Since the last report, a defined media for Liberibacter crescens has been developed. Using the methods previously described, we utilized the literature to break down the undefined the components of the media as best as possible. Changing the ratio of the buffer and alpha-ketoglutarate also made a positive difference. The growth of L. crescens is not 100% comparable to BM7, however the rate of growth is identical up until 5 days, where the defined media cultures begin to die, and BM7 cultures continue to replicate for up to 10 days. Plans to do metabolomics on the two culture conditions is underway; perhaps the defined media is limiting in one or more components, or perhaps an toxic substance is building up. With this new break through, efforts to culture L. asiaticus have continued, integrating information from the other group members. Most prominently, we are using the defined medium as a base for the asiaticus medium, and supplementing with ingredients that the Killiney lab identified in citrus phloem sap. Such ingredients include a significant increase of sucrose and the addition of sugar alcohols such as xylitol and sorbitol, vitamins, and organic acids like ascorbic acid and citric acid. A we suspect a key ingredient in phloem sap to be gamma-aminobutyric acid, as well as cell wall components (lipids and fatty acids). Another vital change is using ferrous chloride instead of ferric chloride, which was discovered in the genome comparison of the Liberibacter species. Las does not have an Iron III transporter, but does contain an Iron II/manganese transporter, therefore we believe adding ferrous chloride will be essential for growth of Las. Although a defined media has been obtained, work to optimize the media formulation is still underway. Plans for optimization include metabolomics, proteomic and trasncriptomic analysis of the ancestral Liberibacter crescens strain BT-0, which we hypothesize to be more similar to Las since it has not been carried in culture at length since its isolation from babaco papaya, unlike the lab strain BT-1, in both the current defined medium as well as in BM7. Clues may be discovered about what nutrients may be missing in the asiaticus medium, or if Las is lacking an essential gene for exogenous culture.New methods for media inoculation and detection have also been explored. Various methods for obtaining cells from infected psyllid guts have been tested. Ideas on how to test for Las growth are also being tested. So far multiple methods are being used in each inoculation attempt, until we can confirm one method is better than any other. These methods include extracting single guts from psyllids and a)pooling multiple in one tube with an amount of the culture media, vortexing, and using the supernatant for inoculation b) using 2 guts per culture tube of 50ul and leaving c) and d) above with the addition of 5ppm cefixime to limit contamination chances. Analysis was done on contaminants that were obtained in one of the culture attempts for antibiotic susceptibility of antibiotics that were shown to not inhibit L. crescens. Cefixime was the only one that inhibited the contaminants of the few antibiotics we decided to test.Direct PCR was investigated to try to assess if Las is present in inoculated cultures. Unfortunately it did not work when taking 2 or 5ul of liquid culture to pcr tube. The use of 16s amplification was used to determine this. Suspicion of the complexity of the media being the inhibitor was then tested by adding known amounts of extracted bacterial DNA to the media and running direct PCR at various dilutions. Results showed amplification at a 1:10 dilution. This needs to be tested again with attempted cultures and 16s to ensure it produces results. If so, this will be an easy first assessment of whether Las is present in culture. More organized meetings would have been beneficial to the group, although there was a lot of great input from all members throughout the project. Work on identifying the components of the hemolymph of the psyllid is underway in the Killiney and Pelz-Stilinski labs. Media formulations still continue with the Davis lab. Hilf had success with isolating viable Las cells and we may still collaborate with the group to test the media with his cell isolation method. The task is to culture Las is very large and complex, however significant progress has been made in the endeavor.