5.5.2. Detection of GFP-expression
For GFP detection in the vegetative recombinant P. larvae isolates ATCC9545 (ERIC I) + pAD43-25 and P. larvae 04-309 (ERIC II) + pAD43-25, clones can be cultivated and analysed during different stages of growth in liquid MYPGP media (Dingman and Stahly, 1983), supplemented with 5 µg/ml chloramphenicol. In each bacterial stage of growth, 10 µl aliquots of the suspension are analysed bright field microscopically using differential interference contrast (DIC). Fluorescence activity is detected by a FITC filter block (e.g. Nikon Ti-E Inverted Microscope). All stages of growth are analysed and it can be observed that the rate of gfpmut3a expressing P. larvae clones remains stable throughout the logarithmic as well as the stationary growth phase, indicating that plasmid pAD43-25 is correctly replicated throughout bacterial growth.
Expression of GFP remained stable even after sporulation and germination. This is an essential prerequisite for using recombinant bacteria in infection assays because only spores can be used for infection, and the vegetative bacteria inside the larvae should still carry and express the introduced gene.