title: Improving acid-fast fluorescent staining for the detection of mycobacteria using a new nucleic acid staining approach.
abstract: Acid fast staining of sputum smears by microscopy remains the prevalent method for detecting Mycobacterium tuberculosis. The sensitivity of microscopy using acid fast stains requires 10(4) bacilli per ml of sputum. Although fluorescent acid fast stains, such as Auramine-O, show improved sensitivity, almost half of culture-positive TB cases are currently estimated to remain smear-negative. These current diagnosis problems provide impetus for improving staining procedures. We evaluated a novel fluorescent acid-fast staining approach using the nucleic acid-binding dye SYBR(®) Gold on mycobacterial in vitro cultures. The SYBR(®) Gold stain detected 99% of MTB in both actively replicating aerobic and non-replicating hypoxic cultures. Transmission light microscopy with Ziehl-Neelsen fuchsin, and fluorescence microscopy with Auramine-O or Auramine-rhodamine detected only 54%-86% of MTB bacilli. SYBR(®) Gold fluoresces more intensely than Auramine-O, and is highly resistant to fading. The signal to noise ratio is exceptionally high due to a >1000-fold enhanced fluorescence after binding to DNA/RNA, thereby reducing most background fluorescence. Although cost and stability of the dye may perhaps limit its clinical use at this time, these results warrant further research into more nucleic acid dye variants. In the meantime, SYBR(®) Gold staining shows great promise for use in numerous research applications.
authors.affiliation: Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523, USA.
authors.forename: Gavin J
authors.initials: GJ
authors.lastname: Ryan
authors.affiliation: The Center for Microbial Cytometry and Howard M. Shapiro, M.D., P.C., 283 Highland Avenue, West Newton, MA 02465-2513, USA.
authors.forename: Howard M
authors.initials: HM
authors.lastname: Shapiro
authors.affiliation: Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523, USA. Electronic address: Lenaerts@colostate.edu.
authors.forename: Anne J
authors.initials: AJ
authors.lastname: Lenaerts
mesh_terms: D001576:Benzophenoneidum; D004269:DNA, Bacterial / Q000235:genetics*; D005456:Fluorescent Dyes; D006801:Humans; D046529:Microscopy, Electron, Transmission; D008856:Microscopy, Fluorescence; D009169:Mycobacterium tuberculosis / Q000235:genetics* / Q000254:growth & development / Q000648:ultrastructure; D009930:Organic Chemicals; D011237:Predictive Value of Tests; D012329:RNA, Bacterial / Q000235:genetics*; D015203:Reproducibility of Results; D012235:Rhodamines; D059629:Signal-To-Noise Ratio; D013183:Sputum / Q000382:microbiology; D013194:Staining and Labeling / Q000379:methods*; D013997:Time Factors; D014397:Tuberculosis, Pulmonary / Q000175:diagnosis* / Q000382:microbiology
chemical_list: D004269:DNA, Bacterial; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D012329:RNA, Bacterial; D012235:Rhodamines; C474470:SYBR Gold nucleic acid gel stain; D001576:Benzophenoneidum
keywords: Acid-fast; Stain; Tuberculosis