Читать книгу Principles of Virology - Jane Flint, S. Jane Flint - Страница 84

To calculate the titer of a virus in plaque-forming units (PFU) per milliliter, 10-fold serial dilutions of a virus stock are prepared in a buffer, and suitable aliquots are inoculated onto susceptible cell monolayers which are covered with an agar overlay (see figure). After a suitable incubation period, the monolayers are stained and the plaques are counted. To minimize error in calculating the virus titer, only plates containing between 10 and 100 plaques are counted, depending on the area of the cell culture vessel. Plates with >100 plaques are generally not counted because the plaques may overlap, causing inaccuracies. According to statistical principles, when 100 plaques are counted, the sample titer varies by ±10%. For accuracy, each dilution is plated in duplicate or triplicate (not shown in the figure). In the example shown, 10 plaques are observed on the plate produced from the 10^–6 dilution. Therefore, the 10^–6 dilution tube contains 10 PFU per 0.1 ml, or 100 PFU per ml, and the titer of the virus stock is 100 × 10⁶ or 1 × 10⁸ PFU/ml.