Viral

Virological electron microscope methods

Faecal (stool) samples.

For electron microscope (EM) examination, viruses contained in faecal samples normally have to be concentrated and partially purified.
Samples of diarrhoea or stool are emulsified in saline and centrifuged at low speed to sediment large pieces of debris and most bacteria. The supernatant is then spun at high speed (high ‘g’) in an ultracentrifuge to sediment any viruses into a pellet at the bottom of the centrifuge tube.

The supernatant from this step is discarded and the pellet resuspended in a small volume of liquid. A drop of this resuspended pellet is placed on an EM specimen grid coated in a very thin layer of plastic, which supports any viruses in the sample. After being allowed to settle, excess fluid is blotted off, the grid washed in distilled water and the adherent viruses stained using a heavy metal salt solution (usually phosphotungstic acid).

The grid is then examined at high magnification (40,000 to 60,000 times) which allows recognition by the electron microscopist of characteristic virus symmetry and morphology.

Skin lesions

In cases where the skin shows lesions or blisters thought to involve a virus infection (such as chicken pox), then some fluid can be aspirated from these lesions and placed on a coated electron microscope specimen grid.

After staining, the grid is examined under an electron microscope for viruses such as Herpesviruses, Poxviruses or Papillomaviruses (wart viruses).

As these viruses are generally much larger than those found in cases of gastroenteritis (diarrhoea and/or vomiting), a lower magnification (about 40,000 times) is used for screening.

The left thin-sectioned image shows where these viruses assemble in infected cells, and the right negatively-stained image allows identification as a polyomavirus. Each virus capsid is about 45nm in diameter.