Fluorescent in situ hybridization of the M. tuberculosis. (A and E) M. tuberculosis dual hybridized with EUB338-FAM (green) (A) and MTB-Cy3 (red) (E) probes. (B and F) M. intracellulare dual hybridized with EUB338-FAM (green) (B) and MTB-Cy3 (red) (F) probes. (C and G) M. avium dual hybridized with EUB338-FAM (green) (C) and MTB-Cy3 (red) (G) probes. (D and H) M. kansasii dual hybridized with EUB338-FAM (green) (D) and MTB-Cy3 (red) (H) probes. Pictures were taken under epifluorescence with a Nikon Eclipse E600 microscope. Original magnification, ×1,000. (I) Fluorescent in situ hybridization utilizing three oligonucleotide probes: MTB-FAM probes (green), MAVP187-Cy3 (red), and EUB338-Cy5 (pseudoblue), hybridized with a mixed culture which contains M. tuberculosis, M. avium, M. intracellulare, and Corynebacterium sp. The micrograph was taken by a Leica confocal laser scanning microscope in which all three channels (FAM, Cy3, and Cy5) were merged. EUB338-Cy5 hybridized with all the bacteria (pseudoblue). Long M. tuberculosis rods are fluorescent green-blue. Short M. avium rods are fluorescent red-blue. Long M. intracellulare rods and short Corynebacterium sp. rods are fluorescent pseudoblue, since they are detected by the EUB338-Cy5 probe but not by the MTB-FAM probe or the MAVP187-Cy3 probe. Original magnification, ×630. (J) Sputum (from a patient with a history of tuberculosis) hybridized with MTB-FAM probes. The arrow indicates a positive Mycobacterium tuberculosis bacillus (green) positively identified by MTB-ISH. (N) A phase-contrast micrograph of the same field shown in panel J. The arrow indicates the same bacillus shown in panel J. (K and L) Brightfield micrographs from AFB staining of Mycobacterium tuberculosis H37Rv-infected guinea pig lung (K) and mouse lung (L) sections. Nuclei were counterstained with methylene blue. (M) Brightfield micrograph from AFB staining of a M. avium subsp. paratuberculosis-infected bovine lymph node. Nuclei counterstained with methylene blue. (O) MTB-FAM probes hybridize with multiple Mycobacterium tuberculosis bacilli (green) in a representative granuloma of the guinea pig lung. Nuclei were counterstained with DAPI (4 ′ ,6 ′ -diamidino-2-phenylindole) (cyan). (P) A representative micrograph of a mouse lung specimen probed with the MTB-FAM oligos and visualized with enzymatic amplification of a colorimetric dye INT-BCIP (reddish-brown bacilli). Nuclei were counterstained with methylene blue. (Q) MTB-FAM probes did not hybridize with the bacilli in the M. avium subsp. paratuberculosis-infected bovine lymph node (fuzzy green background). MAVP187-Cy3 did hybridize to the M. avium subsp. paratuberculosis within the tissue (red bacilli). (K, L, M, and P) Brightfield micrographs taken with a Nikon Eclipse E600 microscope. Original magnification, ×1,000. (O and Q) Epifluorescent micrograph taken with a Leica confocal laser scanning microscope. Original magnification, ×630. Bar = 1 micrometer (for all micrograph scales).