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Huanglongbing disease (aka Citrus Greening Disease) of citrus, associated with infection by the bacterium ‘<i>Candidatus</i> Liberibacter asiaticus’ (LAS), has spread rapidly in Florida since its identification in 2005. Infection with LAS, vectored by the Asian citrus psyllid (<i>Diaphorina citri</i> Kuwayama), is incurable; therefore, knowledge regarding LAS biology and pathogenesis is essential to develop a treatment. However, LAS cannot currently be successfully cultured, limiting its study. To gain insight into the conditions required for growth of LAS in vitro, LAS inoculum obtained from seeds of fruit from infected pomelo trees (<i>Citrus grandis</i> ‘Mato Buntan’) was added to different media, and cell viability was monitored for 18 days using quantitative polymerase chain reaction (qPCR) in conjunction with ethidium monoazide (EMA). Media tested were 100% King’s B (K), King’s B with 50% juice from the infected fruit (J50), King’s B with 50% store-bought grapefruit juice (G50), and 100% store-bought grapefruit juice (G). Results show the J50, G50, and G media all prolong viability compared to K, with G50 and G being the most successful. Slides placed in inoculated media developed biofilm over time, with the most biofilm formed in G50 and G media. Fluorescence in situ hybridization (FISH) of this biofilm with LAS-derived 16S rRNA gene probes showed the biofilm contains aggregates of LAS cells. Results will contribute to future development of a culture medium for LAS.<p><p>Keywords: Bacteria-Phytoplasma-Spiroplasma-Fastidious Prokaryote, Fruits-Nuts, Citrus