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Introduction The mediastinal lymph node (LN) is a key site of Mycobacterium tuberculosis (Mtb)-host interaction. Though many studies have tried to localise and visualise Mtb in tissue samples from the infected human host, this has been elusive due to the challenges of obtaining and adequately preserving human tissue, precluding high resolution ultrastructural approaches such as electron microscopy.Methods Following ethical approval we recruited individuals attending the Royal Free Hospital with suspected tuberculosis despite negative sputum Mtb PCR and culture, who were not on treatment and had planned diagnostic EBUS lymph node (LN) sampling. We consented participants for collection of 1–2 additional cores of mediastinal LN tissue for research analysis during the same diagnostic procedure. Research samples were placed in fixative 4%EM grade PFA with 0.1% glut in HEPES 200 mM pH 7.4 at the time of sampling and transferred to Francis Crick Institute where en bloc staining was performed and the sample embedded in UltraBed ultralow-viscosity resin. Serial-section scanning electron microscope array tomography and microCT were performed with volume reconstruction.Results To date 11 individuals with possible tuberculosis have been recruited. 8 had mediastinal LN sampling, 5 of whom were Mtb PCR-positive and 3 PCR-negative. 3 of the 5 positive cases were AFB culture-positive and 4 demonstrated necrotising granulomatous lymphadenitis by histology. In a Mtb PCR-positive sample that has undergone extensive imaging analysis, extracellular bacteria were identified in areas of debris within the LN tissue. The bacteria themselves contain lipid droplets and granules characteristic of polyphosphates ( figure 1). Analysis of this and other participants’ LN samples is ongoing.Abstract M33 Figure 1Conclusion We have established a clinical cohort leveraging expertise at Royal Free Hospital and Francis Crick Institute to visualise for the first time Mtb within mediastinal LN tissue. This demonstrates the presence of bacteria with an energy source and a potential mechanism to survive oxidative stress in a patient with tuberculosis. This approach offers the possibility of furthering our understanding of clinically relevant aspects of Mtb-host interactions at the tissue, cellular and subcellular levels.