Isolation of Mutant Bacteria
Important advances in genetics were made in the early 1950s through studies of bacteria. As prokaryotic organisms, bacteria have certain advantages over eukaryotic organisms because they are haploid and have an extremely short generation time. Mutant bacteria can be identified easily. The growth of some mutant bacteria depends on whether a certain substance is present in the medium (auxotrophism). Bacterial cultures are well suited for determining mutational events since an almost unlimited number of cells can be tested in a short time. Without great difficulty, it is possible to detect one mutant in 10e7 colonies. Efficiency to this degree is not possible in the genetic analysis of eukaryotic organisms.
A. Replica plating to recognize mutants
In 1952, Joshua and Esther Lederberg developed replica plating of bacterial cultures. With this method, individual colonies on an agar plate can be taken up with a stamp covered with velvet and placed onto other culture dishes with media of different compositions. Some mutant bacteria differ from non mutants in their ability to grow. Here several colonies are shown in the Petri dish of the initial culture. Each of these colonies originated from a single cell. By means of replica plating, the colonies are transferred to two new cultures. One culture (right) contains an antibiotic in the culture medium; the other (left) does not. All colonies grow in normal medium, but only those colonies that are antibiotic resistant owing to a mutation grow in the antibiotic-containing medium. In this manner, mutant colonies can be readily identified.
B. Mutant bacteria identified through an auxotrophic medium
Here it is shown how different mutants can be distinguished, e.g., after exposure to a mutagenic substance. After a colony has been treated with a mutagenic substance, it is first cultivated in normal nutrient medium. Mutants can then be identified by replica plating. The culture with the normal medium serves as the control. In one culture with minimal medium, from which a number of substances are absent, two colonies do not grow (auxotrophic mutants). Initially, it is known for which of the substances the colonies are auxotrophic. If a different amino acid is added to each of two cultures with minimal medium, e.g., threonine (Thr) to one and arginine (Arg) to the other, it can be observed that one of the mutant colonies grows in the threonine-containing minimal medium, but the other does not. The former colony is dependent on the presence of threonine (Thr – ), i.e., it is an auxotroph for threonine. The other culture with minimal medium had arginine added. Only here can the other of the two mutant colonies, an auxotroph for arginine (Arg – ), grow. After the mutant colonies requiring specific conditions for growth have been identified, they can be further characterized. This procedure is relatively simple and makes rapid identification of mutants possible. Many mutant bacteria have been defined by auxotrophism. The wild-type cells that do not have special additional growth requirements are called prototrophs (Figures adapted from Stent & Calendar, 1978).