While testing the specificity and sensitivity of the newly developed probes, each preparation of reference cells from all different bacterial strains were
additionally probed with a generic eubacterial probe (EUB338) and a non-sense nucleotide probe (NonEUB338) to confirm accessibility of the target rRNA as well as to exclude unspecific labelling of bacterial cells or tissue due to preparation artefacts . Probes Bwall1448 and Bwphi1448 were used together to detect all Francisella species IACS-10759 and to discriminate between F. philomiragia and F. tularensis. The combination of probes Bwtume168II and Bwmed1397 was applied in order to identify and discriminate F. tularensis subsp. tularensis (type A) and F. tularensis subsp. PS-341 mouse mediasiatica. Isolates of the subspecies F. tularensis holarctica and F. tularensis subsp. novicida were identified using probes Bwhol1151 or Bwnov168, respectively. The addition of 30, 35 or 50% formamide to the hybridization buffer
resulted in specific hybridization of the oligonucleotides to their respective target organisms. To reduce the amount of toxic waste, formamide was not used in the washing steps following hybridization. As a substitute, the NaCl concentration was decreased in the washing buffer according to the formula of Lathe  to obtain the necessary stringency. Citifluor (Citifluor Ltd., London, United Kingdom) was used as a mounting medium on hybridized slides, and the slides were examined both with a Leica (Heerbrugg, Switzerland) TCS NT scanning confocal microscope equipped with a standard filter set and a conventional fluorescence microscope (Axiostar plus/Axio CAM MR, Zeiss, Jena Germany). For probe excitation, an argonkrypton laser (Leica) or a mercurium-spectral light was used. Three different fluorochromes (DAPI, 6-FAM and Cy3) could be detected simultaneously
with three different photomultipliers utilizing the green (6-FAM), red (Cy3), and blue (DAPI) channels of the TCL Leica Application Suite (Leica) or selleck screening library Axiovision 4.5 (Zeiss) software packages. For the tissue sections, optical sectioning (0.5 to 1.0 μm width) was performed to reveal the three-dimensional localization of the probe-conferred fluorescence within the samples. The standard software delivered by the manufacturers was used to further process the digitized images. Identification of different F. tularensis subspecies in clinical material and infected cell cultures Aerobic BACTEC blood culture bottles (BD, Heidelberg, Germany) were spiked with live bacterial cells from different F. tularensis subspecies. Single cultures were started with inoculums of 10 to 1000 colony forming units (cfu) in 5 ml whole human blood. Additionally, cells from two different subspecies were mixed at ratios of 1:1, 1:10, 1:100, 1:1000 and then cultured under aerobic conditions until the BACTEC instrument reported bacterial growth.