Question

33. Describe an experiment in which you would use replica plating using the following prompts: ( 10 total)

1. What is the question you are trying to answer that would require replica plating?

2. What tools do you need? ( bacterial strains, types of agar, nutrients, etc)

3. Why do you need to use replica plating instead of regular plating?

4. What are you expected results?

5. Is this an example of direct selection or indirect selection? Explain.

Answer 1

a) Replica plating followed for isolation of auxotrophic mutant. Auxotrophic organism means they require specific growth factors ( like vitamin, amino acid, sugar etc) for growth,without them they are unable to grow.

b)for this experiment two media require. One is complete medium and another is minimal medium. Complete medium contain is rich in growth factors. Minimal medium contain less amount of nutrients.

Organism is E.coli used for this experiment.

If an organism has the ability to produce mutant strains resistant to antibiotics, the nature of mutation, whether it is spontaneous or induced have to be tested. It would be a difficult task to identify a few mutant colonies from a vast population of 100-500 colonies. This can be accomplished by a replica plating technique. The technique was developed by Joshua and Esther Lederberg in 1952 for providing the direct evidence for the existence of pre-existing mutations. This technique isolates both nutritional mutants and antibiotic resistant mutants.
Replica plating allows the observation of microbes under a series of growth conditions. The bacteria are grown in an environment that is not selective for given mutation. This technique is used to transfer the members of each colony to a selective environment. A simple velveteen covered colony transfer device is used to transfer the colonies in nutrient agar medium supplemented with or without a particular antibiotic or nutrient( vit .B12 or amino acid). The fibers of velvet act as fine inoculating needles, picking up the bacterial cells from the surface of this master plate. The velvet with its attached microbes is then touched to the surface of a sterile agar plate, inoculating it. In this manner, microbes can be repeatedly stamped onto media of differing composition. By comparing the presence of colonies following incubation we can indirectly determine the mutant colonies by their absence in the selective environment. A colony that develops on a complete medium fail to develop on a minimal medium that lacks a specific growth factor, the occurrence of a nutritional mutant is indicated. The microbes that do not grow on the minimal medium represent auxotrophic strains. A simple velveteen covered colony transfer device is used to transfer the colonies in nutrient agar medium supplemented with or without a particular antibiotic or nutrient. A colony that develops on a complete medium fail to develop on a minimal medium that lacks a specific growth factor, the occurrence of a nutritional mutant is indicated. The microbes that do not grow on the minimal medium represent auxotrophic strains.

c) by using this experiment we get direct results of mutant colonies.

d) Auxotrophic mutant will not grow on minimal media. But there growth were observed on complete medium.

e) It is example of direct selection.