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Utilization of Capsules for Negative Staining of Viral Samples within Biocontainment
Published on: July 19, 2017
A new electron microscope positive staining method for viruses in suspension
J C Doultree1, R E Kiernan, J Y Lee
1Virology Department, Fairfield Hospital, Victoria, Australia.
Journal of Virological Methods
|June 1, 1992
Summary
The Tokuyasu staining procedure (TSP) was evaluated for virus visualization. Methanolic uranyl acetate in TSP improved positive staining for both enveloped and non-enveloped viruses, revealing novel structures.
Area of Science:
- Virology
- Microscopy Techniques
- Biochemistry
Background:
- Accurate visualization of viral morphology is crucial for understanding viral structure and function.
- Traditional staining methods can have limitations in preserving viral integrity and achieving optimal contrast.
Purpose of the Study:
- To evaluate the efficacy of the Tokuyasu staining procedure (TSP) for positive staining of various viruses in suspension.
- To compare the effects of aqueous versus methanolic uranyl acetate (UA) in the TSP.
- To investigate the potential for observing novel morphological features of viruses.
Main Methods:
- The Tokuyasu staining procedure (TSP) was employed, involving initial staining with uranyl acetate (UA) followed by embedding in a UA and polyvinyl alcohol (PVA) mixture.
- Experiments utilized rotavirus (non-enveloped), rubella virus (enveloped), and glutaraldehyde-fixed Human T Cell Lymphotropic Virus Type I (HTLV-I) and Human Immunodeficiency Virus Type 1 (HIV-1) (enveloped).
- Variations in UA (aqueous vs. methanolic) were tested to assess their impact on stain penetration and particle appearance.
Main Results:
- Using aqueous UA in TSP resulted in mixed positive and negative staining for rotavirus and rubella virus.
- Glutaraldehyde-fixed HTLV-I and HIV-1 showed only negative staining with aqueous UA, indicating poor stain penetration.
- Substitution of methanolic UA for aqueous UA in TSP yielded exclusive positive staining for rotavirus and rubella virus.
- Methanolic UA also enabled stain penetration in glutaraldehyde-fixed HTLV-I and HIV-1, resulting in positively stained particles.
- Novel morphological details of rotavirus and rubella virus were observed using the TSP.
Conclusions:
- The modified Tokuyasu staining procedure using methanolic uranyl acetate enhances positive staining of both enveloped and non-enveloped viruses.
- This improved TSP facilitates better visualization of viral structures, including glutaraldehyde-fixed viruses.
- The technique offers potential for discovering new insights into viral morphology.
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