Isolation of Bacteria from the Mosquito Midgut

Abstract

Source: Liu, X., et al. A Bacterial Oral Feeding Assay with Antibiotic-Treated Mosquitoes. J. Vis. Exp. (2020)

This video demonstrates the dissection of the mosquito midgut to isolate gut bacteria. The midgut is extracted from the digestive tract, homogenized to release the bacteria, and plated on nutrient agar to culture bacterial colonies.

Protocol

1. Dissect the mosquito midgut
   1. Transfer the surface-sterilized mosquito onto the glass slide with a drop of sterile 1x phosphate-buffered saline (PBS) buffer. Dissect under the dissecting microscope.
   2. Under the lower magnification of the dissecting microscope, carefully remove the legs, wings, and head of the mosquito to prevent escape. Remove the last somite of the mosquito by cutting directly, rather than pulling it, to prevent the digestive tract from breaking.
   3. Clamp the mosquito's thorax and the end of the abdomen with forceps. Gently pull out the mosquito's digestive tract. The acquired digestive tract usually contains the crop, foregut, midgut, hindgut, and the Malpighian tubes.
   4. Adjust the dissecting microscope to a higher magnification. Remove the crop, foregut, hindgut, and Malpighian tubes from the dissected digestive tract to obtain the mosquito's midgut.
   5. Take care while removing the crop, as the crop inflates and resembles the midgut after the mosquito takes a sugar meal. The Malpighian tubes, a group of long and thin excretory tubes, are located at the boundary between the midgut and the hindgut.
2. Isolate gut bacteria
   1. Transfer the dissected midgut to a sterile 1.5 mL tube with 200 µL of sterile 1x PBS buffer.
   2. Grind the midgut on a sterile bench thoroughly with a sterile pestle to allow complete release of gut bacteria to the buffer.
   3. Serially dilute the homogenate to three 10-fold dilutions. Add 50 µL of each dilution to the LB (Luria broth) agar plate and spread it on the plate. If the midgut contains a high concentration of gut flora, it is essential to make more serial dilutions to pick out single colonies.
   4. Incubate the plate at 37 °C for 1–2 days until single colonies are visible.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Aedes aegypti			Female mosquitoes
Centrifuge tube	Sangon Biotech	F601620-0010	1.5 mL, Natural, graduated, sterile
Disposable tissue grinding pestle	Sangon Biotech	F619072-0001	70 mm Long, conical, blue, sterile
Forceps	RWD	F11029	Dissection
LB agar powder	Sangon Biotech	A507003	Tryptone 10.0 g, Yeast Extract 5.0 g, NaCl 10.0 g, Agar 15.0 g.
Microscope	Zeiss	Stemi508
10X PBS buffer	Sangon Biotech	E607016	This product is a 10X solution. Please dilute it 10 times before use. The pH value is 7.4.