Isolation of Antibiotic-Resistant Bacteria from a Water Sample for Molecular Analysis

Begin with a water sample that contains waterborne bacteria, and filter it to remove debris.

Transfer the diluted filtrate onto low-nutrient agar plates, each supplemented with a specific antibiotic. Spread the filtrate and incubate.

Bacteria carrying a specific antibiotic-resistance gene grow on the corresponding antibiotic plates and form colonies.

Count the colonies to determine the colony-forming units (CFUs), representing the number of antibiotic-resistant bacteria.

Select a single colony from each plate and suspend it in sterile distilled water.

Heat the suspension to lyse the cells and release intracellular contents, including bacterial DNA that contains the antibiotic-resistance gene.

Centrifuge the tubes to separate the debris and collect the supernatant containing DNA.

Add amplification reagents to the extracted DNA.

Place the tubes in a thermal cycler and run the program to amplify a specific region of bacterial DNA.

The amplified DNA is ready for sequence analysis to identify the antibiotic resistance gene.

Process the water sample aseptically by filtering it through a sterile muslin cloth to remove any particulate matter. Then, serially dilute the filtered water sample for furth