To prepare the infecting pen, melt the end of a 200-microliter micropipette tip and insert a 0.15-millimeter insect minutien pin into the molten plastic. Allow the plastic to solidify so that the pin is held in place with 0.5 centimeters of pin extending from the plastic.
Anesthetize flies with carbon dioxide, and prick the flies in the sternal pleural plate of the thorax with the needle avoiding the attachment sites of the wings and the legs. Remove the flies from the minutien pin using soft forceps. Make sure to dip the pin into the bacterial suspension between pricking each fly.
Place the pricked flies into a fresh file with new fly medium laying the vial on its side until all of the flies have recovered from the anesthesia.
