PROKARYOTES CAN BE FOUND GROWING EVEN IN the harshest climates and the most severe conditions. Environments that no unprotected human could survive, such as the ocean depths, volcanic vents, or the polar regions, have thriving species of prokaryotes. Indeed many scientists believe that if life exists on other planets, it may resemble these microorganisms. Each individual species, however, has a limited set of environmental conditions in which it can grow; even then, it will grow only if specific nutrients are available. Some prokaryotes
In nature, many different organisms, including bacteria, live together as a mixed population, jointly contributing to numerous activities and processes in their surroundings. In the laboratory, bacteria are isolated and grown in pure culture in order to study the functions of a particular species. A pure culture is defined as a population of organisms descended from a single cell and is therefore separated from all other species. Only an estimated 1% of all prokaryotes, however, can currently be cultured successfully. This makes it exceedingly difficult to study the vast majority of
84 Chapter 4 Dynamics of Prokaryotic Growth environmental microorganisms. Fortunately for humanity, most known medically significant bacteria can be grown in pure culture.
Pure cultures are obtained using a variety of special techniques. All glassware, media, and instruments must be sterile, or free of microbes, prior to use. These are then handled using aseptic techniques, which are procedures that minimize the chance of other organisms being accidentally introduced. The medium that the cells are grown in, or on, is a mixture of nutrients dissolved in water and may be in a liquid broth or a solidified gel-like form. ■ aseptic techniques, p. 112 ■ sterilization, p. 109
The basic requirements for obtaining a pure culture are a solid medium, a media container that can be maintained in an aseptic condition, and a method to separate individual cells. A single bacterium, supplied with the right nutrients, will multiply on the solid medium in a limited area to form a colony, which is a mass of cells all descended from the original one (figure 4.1). About 1 million cells are required for a colony to be easily visible to the naked eye.
Agar, a polysaccharide extracted from marine algae, is used to solidify a specific nutrient solution. Unlike other gelling agents such as gelatin, very few bacteria can degrade agar. It is not destroyed at high temperatures and can therefore be sterilized by heating, a process that also liquefies it. Melted agar will stay liquid until it is cooled to a temperature below 45°C. Therefore, nutrients that would be destroyed at high temperatures can be added at lower temperatures before the agar hardens. Once solidified, an agar medium will remain so until it is heated above 95°C. Thus, unlike gelatin, which is liquid at 37°C, agar remains solid over the entire temperature range at which the majority of bacteria grow. Agar is also translucent, enabling colonies embedded in the solid medium to be seen more easily. ■ polysaccharide, p. 29
The culture medium is contained in a Petri dish, a two-part, glass or plastic, covered container. While not airtight, the Petri dish does exclude airborne microbial contaminants. Once a Petri dish contains a medium, it is most commonly referred to
as a plate of that medium type—for example, a nutrient agar plate or, more simply, an agar plate.
The streak-plate method is the simplest and most commonly used technique for isolating bacteria (figure 4.2). A sterilized inoculating loop is dipped into a solution containing the organism of interest and is then lightly drawn several times across an agar plate, creating a set of parallel streaks covering approximately one-third of the plate. The loop is then sterilized and a new series of parallel streaks are made across and at an angle to the previous ones, covering another third of the plate. This action drags some of those cells streaked over the first portion of the plate to an uninoculated portion, creating a region containing a more dilute inoculum. The loop is sterilized again, and another set of parallel streaks are made, dragging into a third area some of the organisms that had been moved into the second section. The object of this is to reduce the number of cells being spread with each successive series of streaks, effectively diluting the concentration of cells. By the third set of streaks, cells should be separated enough so that distinct well-isolated colonies will form.
Once a pure culture has been obtained, it can be maintained as a stock culture, a culture stored for use as an inoculum in later procedures. Often, stock cultures are stored in the refrigerator on an agar slant. This is agar medium in a tube that was held at a shallow angle as the medium solidified, creating a larger surface area. For long-term storage, stock cultures can be frozen at -70°C in a glycerol solution. The glycerol prevents ice crystals from damaging cells. Alternatively, cells can be lyophilized, or freeze-dried. ■ lyophilization, p. 124
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