Open AccessProbing Mixed-Genotype Infections I: Extraction and Cloning of Infections from Hosts of the Trypanosomatid Crithidia bombi
Author(s) -
Rahel Salathé,
Martina Tognazzo,
Regula SchmidHempel,
Paul SchmidHempel
Publication year - 2012
Publication title -
plos one
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.99
H-Index - 332
ISSN - 1932-6203
DOI - 10.1371/journal.pone.0049046
Subject(s) - biology , crithidia , cloning (programming) , host (biology) , genotype , parasite hosting , flow cytometry , microbiology and biotechnology , zoology , genetics , gene , protozoa , world wide web , computer science , programming language
We here present an efficient, precise and reliable method to isolate and cultivate healthy and viable single Crithidia bombi cells from bumblebee faeces using flow cytometry. We report a precision of >99% in obtaining single trypanosomatid cells for further culture and analysis (“cloning”). In the study, we have investigated the use of different liquid media to cultivate C. bombi and present an optimal medium for obtaining viable clones from all tested, infected host donors. We show that this method can be applied to genotype a collection of clones from natural infections. Furthermore, we show how to cryo-preserve C. bombi cells to be revived to become infective clones after at least 4 years of storage. Considering the high prevalence of infections in natural populations, our method provides a powerful tool in studying the level and diversity of these infections, and thus enriches the current methodology for the studies of complex host-parasite interactions.