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Plaquing of Herpes Simplex Viruses
* These authors contributed equally
In This Article
Summary
Plaquing is a routine method used to quantify live viruses in a population. Though plaquing is frequently taught in various microbiology curricula with bacteria and bacteriophages, plaquing of mammalian viruses is more complex and time-consuming. This protocol describes the procedures that function reliably for regular work with herpes simplex viruses.
Abstract
There are numerous published protocols for plaquing viruses, including references within primary literature for methodology. However, plaquing viruses can be difficult to perform, requiring focus on its specifications and refinement. It is an incredibly challenging method for new students to master, mainly because it requires meticulous attention to the most minute details. This demonstration of plaquing herpes simplex viruses should help those who have struggled with visualizing the method, especially its nuances, over the years. While this manuscript is based on the same principles of standard plaquing methodology, it differs in that it contains a detailed description of (1) how best to handle host cells to avoid disruption during the process, (2) a more useful viscous medium than agarose to limit the diffusion of virions, and (3) a simple fixation and staining procedure that produces reliably reproducible results. Furthermore, the accompanying video helps demonstrate the finer distinctions in the process, which are frequently missed when instructing others on conducting plaque assays.
Introduction
The beginnings of virus plaque assays go back to the first discoveries of viruses in the 1890s1. Tobacco mosaic virus was first isolated and passed on tobacco leaves, where individual spots of infection could be recognized and quantified as originating from a single, live virus entity2, later identified as a virion2. Later seminal studies with bacteria and bacteriophages perfected the techniques used to plaque these viruses, including bacteria at the mid-log phase of growth, serial dilution of bacteriophage samples, and top agar with subsequent visualization of literal holes (named plaques) in the....
Protocol
All procedures with the Vero cells and live herpesviruses have been approved by the Towson University Institutional Biosafety Committee. A generalized scheme of these procedures is represented in Figure 1.
1. Seeding of the Vero cells
- The day before initiating the plaque assay, trypsinize Vero cells and resuspend them in regular Dulbecco's Modified Eagles Medium (DMEM) and supplement as per standard cell culture methodology
Representative Results
Table 1 shows an experiment that has optimal results. All 10-fold dilutions follow an approximately 10-fold decrease in plaque counts. These kinds of data can also be seen in Figure 2, an actual plaque assay where the countable number of plaques fell in the 10-4 range for all three replicates. The same can be seen in Figure 3, the top row, where the countable number of plaques was in the 10-3 dilution.
Discussion
While plaque assays are almost as old as mammalian cell culture itself, it seems that each lab has its own set of protocols to execute this basic assay5,6,10,11,12,13,14,15,16,20. Althoug.......
Acknowledgements
We thank countless students in our labs (PJD and BJM) who have worked with us over the years refining these methods. A special thanks to Stan Person, under whose tutelage this methodology was first developed. This work was partially supported by the Towson University Fisher College of Science and Math Undergraduate Research Support fund and NIGMS Bridges to the Baccalaureate grant 5R25GM058264. This content is solely the authors' responsibility and does not necessarily represent the official views of the National Institutes of Health's National Institute of General Medical Sciences.
....Materials
| Name | Company | Catalog Number | Comments |
| 12-well plates | Corning | 3512 | |
| 6-well plates | Corning | 3516 | |
| Alpha-MEM | Lonza | 12169F | |
| Antibiotic/antimycotic | Gibco | 15240096 | |
| Crystal violet | Alfa Aesar | B2193214 | |
| DMEM | Gibco | 11965092 | |
| Dulbecco's PBS (no Mg++ or Ca++) | Gibco | 14190144 | |
| Fetal calf serum | Millipore-Sigma | TMS-013-B | |
| L-alanyl-L-glutamine (Glutamax) | Gibco | GS07F161BA | |
| Hemacytometer | Thermo Fisher | 02-671-54 | |
| Methylcellulose | Millipore-Sigma | 27-441-0 | |
| Quaternary agent (Lysol I.C.) | Thermo Fisher | NC9645698 | |
| Trypan Blue | Corning | 25900CI | |
| Trypsin | Cytiva | SH30042.01 | |
| Vero cells | ATCC | CCL-81 |
References
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