Exp. No. 9 Isolation of drug resistant mutant – Gradient plate technique

 Isolation of drug resistant mutant – Gradient plate technique

Introduction

Mutation is a heritable change in the nucleotide sequence of DNA. Mutations may be characterized according to either the kind of genotypic change that has occurred or their phenotypic consequences. Mutations can alter the phenotype of a microorganism in several different ways. Morphological mutations change the microorganism’s colonial or cellular morphology. Nutritional or biochemical variation may occur in a gene that encodes an enzyme involved in a metabolic pathway of amino acid synthesis. Changes in gene regulation occurs when mutation occur in a gene encoding a transcription factor. Lethal mutations prevent the reproducing capability of the organism, and when expressed, it results in the death of the microorganism.

Mutations often inactivate a biosynthetic pathway of the microorganism, and frequently make a microorganism unable to grow on a medium lacking an adequate supply of the pathway’s end product. Based on this principle microorganism are classified as Prototrophic and Auxotrophic. Prototrophic organisms (wild type) have the same nutritional requirements as that of their ancestors. They need only inorganic salts, an organic energy source such as sugar, fat, protein and water to survive and grow. That is, the Prototroph’s need only "Minimal medium" for their growth and survival. Auxotrophic mutants are unable to grow without one or more essential nutrients. Auxotrophs are mutant for particular nutrient synthesis pathway enzymes. Such an error is known as an inborn error of metabolism, whether it occurs in a bacterium or a eukaryote. An auxotroph can be grown only on an enriched medium that provides the particular nutrient that the mutant cannot metabolize on its own.

There are two classes of mutations: Spontaneous mutations and induced mutations.

            Spontaneous Mutations: A mutation without a known cause is called Spontaneous mutations. This occurs at low frequency leading to the chemical instability of purine and pyrimidine bases and also due to low level of metabolic errors, or mistakes during the DNA replication.

            Induced Mutations: Mutations that results from exposure of organisms to mutagenic agents such as ionizing irradiation, ultraviolet light or various chemicals that react with nucleic acids. In experimental organisms, researchers often treat them with these mutagens in order to increase the frequency of mutation in them. Generally, chemical mutagens induce point mutations, whereas ionizing radiations gives rise to large chromosomal abnormalities. Point mutations are simple changes in single base-pairs, the substitution of one base-pair for another, or duplication or deletion of single base-pairs. Point mutations occur at a single point on a chromosome. Missense mutation is a type of point mutation, in which a single nucleotide is changed that leads to substitution of a different amino acid and a nonsense mutation, is a point mutation, that changes a normal codon into a stop codon that does not code for an amino acid and arrest peptide synthesis without amino acid insertion resulting in a non functional protein product. Frame shift mutation, is a kind of mutation caused by the addition or deletion of nucleotides which is not a multiple of three so that the codon is read incorrectly during translation. A silent mutation causes base substitution without amino acid substitution and thus has no effect. Such substitutions will not cause any change in their product and cannot be detected without genome sequencing. In any case, the mutation events are often reversible. The subsequent mutations in the nucleotide pair restore the original wild type of phenotype. That is, a gene that has undergone mutation reverses to its original base composition. This is referred to as back-mutation, reverse mutation or reversion. Genetic and biochemical investigations in bacteriology are often initiated by isolation of mutant strains. The spontaneous mutations due to resistance in antibiotics such as Streptomycin are easily detected because they grow in the presence of antibiotic concentrations that inhibit the growth of normal bacteria.

Aim

To induce mutation and to isolate streptomycin resistant strains of bacteria (E. coli).

Materials and Methods

24 hour old nutrient broth culture of Escherichia coli, two nutrient agar deep tubes (10 ml per tube/culture), 1% Streptomycin sulphate solution (100 µg/ml), a beaker with 90% ethanol, sterile Petri plates, sterile 1 ml pipette, glass rod spreader, water bath.

Procedure

I) Preparation of gradient plate:

1.      Melt two nutrient agar plates maintained at 96ºC and cool to 55ºC

2.      Pour the contents of one agar tube into a sterile petriplate. Allow the medium to solidify in a slanting position by placing either a glass rod under one side.

3.      After the agar medium is solidified remove the glass rod and place the plate in the horizontal position.

4.      Pipette out 0.1mL of 1% Streptomycin solution into the second tube of the second nutrient agar medium.

5.      Rotate the tube between the palms and pour contents to cover the gradient layer agar and allow to the medium to solidify on a level table.

6.      Label the low and high antibiotic concentration area on the bottom of the plate.

II) Inoculation of culture:

1.      Pipette out 200µl (0.2ml) of the overnight Escherichia coli culture onto the gradient plate after 24 hours of its preparation.

2.      Spread the inoculums evenly over the agar surface with a sterile bent glass rod by rotating the plate.

3.      Incubate the inoculated plate in an inverted position at 37ºC for 48-72 hours.

4.      Observe the plate for appearance of E.coli colonies in the area of low streptomycin concentration (LSC) and high streptomycin concentration (HSC) and record the results.

Inference:

If colonies which appear in the area of high concentration streptomycin region, it will be streptomycin resistant mutants.

III. Confirming presence of Streptomycin resistant colonies of E.coli

1.      Select and mark an isolated colony of E.coli in the HSC region of the Nutrient agar plate.

2.      Pick the selected colony with a sterile inoculating loop and streak on to second gradient plate towards the HSC region.

3.      Repeat this step with one or two colonies of streptomycin resistant mutants from the HSC region.

4.      Incubate the inoculated plates in an inverted position at 37ºC for 24-72 hours.

5.      Observe the growth of streaked colonies towards the HSC region.

Result

Growth of E.coli colonies in HSC area indicates the successful isolation of streptomycin resistant mutants.