contrast to higher organisms, the metabolism of microorgansims is dependent on the presence of liquid water. The requirements of microorganisms with respect to available water differ widely. In order to compare the available water content of solids and solutions, water activity or relative humidity are useful parameters.

Carbon Dioxide

In autotrophic metabolisms, microbes tap various sources of energy and reducing power, which they use to reduce CO₂ to organic compounds. Sodium hydrogencarbonate is usually added to the culture media if autotrophic CO₂-fixing microorganisms are to be grown, and incubation is performed in a carbon dioxide-containing atmosphere in closed vessels or, alternatively, air or carbon dioxide-enriched air is circulated through the vessel. While some chemoautotrophs are aerobic, using oxygen as the ultimate electron acceptor and deriving energy from the respiration of various inorganic electron donors, other microorganisms engage in anaerobic respiration, using an inorganic terminal electron acceptor other than oxygen. Heterotrophic (= assimilating organic carbon sources) microorganisms require carbon dioxide as well. Many bacteria living in blood, tissue or in the intestinal tract are adapted to a carbon dioxide content higher than that of normal air. These bacteria are therefore incubated in an atmosphere containing 10%(vol) carbon dioxide. Phototrophic bacteria are obligate anaerobes and use energy from light for a succession of reactions that convert carbon dioxide to triosephosphate and other cell constituents. Even though carbon dioxide is recycled rather than assimilated, nearly all growing cells have an absolute requirement for an adequate pCO₂. It is therefore important to note that the removal of carbon dioxide e.g. by KOH-absorption, inhibits the growth of nearly all bacteria.

Microbiological Culture Methods I

[1] [2] 下一页