Visualization and Size Assessment of Faustovirus Using Electron Microscopy

Abstract

Source: Bou Khalil, J. Y., et al. A Rapid Strategy for the Isolation of New Faustoviruses from Environmental Samples Using Vermamoeba vermiformis. J. Vis. Exp. (2016).

This video demonstrates imaging of faustovirus particles from an infected amoeba culture using transmission electron microscopy. It covers grid preparation, negative staining with ammonium molybdate to enhance contrast, and visualization of the virus under the microscope. The protocol concludes with measuring the size of the faustovirus using the microscope's integrated software tools.

Protocol

1. Electron Microscopy
   1. After the first identification on the slides, assess the presence of the giant virus through electron microscopy observation of supernatant cultures.
   2. Deposit 5 µl of the positive sample onto the glow-discharged grid. Wait for approximately 20 minutes at room temperature.
   3. Dry the grid carefully and deposit on it a small drop of 1% ammonium molybdate for 10 sec. Leave the grid to dry for 5 min.
   4. Proceed to electron microscopy at 200 keV.
   5. Place the grid on the holder; introduce the holder into the microscope. Check the vacuum overview by clicking on the corresponding icon. Close valves and begin observation at a spot size 5, and magnification of x 25,000.
   6. Measure the giant virus using ImageJ or directly on the microscope using the measure tool of the microscope located on the acquisition screen.
      NOTE: Classify the sample into the Faustovirus group with a capsid size of approximately 200 nm.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Amoeba strain CDC-19	ATCC	France	50237
Plates	Cellstar	France	655180
Vacuum driven filters	Thermo scientific	France	BPV4550 / 20170115
Phosphate-Buffered Saline	Thermo Fisher scientific	France	10010-023
DAPI stain	Life Technologies	France	D1306
Carbon grids	Euromedex	France	FCF400NI
Ammonium molibdate	VWR internationanl	France	21276185
Flasks	SARSTEDT	Germany	833911
0.22 μm Filters	Milex millipor	France	SE2M229104
Ultracentrifuge Sorval WX 80	Thermo scientific	France	9102448
Rapid-flow filters	Nalgene	France	450-0020