Virus Particle Detection and Quantification
Negative Stain Electron Microscopy (NSEM) has been used extensively for over 50 years to identify and characterise virus particles. The negative staining technique is particularly useful for detecting contaminants such as mycoplasma, yeasts, bacteria and viruses in vaccines, bulk harvests and culture supernatants. Negative staining is also used for the monitoring of retroviral load quantities in fermenter bulk harvest material prior to downstream purification/processing.

Direct Negative Stain Electron Microscopy (D-NSEM) is used where counts are expected to be >10⁷ particles/ml, such as high titre virus preparations. Two other typical procedures are NSEM after both sucrose density purification and ultra-centrifugation (SD-NSEM), and NSEM after only ultra-centrifugation (UC-NSEM). Both these methods improve the detection limit (DL) to ~10⁵particles/ml, however, there are some drawbacks to using these methods where the SD-NSEM suffers a loss of virus and the UC-NSEM method concentrates debris (if present) with the virus, compromising visualisation thus impacting on the DL. To combat these disadvantages, a Thin Section Transmission Electron Microscopy (TS-TEM) method is offered which provides a consistent, accurate and repeatable examination of bulk harvest/culture supernatant samples with a DL of 10⁵ particles/ml. This improved thin sectioning methodology has been applied to the testing of fermenter bulk harvests (Reid et al., 2003; EW Milne 2003). In addition, thin sectioning studies of cellular material are also excellent for revealing intra-, and extra-cellular agents such as mycoplasma and protozoa.