Isolation of antibiotic resistance mutant by replica plating

what is mutants and how many mutants are there?
how to isolate antibiotic resistance mutants?

1. Isolation of antibiotic resistance mutant by replica plating technique Presented by Fathima Hameed

2. WHAT IS MUTATION ? Mutation is a heritable change in the nucleotide sequence of DNA. Characterized as either genotype / phenotype Changes in gene regulation Changes in cellular morphology Lacking of nutrients, microbes unable to grow on the medium. Based on this category, microorganisms divided into 2 types; Prototrophic - unable to grow, lacking same nutrition's like wild type Auxotrophic -unable to grow, lacking some essential nutrients.

3. CLASSES OF MUTATIONS Mutation

4. WHAT IS ANTIBIOTIC RESISTANCE MUTANTS ? Heritable change of mutant strains are mostly resistant to antibiotics. Natural selection of antibiotic resistance mutants: Large bacterial population is mainly consists of susceptible and only pair of bacteria that are resistant to antibiotics. When Antibiotics are added, susceptible bacterium will be killed and resistant bacteria will be multiplied.

5. REPLICA PLATING It was developed by Joshua and Esther Lederberg in 1952. This technique is used to identify mutant colonies from large population. it also used to isolate both nutritional and antibiotic resistance mutants. Master plate Sterilized velveteen Wooden holder

6. What are the Materials were used? 24 hrs old bacterial culture Nutrient agar medium Streptomycin Ethanol Sterile petridishes Sterile velveteen cloth Glass rod Quebec colony counter

7. Procedure for isolation of antibiotic resistance mutants by replica plating method Preparation Of Master Plate: The nutrient agar medium plate was prepared Pour the medium into sterile petriplate in a horizontal position. Add 0.1 % of streptomycin in the nutrient agar, mixed well and poured into the another side of the plate. Add 200 µl of bacterial culture on the top of the nutrient medium. Evenly spread out the culture on the surface of the medium using glass rod. Incubate the plates in an inverted position at 37ºC for 24-48 hrs. After incubation, observed the colony growth on the master plate.

8. Procedure for isolation of antibiotic resistance mutants by replica plating method Preparation Of Replica Plate: The bottom of the master plate was note down nutrient agar and nutrient agar supplemented with streptomycin. Take a sterile velveteen cover was carefully press down on the nutrient as well as the streptomycin plate . Master plate was stored in refrigerator. Inoculated plates were incubated in an inverted position at 37ºC for 48-72 hrs . After incubation period, observed colony growth on the replica plate and counted the colonies using Quebec colony counter. To comparing the colonies on both nutrient and streptomycin plate.

9. Replica plating method

10. Results The bacterial colonies which appeared in the streptomycin supplemented plate, it indicates the presence of streptomycin resistance mutants and the number of colonies were counted using colony counter.

11. References http://vlab.amrita.edu/?sub=3&brch=76&sim=1089&cnt=1#:~: text=Replica%20Plating%20Method%3A&text=The%20technique%20was%20developed%20by,mutants%20and%20antibiotic%20resistant%20mutants . https://www.reactgroup.org/toolbox/understand/antibiotic-resistance/mutation-and-selection / https:// www.google.com/url?sa=i&url=https%3A%2F%2Fwww.researchgate.net%2Ffigure%2FReplica-plates-of-Streptomyces-griseus-WAc-86-A-Original-plate-on-complete agar_fig1_10199384&psig=AOvVaw1mr1nlfaIRMGgiCeiWfdM9&ust=1601123047868000&source= images&cd = vfe&ved =0CA0QjhxqFwoTCJi-gKqmhOwCFQAAAAAdAAAAABAK

12. Thank you…,