Obtaining a pure culture

Bacteria may be cultured using either liquid or solid media. Solid media are particularly useful in the isolation of bacteria; they are also used for their long-term storage. Liquid (broth) cultures are used for rapid and large-scale production of bacteria.

A culture consisting entirely of one strain of organism is called a pure or axenic culture. In theory, such a culture represents the descendants of a single cell.

Microorganisms in the natural world do not live in pure cultures; they exist as part of complex ecosystems comprising numerous other organisms. The first step in the cultivation of microorganisms is therefore the creation of a pure culture. A key development for the production of pure cultures was the ability to grow microorganisms on a solid medium. Koch had noticed that when a nutrient surface such as cut potato was exposed to air, individual microbial colonies grew up, and he inferred from this that these had each arisen from the numerous divisions of single cells.

It soon became apparent that a number of organisms would not grow on potatoes, so Koch and his colleagues turned to gelatin as a means of solidifying a synthetic nutrient growth medium. Horizontal slabs were cut, and covered to help keep them free from atmospheric contaminants. Gelatin was a convenient means of solidifying media, as it could be boiled and then allowed to set in the desired vessel. There were two main drawbacks to its use, however; many organisms needed to be incubated at around body temperature (37 °C), and gelatin melted before this temperature was reached. Also, it was found that a number of bacteria were capable of utilising gelatin as a nutrient source, resulting in the liquefaction of the gel.

A more suitable alternative was soon found in the form of agar. This is a complex polysaccharide derived from seaweeds, and was suggested by the wife of one of Koch's colleagues, who had used it as a setting agent in jam making. Agar does not melt until near boiling point; this means that cultures can be incubated at 37 °C or above without the medium melting. Moreover, when it cools, agar remains molten until just over 40 °C, allowing heat-sensitive media components such as blood to be added. In addition, most bacteria can tolerate a short exposure to temperatures in this range, so they too can be inoculated into molten agar (see pour plate method below). Crucially, agar is more or less inert nutritionally; only a very few organisms are known that are able to use agar as a food source; consequently, it is the near ideal setting agent, resisting both thermal and microbial breakdown. Agar soon became the setting agent of choice, and has remained so ever since; shortly afterwards, Richard Petri developed the two-part culture dish that was named after him, and which could be sterilised separately from the medium and provided protection from contamination by means of its lid,. This again is still standard equipment today, although the original glass has been largely replaced by presterilised, disposable plastic.

The standard method of obtaining a pure bacterial culture is the creation of a streak plate (Figure 4.2). A wire inoculating loop is used to spread out a drop of bacterial suspension on an agar plate in such a way that it becomes progressively more dilute;

A petri dish is the standard vessel for short-term growth of solid medium cultures in the laboratory. It comprises a circular dish with overlapping lid.

loop in Bunsen

Streak

Pick up a bacterial colony on sterile loop

Petri dish containing agar Flame loop

Figure 4.2 The streak plate. Streaking the sample across the agar surface eventually results in individual cells being deposited. Repeated cycles of cell division lead to the production of visible, isolated colonies. From Nicklin, J, Graeme-Cook, K & Killington, R: Instant Notes in Microbiology, 2nd edn, Bios Scientific Publishers, 2002. Reproduced by permission of Thomson Publishing Services

Figure 4.2 The streak plate. Streaking the sample across the agar surface eventually results in individual cells being deposited. Repeated cycles of cell division lead to the production of visible, isolated colonies. From Nicklin, J, Graeme-Cook, K & Killington, R: Instant Notes in Microbiology, 2nd edn, Bios Scientific Publishers, 2002. Reproduced by permission of Thomson Publishing Services

Streak

Pick up a bacterial colony on sterile loop

Petri dish containing agar Flame loop eventually, individual cells will be deposited on the agar surface. Following incubation at an appropriate temperature, a succession of cell divisions occurs, resulting in the formation of a bacterial colony, visible to the naked eye. Colonies arise because movement is not possible on the solid surface and all the progeny stay in the same place. A colony represents, in theory at least, the offspring of a single cell and its members are therefore genetically identical. (In reality, a clump of cells may be deposited together and give rise to a colony; this problem can be overcome by repeated isolation and restreaking of single colonies.)

An alternative method for the isolation of pure cultures is the pour plate (Figure 4.3). In this method, a dilute suspension of bacteria is mixed with warm molten agar, and poured into an empty petri plate. As the agar sets, cells are immobilised, and once again their progeny are all kept together, often within, as well as on, the agar. This method is especially useful for the isolation of bacteria that are unable to tolerate atmospheric levels of oxygen.

Molten agar at 45-50°

Bacterial suspension

Molten agar at 45-50°

Bacterial suspension

Colonies grow on surface and within agar

Colonies grow on surface and within agar

Figure 4.3 The pour plate. A sample of diluted bacterial suspension is mixed with molten agar and poured into a petri dish. Most bacteria can tolerate a short exposure to the agar, which is held at a temperature just above its setting point

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  • Phillipp
    WHAT IS THE FIRST STEP IN OBTAINING A PURE CULTURE?
    8 years ago

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