replica plating experiment was performed to prove the

Ame’s Test, Replica plate technique and Fluctuation test

Fluctuation test

This test was devised by Luria and Delbruck (1943) for investigating the response of bacterial populations to changes in the environment. At that time two hypotheses co-existed-

1.  Genetic changes occur adaptively, i.e. as a result of environmental Influences on the cells.

2.  Genetic changes occur spontaneously i.e. independently of the environment. If mutants arose spontaneously and randomly, the number of mutant cells should vary considerably from culture to culture. This was resolved by an experiment called the fluctuation test that may be performed in the following manner.

• A young bacterial suspension containing about 500 E. coli cells per mL is divided into two cultures, A and B of 10 ml each. Culture A is then further subdivided into a series of 50 tubes, each containing 0.2 ml.
• Both cultures are incubated until a predetermined population density is attained. Under these conditions, the cell concentration in the two cultures will be approximately equal.
• The number of streptomycin-resistant colonies which develop when the contents of each of the small tubes are poured onto the surface streptomycin-containing agar plates is then compared with the number which develops when a similar series of 0.2 ml samples from culture B are pipetted onto the surface of streptomycin containing agar.
• If bacterial resistance developed by direct adaptation to the streptomycin, there should be no significant difference in the numbers of resistant colonies on any of the plates derived from the two cultures.
• By this theory, it should make no difference whether the cultures were incubated in a series of separate tubes or were all grown together in one large tube since they are all presumed to be sensitive until brought into contact with streptomycin.

• If on the other hand, streptomycin resistance resulted from spontaneous mutations during growth in streptomycin-free medium: there should be a large fluctuation between the numbers of resistant colonies, which developed from each of the 50 small tubes.
• This fluctuation in the number of resistant bacteria in a series of parallel cultures is a consequence of the randomness in time of the occurrence of spontaneous mutation to resistance occurred early during cultural growth, the resistant mutant would pass through a large number of cell divisions, and there would be many resistant bacteria present at the time of testing.
• If the mutation to resistance occurred just before the cells were added to streptomycin, only one resistant colony should appear on the plate.
• A similar series of samples taken from the same container (culture B) serves as a control on the experimental technique. By either hypothesis, random samples taken from it should contain the same number of resistant bacteria.
• The result obtained from the fluctuation test supports the spontaneous mutation theory.

Replica plate technique

• This technique was devised by Lederberg and Lederberg (1952). The replica plating technique was used to verify the spontaneous origin of bacterial resistance.
• By this procedure samples from all the colonies on a plate may be transferred simultaneously to another plate by means of velveteen covered stamp pad.
• Colonies picked up from first plate this plate is known as ‘master plate’, colonies transferred on second plate first plate is known as ‘replica plate’.

• Several million streptomycin-sensitive bacteria were spread onto the surface of a streptomycin-free agar plate and incubated for a few hours to allow microcolonies.
• The sterile velveteen stamp was then pressed onto the plate containing these microcolonies so that a few of the bacteria from each of them adhered to the fibers.
• Next, the pad was pressed against the surface of the streptomycin-containing plate, and the bacteria transferred to it were incubated until resistant colonies appeared.
• These colonies were then superimposed over the original plate, and the bacterial growth in the area corresponding to the resistant colonies was picked into a fresh nutrient broth, there will be almost a hundredfold increase in the proportion of resistant mutants after incubation of this fresh culture.
• After overnight incubation of this new culture, diluted aliquots of it were spread onto the surface of the fresh, drug (streptomycin) free agar plates, and the same procedure of incubation and replication was repeated as in the previous step.
• This time more resistant colonies were found on the drug-containing plates than in the first cycle. A colony was located on the plain agar plate (without streptomycin) or master plate which exactly corresponded to a colony on the drug-containing replica plate. The colony on the plain agar was picked and proved to consist entirely of resistant cells.
• The Ames test is a widely employed method that uses bacteria to test whether a given chemical can cause mutations in the DNA of the test organism. More formally, it is a biological assay to assess the mutagenic potential of chemical compounds.
• The Ames test uses several strains of the bacterium Salmonella typhimurium that carry mutations in genes involved in histidine synthesis.
• These strains are auxotrophic mutants, i.e. they require histidine for growth, but cannot produce it. The method tests the capability of the tested substance in creating mutations that result in a return to a “prototrophic” state so that the cells can grow on a histidine-free medium.
• The tester strains are specially constructed to detect mutations in the genes required to synthesize histidine, so that mutagens acting via different mechanisms may be identified. The bacteria are spread on an agar plate with a small amount of histidine.
• This small amount of histidine in the growth medium allows the bacteria to grow for an initial time and have the opportunity to mutate.
• When the histidine is depleted only bacteria that have mutated to gain the ability to produce their own histidine will survive. The plate is incubated for 48 hours. The mutagenicity of a substance is proportional to the number of colonies observed.
• Isolate an auxotrophic strain of Salmonella typhimurium for histidine. (ie. His-ve)
• Prepare a test suspension of his—ve Salmonella typhimurium in a plain buffer with test chemical (e.g. 2—-aminofluorene). Also add a small amount of histidine. Note: small amount of histidine is required so bacteria start growing. Once histidine is depleted only those bacteria mutated to gain the ability to synthesize histidine form colonies.
• Prepare a control suspension of His-ve Salmonella typhimurium but without test chemicals.
• Incubate the suspensions at 37°C for 20 minutes
• Prepare the two agar plate and spread the suspension on agar plate.
• Incubate the plates at 37°C for 48 hours.
• After 48 hours count the number of colonies in each plate.

Result Interpretation

• The mutagenicity of chemicals is proportional to a number of colonies observed.
• If there is a large number of colonies on the test plate in comparison to control, then such chemical are said to be mutagens.
• Very few numbers of colonies can be seen on control plate also. This may be due to spontaneous point mutation on hisidine encoding gene.