Isolation of Bacteria from the Mosquito Midgut

Begin with a slide containing an anesthetized adult mosquito in a drop of phosphate-buffered saline or PBS.

Under a dissecting microscope, remove the mosquito’s legs, wings, and head.

Cut off the tip of the abdomen to avoid damaging the digestive tract.

Secure the thorax and end of the abdomen, then gently pull out the digestive tract to extract it.

The extracted digestive tract includes the midgut, which is responsible for digestion and harbors diverse gut bacteria that influence mosquito physiology.

Under higher magnification, carefully remove the surrounding structures to isolate the midgut.

Transfer the isolated midgut into a fresh tube containing PBS.

Homogenize the midgut to release the bacteria into the PBS.

Spread the diluted homogenate onto a nutrient agar plate.

Incubate the plate to allow bacterial colonies to grow, representing the microbial flora of the mosquito midgut.

Dissect the mosquito separately on a sterile glass slide containing a drop of PBS. Carefully remove the legs, wings, and head of the mosquito using forceps.

Clamp the mosquito's chest and the last section of abdomen with forceps and pull apart to isolate the digestive tract.

Use forceps to remove the crop and the malpighian tube from the digestive tract. The crop is positioned at the anterior of midgut and bulges after ingesting sugar water. Malpighian tubes are group of slender excretory tubes on the junction of midgut and hindgut.

Place the dissected midgut into a EP tube with 200 microliters sterile PBS buffer and grind it with the sterile grinding pestle in the super clean bench. Serial dilute the homogenate to three 10-fold dilutions at 50 microliters of each dilution to the LB agar plate and then spread plate.

Incubate the plate at 37 degrees for one to two days until single colonies are visible.