- Flea Crushing
Beforehand, gather infected fleas into an microcentrifuge tube and place in -80 freezer to kill fleas. Remove fleas from freezer. Sterilize fleas with two washes of 95% ethanol (add ethanol to tube and pipet up and down to wash fleas). Remove ethanol from fleas and wash with sterile water. Dump fleas on an empty, sterile Petri plate and remove remove as much water as possible. Spread the fleas out and allow them to dry for 15 minutes. While the fleas are drying, label and number microcentrifuge tubes, one for each flea. Using wide bore pipet tips, measure out and place 20 uL of glass sand in each tube. Using ethanol-sterilized forceps, transfer one flea to each tube, sterilizing the forceps each time you transfer fleas infected with a different substance. Stick the end of a 1000 uL plastic pipet tip in a flame to soften the plastic. Remove from the flame and allow the plastic to cool or press it against the inside of a microcentrifuge tube to blunt it (just make sure that there is no longer an opening at the end of the tip). Use the tip to grind a flea into the glass sand. Leave the pipet tip in the glass sand. Use a new tip for each flea. Add 500 uL of broth (PBS or TB) to each tube, rinsing off and discarding the melted pipet tips. Vortex each tube and plate 100 uL of each flea onto its own plate (TB with any necessary antibiotics). Incubate plates at 30 degrees C. Save fleas in 4 degree C fridge. After 24 hours count the colonies on the plates.