Optimization and Development of Protocol for Detection of Foot-And-Mouth Disease Virus by Negative Staining Using Transmission Electron Microscopy

Using transmission electron microscopy (TEM), a specimen can be ready for examination within 10 minutes, and an experienced virologist, pathologist, or technologist can identify a pathogen morphologically.There is paucity of literature with respect to study of TEM as a tool for the detection of foot-and-mouth disease (FMD) virus in suspension in diagnostic pathology.The current study aimed to optimize and develop the process of detection of foot-and-mouth disease virus and its capsid in TEM by negative staining methods.FMD virus infected BHK-21 cell and mock infected cells were centrifuged for 5 minutes at 12000 rpm, and the supernatant was used for negative staining.The supernatant was placed on the TEM grid using either single droplet touch methods or direct application of a single drop to the TEM grid and washed three times with water.After washing, the TEM grid was stained with 1% aq.Phosphotungstic acid, PTA (negative stain), then air dried and observed under TEM for imaging.The image was captured with a Jeol 1400 Plus TEM at 120 kV and 15000X magnification.The positive cell suspension was found positive for FMD virus under TEM in which virion capsid appears to be icosahedral, whereas in mock infected cell suspension samples no FMD virus particles were observed.The current study describes the process of demonstration of FMD virion in cell suspension using a negative staining method.This method can also be used to detect FMD virus in vesicular fluid, other fluids suspected for FMD virus, and other viruses.