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BACKGROUND
A heritable alteration in the nucleotide sequence of DNA is called mutation and is characterized either by the type of genotypic change that has taken place or the phenotypic consequences. The phenotype of an organism can change in several ways because of mutation. Morphological mutation alters the colonial or cellular morphology of the microorganism. Any changes in the nutritional or biochemical properties in the gene indicate a particular enzyme that is involved in the metabolic pathway of amino acid synthesis. Gene regulation alteration occurs whenever there is mutation occurring in a gene that is encoding transcription later. Lethal mutations prohibits the reproducing calibre of the microorganism and when expressed it causes death of the organism. Mutation disturbs the biosynthetic pathway of the microorganism and prevents the growth of the organism in the medium that lacks the required supply of pathways end product. Depending on this microorganism are differentiated into phototrophic and Auxotrophic organisms. Phototrophic organisms are those with same nutritional requirements as their ancestors and require minimal medium for their growth. Auxotrophic mutants are not capable of growing without required nutrients. They are the mutants for specific nutrient synthesis pathway enzymes. An auxotroph can only grow in an enriched medium that offers specific nutrient that the mutant cannot metabolize on its own.
There are two kinds of mutation that is spontaneous mutation and induced mutation. Spontaneous mutation is one that occurs without any known cause. It occurs at very low frequency thereby making purine and pyrimidine unstable chemically. Whereas induced mutation occurs because of exposure of organisms to some mutagenic agents like ionizing irradiation or other chemicals that react with nucleic acids.
In bacteriology, genetic and biochemical research are started by isolating mutant strains. Spontaneous mutations can be easily recognized because of resistance to antibiotics and they can grow in the presence of an antibiotic that blocks the growth of regular bacteria.
Replica plating method: When an organism is capable of producing mutant strains that are resistant to antibiotics, it is important to study the type of mutation whether it is induced or spontaneous. From a large number of colonies it is difficult to identify mutant colonies but it becomes possible by replica plating method. This method helps in isolation of both nutritional mutants as well as antibiotic resistant mutants. It involves replicating the original plate having sensitive cells into two or more plates having either T1 phage or streptomycin.
Replica plating assists in observing microbes under various growth conditions. The bacteria are allowed to grow in a condition that doesn’t support the mutation. With these technique members of each colony is shifted to selective environment. Microbes that fail to grow on minimal medium are auxotrophic strains.
The basic objective of this experiment is to isolate auxotrophic antibiotic resistant mutant bu induced mutagenesis in bacteria by method of replica plating.
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REQUIREMENTS
Sample: 24 hour nutrient broth culture of E.coli.
Chemicals: Minimal salt sugar with glucose.
3 10ml nutrient agar deeps
1% streptomycin sulphate solution.
Others: Sterile petridishes
Glass rod
Beaker containing 95% ethanol
Bent glass rod
Colony counter
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PROCEDURE
Take the nutrient agar deep tubes and melt it in a hot water both that is maintained at a temperature of 960C. Then allow it to cool down to a temperature of 550C.1 Now pour it on two sterile petriplates and leave it for some hours to get solidified in a horizontal position. Now add 0.1% streptomycin with a help of sterile pipettes into the third tube of the molten nutrient agar and mix it properly and pour it into sterile petriplate and allow it to get solidified.2 Now take E.coli test culture and add 200 microlitre of it to the surface of the nutrient agar plate. With the help of alcohol dipped bent glass shown in little flame spread the innoculum throughout the plate evenly. Now keep the plate in incubator for about 24-48 hours at 370C.3 After incubation period observe the E.coli colonies on the plate and it is the master plate. Now next day sterile velvet colony was lowered carefully and then pressed into the colonies of the E.coli present in the master plate. By avoiding changing the carrier position the velveteen colony was pressed slowly into the nutrient agar plate followed by nutrient agar plate that is supplemented with streptomycin.4 Now keep the inoculated plates, nutrient agar and the streptomycin agar plates in an inverted position for about 48-72 hour at a temperature of 370C. Keep the master plate in the refrigerator.5 After incubation count the total number of colonies present in the replica plates with nutrient agar and the streptomycin agar by using colony counter. The colony that appears on the nutrient agar plates and the streptomycin plates were seen and compared.6
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CONCLUSION
This is one of the best methods for isolating Auxotrophic antibiotic resistant bacteria for studying its further properties.
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REFERENCES
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Brown E. Alfred, Benson’s Microbiological Applications, ninth edition, McGraw Hill Publication
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Prescott M. Lansing, Harley P. John, Klein A. Donald, Laboratory Exercises in Microbiology, fifth edition, McGraw-Hill college division.
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Aneja K R. Experiments in microbiology, plant pathology and biotechnology, fourth edition, New Age International (P) Limited .Publishers.
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Prescott M. Lansing, Harley P. John, Klein A. Donald, Microbiology, sixth edition, McGraw-Hill Higher Education
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Lederberg, J and Lederberg, EM (1952) Replica plating and indirect selection of bacterial mutants. J Bacteriol. 63: 399–406
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Madigan & Martinko, Brock Biology of Microorganisms, Eleventh Edition, Pearson Prentice Hall, Inc.
