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[DESCRIPTION]
<strong>Purpose:</strong> This protocol describes how to negatively or positively stain viruses that have been deposited onto TEM grids so that they can be imaged using transmission electron microscopy (TEM). Positive staining stains the virus itself such that the virus is dark against a lighter background (Figure 1A). This is the easiest staining to do and yields results suitable for analyzing the morphological diversity of viruses from aquatic samples. Negative staining relies on some stain remaining around the edges of a virus such that the details of the virus are clearly defined (Figure 1B). Negative staining is more difficult and is used to obtain publication quality micrographs of viruses.<br /><br /><img src="https://s3.amazonaws.com/pr-journal/f7c2w6.jpg" alt="" width="500" height="132" /><br /><br /><strong>Note:</strong> To deposit viruses onto the grids prior to staining, use either the &ldquo;<a href="http://protocols.io/view/Adsorbing-Viruses-on-TEM-Grids-dar2d5" target="_blank">Adsorbing Viruses onto TEM Grids</a>&rdquo; protocol or the &ldquo;<a href="https://www.protocols.io/view/Quantitatively-Depositing-Viruses-onto-TEM-Grids-cwzxf5" target="_blank">Quantitatively Depositing Viruses onto TEM Grids</a>&rdquo; protocol. Grids should be stained immediately after depositing viruses onto them.

[GUIDELINES]
<strong>Materials required:</strong> <br />- EM grid-grade tweezers (2 or more pairs) <br />- uranyl acetate <br />- ultra-purified water (e.g. Milli-Q water) <br />- petri dish <br />- 4x 2 ml microcentrifuge tubes with screw caps <br />- 4 ml plastic culture tube with cap <br />- small stir bar <br />- stir plate <br />- filter paper (cut into wedges) <br />- 0.02 &micro;m syringe filter and syringe (3-5 ml) <br />- lab coat, respiratory protection (mask), eye protection <br />- timer <br />- waste container for uranyl acetate<br /><br /><br /><strong>Positive Staining of Viruses on Grids:</strong> This is the easiest staining to do. It is &ldquo;quick and dirty&rdquo;. It provides less detail of your viruses, but will allow you to measure their dimensions. It is not the proper staining method if you want really pretty micrographs of your viruses.<br /><br /><br /><strong>Negative Staining of Viruses on Grids:</strong> Negative staining is equal parts magic, art, luck, and skill. If done correctly, it will yield detailed views of your viruses and if you are very lucky, it will yield a few pretty micrographs of your viruses.<br /><br /><br /><strong>Applicable References:</strong> <br /><br />Ackermann and Heldal, 2010 &ndash; An excellent reference for learning about imaging of viruses. <br /><br />Brum and Steward, 2010 &ndash; A reference for the positive staining protocol. <br /><br />Ackermann, H.-W., and Heldal, M. (2010) Basic electron microscopy of aquatic viruses. In Manual of Aquatic Viral Ecology. Wilhelm, S.W., Weinbauer, M.G., and Suttle, C.A. (eds): ASLO, pp. 182-192. <br /><br />Brum, J.R., and Steward, G.F. (2010) Morphological characterization of viruses in the stratified water column of alkaline, hypersaline Mono Lake. Microbial Ecology 60: 636-643.

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