Preparation of mycetocytes for culture in vitro

Procedures for isolating the mycetocytes of cockroaches were developed as a preparatory step for culturing these cells which are infected with a symbiotic bacterium. Of several enzymes tested, trypsin was the most effective for disaggregating fat body tissues of three species, Blattella germanica, Periplaneta americana, and Blaberus craniifer. A thorough mincing of the fat body was necessary because none of the enzymes completely digested connective tissue. Three types of cells were recovered: fat cells, urate cells, and mycetocytes. Fat cells of B. germanica were dispersed by trypsin in 30 min, but longer treatments were required for mycetocytes. Even after 45 min of trypsinization, half of the mycetocytes were still associated with urate cells, indicating a strong membrane association between the two cell types. Mycetocytes were separated from urate cells of B. craniifer with a two-step gradient of sucrose. Centrifugation of heterogeneous cell populations at 275g separated most of the fat cells, which formed a pellicle, from the mycetocytes, which precipitated. A maximum of 9.3 × 10⁴ mycetocytes were isolated from individual, newly emerged adult females of B. germanica. A linear Ficoll gradient was used to determine the specific gravity of individual mycetocytes, which ranged from 1.050 to 1.145 g/cm³.