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Lodish H, Berk A, Zipursky SL, et al. Molecular Cell Biology. 4th edition. New York: W. H. Freeman; 2000.

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Molecular Cell Biology. 4th edition.

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Section 6.1Growth of Microorganisms in Culture

Cultured cells have several advantages over intact organisms for research on fundamental aspects of cell biology. First, most animal and plant tissues comprise a variety of different types of cells, whereas cells of a specific type can be grown in culture; thus cultured cells are more homogeneous in their properties than in vivo cells in tissues. Second, experimental conditions can be controlled much more rigorously in culture than in an organism. By manipulating the growth conditions, for example, one can measure the effects of specific chemicals and growth factors on a particular cell type in culture. A third reason for preferring cultured cells is that in many cases a single cell can be readily grown into a colony, a process called cell cloning, or simply cloning. The resulting strain of cells, which is genetically homogeneous, is called a clone. This simple technique, which is commonly used with many bacteria, yeasts, and mammalian cell types, makes it easy to isolate genetically distinct clones of cells.

Many Microorganisms Can Be Grown in Minimal Medium

Among the advantages of using microorganisms such as the bacterium Escherichia coli and the yeast Saccharomyces cerevisiae are their rapid growth rate and simple nutritional requirements, which can be met with a minimal medium (Table 6-1, top). A minimal medium for such microorganisms can contain glucose as the sole source of carbon; metabolism of glucose to smaller molecules (e.g., CO2, ethanol, or acetic acid) can generate the ATP necessary for energy-requiring activities of the cells. The sole nitrogen source in a minimal medium can be ammonium (NH4+), from which the cells can synthesize all the necessary amino acids and other nitrogen-containing metabolites. Salts and trace elements are the only other components of a minimal medium.

Table 6-1. Growth Media for Common Bacteria and Yeasts.

Table 6-1

Growth Media for Common Bacteria and Yeasts.

Many prokaryotes (i.e., bacteria) and single-celled eukaryotes such as yeast, both of which grow in nature as single cells, are easily grown in culture dishes — usually on top of agar, a semisolid base of plant polysaccharides. The agar is first dissolved in a heated nutrient medium, and the solution is poured into petri dishes; as it cools, the solution solidifies. A dilute suspension of cells then is dispersed on top of the agar; in time each cell grows into a discrete colony (Figure 6-1a). Since the cells in a colony all derive from a single cell, they form a clone and have identical genomes (DNA). All the cells in a clone generally express the same set of genes and contain the same enzymes and other constituents in similar proportions. The division time for E. coli and similar microorganisms ranges from 20 minutes to 1 hour. Thus a single E. coli cell, which divides approximately every 30 minutes, can grow into a colony containing 107 – 108 cells in 12 hours (224 = 1.7 × 107).

Figure 6-1. Plating of the yeast Saccharomyces cerevisiae on agar plates.

Figure 6-1

Plating of the yeast Saccharomyces cerevisiae on agar plates. (a) Colonies of the yeast S. cerevisiae growing on a plate of agar containing only glucose, adenine, and salts. All the cells in a colony are descendants of a single cell and thus form a clone. (more...)

Because yeasts, unlike most eukaryotic organisms, grow as single cells and can be grown in a simple defined medium similarly to bacteria, they are a popular choice for studies of eukaryotic cell function. The entire life cycle of yeasts can be studied in culture, and colonies can be grown from a single vegetative cell (a growing cell) or from a single spore (a dormant cell). Studies with yeasts have provided valuable information on such subjects as the cellular mechanisms for controlling DNA synthesis, genetic recombination, and the gene products that regulate the cycle of events leading to replication and segregation of chromosomes and ultimately cell division (Chapter 13).

Mutant Strains of Bacteria and Yeast Can Be Isolated by Replica Plating

If a random mutation in a microorganism cultured, or plated, on a minimal agar medium renders cells unable to synthesize a necessary metabolite, and this metabolite is not supplied in the medium (e.g., the amino acid arginine), then the cells will die. But if the cells are grown on a rich medium that supplies the metabolite whose synthesis is prevented by a mutation, then the mutant cell can survive and grow into a colony in which all the cells have the same mutation (Table 6-1, bottom).

A mutant colony, say, an Arg colony, can be detected by replica plating, a technique developed by Joshua and Esther Lederberg in the 1940s. The initial plate of multiple colonies growing on rich medium is called the master plate (Figure 6-2). A circular, velvet-covered stamp equal in size to the master plate is pressed onto it, so that the stamp picks up cells from each colony. The stamp is then used to deposit cells onto a second plate containing minimal agar medium without arginine (Arg medium) and also onto a third plate containing minimal agar medium with arginine (Arg+ medium). Cells that can synthesize arginine grow into colonies on both the Arg and Arg+ plates. However, a clone of cells on the master plate with a mutation in a gene required for arginine synthesis cannot grow on the Arg medium, resulting in a space on the Arg plate corresponding to the position of this clone on the master plate and Arg+ plate. This clone of cells, thus identified as being defective in the synthesis of arginine, then can be isolated from the master plate.

Figure 6-2. Replica plating is used to detect random mutations in bacterial and yeast cultures that produce cells differing in a single genetic trait from other cells in the culture.

Figure 6-2

Replica plating is used to detect random mutations in bacterial and yeast cultures that produce cells differing in a single genetic trait from other cells in the culture. A velvet-covered stamp equal in size to a petri dish is pressed onto the master (more...)

Repeated replica plating of a master plate with paired minimal-medium plates containing or lacking different specific nutrients (e.g., amino acids, nucleic acid bases, vitamins) can identify clones of cells with mutations in the genes required for synthesis of different biological molecules. This approach has been used to isolate thousands of different bacterial and yeast nutritional auxotrophs; these are strains that are unable to grow in the absence of a specific nutrient.

Under standard culture conditions, random mutations occur in E. coli genes at a frequency of 1 in 106 – 108 base pairs per generation. Thus, by the time a single cell has divided to produce a few hundred or thousand cells, some individual genes will differ among cells in the clone (see Figure 6-1b). To maintain the genetic homogeneity of a clone, it must be recloned frequently under selective conditions that permit only cells with a particular set of desired characteristics to survive and grow. For example, to maintain an Arg clone, isolated as described above, the cells are replated on minimal medium plus arginine; only cells that can synthesize all the metabolites required for growth except arginine will grow on this selective medium. The use of mutant clones both to map the position of genes on E. coli and yeast chromosomes and to unravel various biosynthetic pathways is discussed in Chapter 8.

SUMMARY

  •  Free-living microorganisms like the bacterium E. coli and the budding yeast S. cerevisiae can be grown in minimal media consisting of glucose as a source of carbon and energy, ammonium as a source of nitrogen, and salts and trace elements.
  •  Under suitable conditions, a single cell grows into a colony of cells on agar medium. The resulting population of genetically identical cells derived from a single parent cell is called a clone.
  •  Replica plating of colonies of microorganisms on two different types of agar medium allows detection of mutant clones that can form colonies on one type of medium but not another (see Figure 6-2).
  •  Nutritional auxotrophs, a common type of mutant, cannot synthesize an essential metabolite that wild-type cells produce.

By agreement with the publisher, this book is accessible by the search feature, but cannot be browsed.

Copyright © 2000, W. H. Freeman and Company.
Bookshelf ID: NBK21593

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