Stamp technology (microbiology)

from Wikipedia, the free encyclopedia

The stamp technology ( English replica plating , in German about replication plating ) is used in microbiology to transfer some material from every bacterial colony that has grown on the surface of one gel culture medium simultaneously and in the same arrangement onto the surface of another gel culture medium (stamp imprint) . Joshua Lederberg and his wife Esther Lederberg were the first to describe and use this technique.

execution

With the stamp technique, a little material from all colonies that have formed after a smear on the surface of a gel-like nutrient medium (so-called "plate") is transferred as inoculation material in the same arrangement of the individual colonies to the surface of another gel nutrient medium. As a rule, round Petri dishes are used as culture vessels in which the gel culture media are located . A so-called Lederberg stamp is used for the transfer : a sterile velvet cloth is stretched over a cylinder with the same diameter as the inner diameter of the Petri dish . First, this stamp is brought into contact with the plate overgrown with colonies and then with the plate that is not yet overgrown by gently pressing it on. As a result, a little material from each colony is transferred in the same order to the as yet bare plate. This "replica plate" is incubated for the colonies to grow. The pile fibers of the velvet prevent the colonies from smearing. Before using velvet, colonies were transferred with sterile toothpicks (method according to E. Tatum) or with sterile filter paper (method according to N. Visconti). From a starting plate (matrix) overgrown with colonies, several impressions can be made on different gel media.

Application examples

Deficiency mutants

The stamping technique can be used to find and isolate deficiency mutants, i.e. mutants of microorganisms which, unlike the original strain , cannot produce certain substances required for their growth and reproduction and are therefore dependent on the presence of these substances in the culture medium . For this purpose, colonies are created on the surface of a suitable rich gel nutrient medium (complete medium ) of isolated individuals of a population , which colonies (in the ideal case) each emerged from a single individual through its reproduction. Ideally, each colony consists of many genetically uniform individuals, i.e. they form a clone . A replica plate is created using the stamping technique. The composition of this replica plate lacks a certain nutrient ( deficiency medium ) which the deficiency mutants in search of need because, in contrast to the original strain, they cannot produce them themselves. In contrast to the original strain, the deficiency mutants sought cannot grow or reproduce here. On the surface of the replica plate, the colonies of the mutants sought are missing in the colony pattern. By comparing the colony patterns of the master plate and the replica plate, one can see which of the colonies on the master plate consist of the deficiency mutants sought and from which the deficiency mutants can be inoculated for further cultivation.

Resistant (transgenic) strains

When generating transgenic bacteria in genetic engineering , the success rate is relatively low and the transgenic bacterial strains generated must be isolated. If you also plant antibiotic resistance ( e.g. against methicillin ) with the alien gene used , transgenic bacterial strains can be easily isolated. If there are several colonies on a nutrient medium, among which there could be a transgenic colony, a copy with all colonies is stamped on a nutrient medium mixed with the antibiotic (in this case methicillin). Every colony that can grow on this nutrient medium contains the desired transgenic bacteria.

literature

Individual evidence

  1. ↑ Lederberg, J and Lederberg, EM (1952) Replica plating and indirect selection of bacterial mutants. J Bacteriol . 63 : 399-406. ( Full text )
  2. ↑ J. Lederberg, EM Lederberg: Replica plating and indirect selection of bacterial mutants. In: J Bacteriol. (1952), Vol. 63 (3), pp. 399-406. PMID 14927572 ; PMC 169282 (free full text)