Imperial College London

Professor George K. Christophides

Faculty of Natural SciencesDepartment of Life Sciences

Professor of Infectious Diseases & Immunity
 
 
 
//

Contact

 

+44 (0)20 7594 5342g.christophides

 
 
//

Location

 

6167Sir Alexander Fleming BuildingSouth Kensington Campus

//

Summary

 

Publications

Citation

BibTex format

@article{Habtewold:2016:10.1186/s13071-016-1438-0,
author = {Habtewold, T and Duchateau, L and Christophides, GK},
doi = {10.1186/s13071-016-1438-0},
journal = {Parasites & Vectors},
title = {Flow cytometry analysis of the microbiota associated with the midguts of vector mosquitoes},
url = {http://dx.doi.org/10.1186/s13071-016-1438-0},
volume = {9},
year = {2016}
}

RIS format (EndNote, RefMan)

TY  - JOUR
AB - BackgroundThe scientific interest to understand the function and structure of the microbiota associated with the midgut of mosquito disease vectors is increasing. The advancement of such a knowledge has encountered challenges and limitations associated with conventional culture-based and PCR techniques. MethodFlow cytometry (FCM) combined with various cell marking dyes have been successfully applied in the field of ecological microbiology to circumvent the above shortcomings. Here, we describe FCM technique coupled with live/dead differential staining dyes SYBR Green I (SGI) and Propidium Iodide (PI) to quantify and study other essential characteristics of the mosquito gut microbiota. Results A clear discrimination between cells and debris, as well as between live and dead cells was achieved when the midgut homogenate was stained with 5x103 dilution of the SGI and 30 µM concentration of the PI. Reproducibly, FCM event collections produced discrete populations including non-fluorescent cells, SYBR positive cells, PI fluorescing cells and cells that fluoresce both in SYBR and PI, all these cell populations representing, respectively, background noise, live bacterial, dead cells and inactive cells with partial permeability to PI. The FCM produced a strong linear relationship between cell counts and their corresponding dilution factors (R² = 0.987), and the technique has a better precision compared to qRT-PCR. The FCM count of the microbiota reached a peak load at 18 h post-feeding and started declining at 24 h. The present FCM technique also successfully applied to quantify bacterial cells in fixed midgut samples that were homogenized in 4% PFA. ConclusionThe FCM technique described here offers enormous potential and possibilities of integration with advanced molecular biochemical techniques for the study of the microbiota community in disease vector mosquitoes.
AU - Habtewold,T
AU - Duchateau,L
AU - Christophides,GK
DO - 10.1186/s13071-016-1438-0
PY - 2016///
SN - 1756-3305
TI - Flow cytometry analysis of the microbiota associated with the midguts of vector mosquitoes
T2 - Parasites & Vectors
UR - http://dx.doi.org/10.1186/s13071-016-1438-0
UR - http://hdl.handle.net/10044/1/30467
VL - 9
ER -