Sodalis glossinidius is known exclusively in endosymbiosis with tsetse flies (Genus: Glossina) and is one of the few insect bacterial symbionts that have been successfully cultured in vitro. This study details improved isolation and solid culture protocols that allow for a standardised and rapid preparation/maintenance of clonal material from individual flies. The isolation and culture of S. glossinidius was confirmed by partial sequencing of the 16S rDNA gene and specific PCR. In addition, the growth dynamics and changes in cell viability during liquid culture are described. The potential for culture of other endosymbiont taxa is discussed.