Analyse vibratoire de structures sandwiches stratifiees

SIGLECNRS AR 11469 / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Effects of temperature and treatment time on inactivation of BCG cells in PBS.

1<p>BCG cells were suspended in PBS at concentrations of approximately 1×10⁷ CFU/mL. After treatment of 0.1 M NaOH and 1% SDS, 0.1 mL aliquots were plated on triplicate Middlebrook 7H10 plates with OADC enrichment. Numbers shown are total counts for the three plates after 35 days of incubation at 37°C.</p>2<p>TNTC, too numerous to count.</p

Semi-Automated, Occupationally Safe Immunofluorescence Microtip Sensor for Rapid Detection of <i>Mycobacterium</i> Cells in Sputum

<div><p>An occupationally safe (biosafe) sputum liquefaction protocol was developed for use with a semi-automated antibody-based microtip immunofluorescence sensor. The protocol effectively liquefied sputum and inactivated microorganisms including <i>Mycobacterium tuberculosis</i>, while preserving the antibody-binding activity of <i>Mycobacterium</i> cell surface antigens. Sputum was treated with a synergistic chemical-thermal protocol that included moderate concentrations of NaOH and detergent at 60°C for 5 to 10 min. Samples spiked with <i>M. tuberculosis</i> complex cells showed approximately 10⁶-fold inactivation of the pathogen after treatment. Antibody binding was retained post-treatment, as determined by analysis with a microtip immunosensor. The sensor correctly distinguished between <i>Mycobacterium</i> species and other cell types naturally present in biosafe-treated sputum, with a detection limit of 100 CFU/mL for <i>M. tuberculosis</i>, in a 30-minute sample-to-result process. The microtip device was also semi-automated and shown to be compatible with low-cost, LED-powered fluorescence microscopy. The device and biosafe sputum liquefaction method opens the door to rapid detection of tuberculosis in settings with limited laboratory infrastructure.</p></div

Preliminary comparison between Olympus and Lumin raw intensity values obtained from microtip measurement.

<p>The microtips were evaluated in treated sputum samples spiked with either PBS alone (control) or PBS containing BCG cells at 10⁴ CFU/mL.</p

Normalized fluorescence intensity results from the microtip assay.

<p>(A) Comparison of different species of <i>Mycobacterium</i> and <i>S. epidermidis</i> at 10⁴ CFU/mL. (B) Microtip detection of treated sputum samples spiked with BCG at densities ranging from 10² to 10⁵ CFU/mL. (C) Microtip detection of treated sputum samples spiked with H37Ra at densities ranging from 10² to 10⁵ CFU/mL.</p

Raw fluorescent images (20X objective) of microtips after capture from spiked sputum samples with BCG at densities ranging from 0 (control) to 10⁵ CFU/mL.

<p>Raw fluorescent images (20X objective) of microtips after capture from spiked sputum samples with BCG at densities ranging from 0 (control) to 10⁵ CFU/mL.</p

Inactivation of <i>M. tuberculosis</i> H37Rv in sputum by using the biosafe protocol.

1<p>H37Rv cells were suspended in sputum samples at concentrations of approximately 1×10⁷ CFU/mL. After treatment, 0.1 mL aliquots were plated on triplicate Middlebrook 7H10 plates with OADC supplement. Numbers shown are total counts for the three plates after 33 days of incubation at 37°C.</p>2<p>Because untreated sputum samples rapidly overgrew plates due to the natural flora of sputum, no-treatment controls consisted of <i>M. tuberculosis</i> cells suspended in PBS.</p>3<p>TNTC, too numerous to count.</p

Comparison of the previous protocol (top) and biosafe sputum processing protocol (bottom).

<p>Comparison of the previous protocol (top) and biosafe sputum processing protocol (bottom).</p

Semi-automated microtip system for concentration and detection of <i>M. tuberculosis</i>.

<p>The microtip used in the device is shown with scale bar of 250 µm. Components are as follows: (1) Sample well in which cells are concentrated and captured onto the microtip, (2) initial rinsing well with 1% SDS solution, (3) labeling well in which cells captured on microtip are bound to fluorescent antibody, (4) final rinsing well with DI water.</p