Summary
Highlights
To prepare a sufficient quantity of bacteria for antibiotic extraction, a single colony from a pure culture streak plate is used to completely cover a new agar plate. A sterile swab, possibly pre-wetted with PBS or water, is used to pick up the bacteria and then spread it evenly across the surface of the new plate using a four-quarter turn method. The plate is then incubated for 24 hours to grow a dense 'lawn' of bacteria, visible as a translucent film.
Once a dense bacterial lawn has grown, the next step is to extract the antibiotics. The agar containing the bacteria is carefully cut into 1 cm pieces, typically using toothpicks, and transferred into a labeled wide-mouth bottle. Safety precautions are emphasized for handling bacteria. The bacteria are then quickly killed and their cell membranes broken open in an ethanol-water bath supercooled with dry ice. This process takes about 20 minutes to completely freeze the agar and lyse the bacteria.
The next stage of extraction takes place in a chemical fume hood due to the flammability and irritant properties of ethyl acetate. Personal protective equipment, specifically eyewear and a splash guard, must be worn. 15 milliliters of ethyl acetate are added to the frozen agar in the bottle using a specialized pipette. Subsequently, 10 milliliters of water are added. The bottle is capped and placed on a shaker overnight. This process separates the mixture into two layers: an organic layer (top) containing the antibiotics and an aqueous layer (bottom). Lab technicians will then extract the organic layer, dry it, and provide it as a powdered chemical.
The dried antibiotic powder is reconstituted by adding 100 microliters of methanol, ensuring it completely dissolves. This solution is then tested against 'escaped pathogens'. A new agar plate is divided into sections (halves or quarters) based on the number of pathogens to be tested. Each section is inoculated with a different escaped pathogen using a sterile swab to create a uniform spread. Ten microliters of the reconstituted antibiotic solution are then pipetted directly onto the bacteria in each section. A control is also set up by adding ten microliters of pure methanol to a separate area to assess the antibiotic's activity beyond the effects of the solvent itself. Alternatively, paper disks soaked in the antibiotic or methanol can be placed on the plate.