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Heat fixation inactivates viral and bacterial pathogens and is compatible with downstream MALDI mass spectrometry tissue imaging - Descarga este documento en PDF. Documentación en PDF para descargar gratis. Disponible también para leer online.

BMC Microbiology

, 15:101

First Online: 13 May 2015Received: 11 August 2014Accepted: 23 April 2015DOI: 10.1186-s12866-015-0431-7

Cite this article as: Cazares, L.H., Van Tongeren, S.A., Costantino, J. et al. BMC Microbiol 2015 15: 101. doi:10.1186-s12866-015-0431-7

Abstract

BackgroundTissue samples should be fixed and permanently stabilized as soon as possible ex-vivo to avoid variations in proteomic content. Tissues collected from studies involving infectious microorganisms, must face the additional challenge of pathogen inactivation before downstream proteomic analysis can be safely performed. Heat fixation using the Denator Stabilizor System Gothenburg, Sweden utilizes conductive heating, under a mild vacuum, to rapidly eliminate enzymatic degradation in tissue samples. Although many studies have reported on the ability of this method to stop proteolytic degradation and other sample changes immediately and permanently, pathogen inactivation has not been studied.

ResultsWe examined the ability of the heat fixation workflow to inactivate bacterial and viral pathogens and the suitability of this tissue for Matrix Assisted Laser Desorption Ionization mass spectrometry imaging MALDI-MSI. Mice were infected with viral or bacterial pathogens representing two strains of Venezuelan Equine Encephalitis virus VEEV and two strains of Burkholderia. Additionally, a tissue mimetic model was employed using Escherichia, Klebsiella and Acinetobacter isolates. Infected tissue samples harvested from each animal or mimetic model were sectioned in half. One half was heat fixed and the other remained untreated. Lysates from each sample were checked for organism viability by performing plaque infectivity assays or plating on nutrient agar for colony forming unit CFU calculation. Untreated infected control tissue demonstrated the presence of each viable pathogen by positive plaque or colony formation, whereas heat fixation resulted in complete inactivation of both the viral and bacterial pathogens. MALDI-MSI images produced from heat fixed tissue were reflective of molecular distributions within brain, spleen and lung tissue structures.

ConclusionsWe conclude that heat fixation inactivates viral and bacterial pathogens and is compatible with proteomic analysis by MALDI-MSI. This treatment will enable the use of infected tissue from studies performed in bio-safety level 3 laboratories with VEEV and Burkholderia to be safely used for proteomic, small molecule drug detection, and imaging mass spectrometry analysis.

KeywordsTissue fixation Proteomics Microbial inactivation Matrix assisted laser desorption ionization mass spectrometry MALDI-MS Tissue imaging AbbreviationsMALDI-MSIMatrix assisted laser desorption ionization mass spectrometry imaging

VEEVVenezuelan equine encephalitis virus

PFUPlaque forming unit

CFUColony forming unit

OCTOptimum cutting temperature

ITOIndium tin oxide

BSL-3,4Biosafety laboratory level 3 or 4

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Autor: Lisa H Cazares - Sean A Van Tongeren - Julie Costantino - Tara Kenny - Nicole L Garza - Ginger Donnelly - Douglas Lane -

Fuente: https://link.springer.com/







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