16 Jul Preparing viruses on TEM grids

Posted at 19:31h in by Bobbi Tannenbaum 0 Comments

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The mPrep system facilitates preparation of viruses and other microorganisms for TEM examination, including those prepared in Biological Safety Level 3 or 4 laboratory suites.

Problems

Virus sample preparation for TEM requires using fine forceps to transfer filmed grids throughout the process. Grids boxes may require decontamination prior to transfer to the TEM.

Grids and support films are easily damaged
Grids are easily lost or mixed up
Sharp forceps are a puncture risk to protective clothing
Grid handling is especially difficult wearing BSL3/4 positive pressure suits
Limited quantities of specimen solutions may be available
Specimen and reagent solutions exposed to air may evaporate, precipitate or be contaminated
Grids and grid storage containers require decontamination
Adsorption and staining times may be inconsistent across multiple grids

mPrep solutions

Encapsulated grids reduce handling and damage
Grids in mPrep/g capsules are always labeled
Forceps are not used for virus preparation
Direct handling of grids may require handling only at the microscope
Capsules require only 35 μl to prepare two grids
Enclosed capsules reduce or eliminate air exposure
Grids in capsules are decontaminated as a unit
Simultaneous processing assures uniform timing

T6 Phage (negative stain)

Ebola virus (negative stain)

Application Notes

AN506: Virus Specimen Preparation in Biosafety Level 3-4 Laboratories

Shows the negative stain TEM preparation of pathogenic Ebola virus in high containment BSL4 labs. Scientists used mPrep/g capsules to eliminate grid handling in the BSL suite. This resulted in easier, safer processing and more reproducible sample preparation. Specimen preparation and microscopy were performed by the US Army Medical Research Institute of Infectious Diseases (USAMRIID) Pathology Division, at Ft. Detrick, MD.

Required

mPrep/g capsules to hold TEM grids
mPrep/f 13 extreme couplers to prevent introducing damaging reagents or viruses into pipettors
Pipettor (such as a Gilson P200 volume) to enable reagent pipetting into mPrep/s capsules

Product Descriptions

mPrep/g Capsules product description

Publications

Easier and Safer Biological Staining: High Contrast UranyLess Staining of TEM Grids using mPrep/g Capsules (Microscopy & Microanalysis 2015)

Abstract

Scanning Transmission Electron Microscopy (STEM) for Accurate Virus Quantification (Defense Technical Information Center 2018)

Publication

Preparation of viral samples within biocontainment for ultrastructural analysis: Utilization of an innovative processing capsule for negative staining (Ft. Detrick Science Fair 2016)

Presented poster

Improved Virus Specimen Preparation for Transmission Electron Microscopy using mPrep/g Capsules: Applications in BSL3-4 Laboratories (Microscopy & Microanalysis 2016)

Abstract

Using Scanning Transmission Electron Microscopy (STEM) for Accurate Virus Dosing Quantification (Microscopy & Microanalysis 2016)

Abstract

Preparation of viral samples within biocontainment for ultrastructural analysis: Utilization of an innovative processing capsule for negative staining (J Virological Methods 2016)

Journal publication

Accurate virus quantitation using a Scanning Transmission Electron Microscopy (STEM) detector in a scanning electron microscope (J. Virological Methods 2017)

Journal publication

Utilization of Capsules for Negative Staining of Viral Samples within Biocontainment (J Visualized Experiments 2017)

Journal publication

Instructions

mPrep/g Instructions

Instructions for using mPrep/g capsules for grid handling and processing.

Template for laying out reagents

Print this 96-well plate template to organize protocols into columns and rows with a microtiter 96-well plate.

Quick Guide mPrep/g

A graphical display illustrating how to load grids into mPrep/g capsules, and how to use pipettors to stain or label grids. Use this guide as a lab reference poster.

Quick start for virus preparation

A graphical display that illustrates how to easily prepare virus specimens for negative stain TEM in Biological Safety Level 3-4, and other laboratories. Use this guide as a lab reference poster.