This study describes a method that allows the rapid and clear diagnosis of plant virus diseases in about half a day by using a combination of microwave assisted plant sample preparation for transmission electron microscopy and negative staining methods.
Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample preparation for transmission electron microscopy (TEM) such investigations can take several days1,2 and are therefore not suited for a rapid diagnosis of plant virus diseases. Microwave fixation can be used to drastically reduce sample preparation time for TEM investigations with similar ultrastructural results as observed after conventionally sample preparation3-5. Many different custom made microwave devices are currently available which can be used for the successful fixation and embedding of biological samples for TEM investigations5-8. In this study we demonstrate on Tobacco Mosaic Virus (TMV) infected Nicotiana tabacum plants that it is possible to diagnose ultrastructural alterations in leaves in about half a day by using microwave assisted sample preparation for TEM. We have chosen to perform this study with a commercially available microwave device as it performs sample preparation almost fully automatically5 in contrast to the other available devices where many steps still have to be performed manually6-8 and are therefore more time and labor consuming. As sample preparation is performed fully automatically negative staining of viral particles in the sap of the remaining TMV-infected leaves and the following examination of ultrastructure and size can be performed during fixation and embedding.
Microwave assisted sample preparation
2. Trimming and sectioning
3. Negative staining
4. Image analysis
5. Representative results:
After microwave assisted sample preparation typical TMV-induced ultrastructural alterations such as large areas containing virions aligned in parallel form could be observed with the transmission electron microscope in the cytosol of infected Nicotiana tabacum cells (Fig 1A). Additionally, in the crude sap of TMV-infected leaves TMV particles could be observed as flexuous, rod-shaped structures after negative staining (Fig 1B). Image analysis of 100 virus particles revealed an average size for TMV of 280nm in length and 17nm in width (Fig 2). The ultrastructure in TMV-infected cells and the size of virions observed in this study were found to be in accordance with TMV-induced ultrastructural properties in tobacco and the size range of TMV particles previously reported in the literature9-15.
Figure 1. Transmission electron micrographs of TMV-infected leaf cells and virions. A) Image shows the ultrastructure of TMV-infected mesophyll leaf cells of Nicotiana tabacum after microwave assisted plant sample preparation. Note the large area of parallel aligned virions which accumulated in the cytosol. C=chloroplast with starch (St), M=mitochondrion, N=nucleus, V=vacuole, Bar=2μm. B) Image shows virions which were detected by negative staining in the sap of infected leaves.
Figure 2. Relative size of virions. Relative distribution of length and width of TMV-particles after negative staining the sap of infected leaves as they appeared in the electron microscope. Mean values (mean length/width ± standard deviation) were calculated from 100 virions.
Microwave assisted plant sample preparation for TEM has been proven to supply fast and reliable ultrastructural data within a few hours3,4,16. The fine structural preservation of organelles and membranes achieved with the method used in this study was similar to conventional and cryofixed samples5,15 with the advantage of a massive reduction in sample fixation and embedding time from 3 days or longer to about 2 hours. This represents the fastest sample preparation protocol for TEM currently available in the literature. The method described in this study combined microwave assisted plant sample preparation for TEM with negative staining methods which allowed a clear and rapid identification of TMV-induced ultrastructural alterations and the viral agent itself. TMV induced ultrastructural alterations could be investigated after trimming, sectioning and post-staining within about 4 hours after the beginning of fixation in the transmission electron microscope. Using the fully automatically specimen preparation mode freed the researcher to conduct negative staining in the interim in order to determine the size and width of the viral agent. Thus, we can conclude that this method allows a clear and rapid diagnosis of plant virus diseases in about half a day which is of great importance for future use in agriculture and scientific experiments in plant phytopathology. As this method could also be used for the rapid diagnosis of animal and human diseases it has a large potential for future application in medical and veterinary pathology.
The authors have nothing to disclose.
This work was supported by the Austrian Science Fund (FWF, P20619 and P22988 to B.Z.).
Name of the reagent/equipment | Company | Catalogue number | Comments |
Glutaraldehyde | Agar Scientific Ltd. | R1312 | |
Osmium tetroxide | Agar Scientific Ltd. | R1022 | |
Agar 100 resin | Agar Scientific Ltd. | R1043 | |
Dodecenyl succinic anhydride | Agar Scientific Ltd. | R1051 | |
Methyl nadic anhydride | Agar Scientific Ltd. | R1081 | |
Benzyl dimethylamine | Agar Scientific Ltd. | R1060 | |
Lead citrate | Agar Scientific Ltd. | R1210 | |
Uranyl acetate | Agar Scientific Ltd. | R1260A | |
Phosphotungstic acid | Agar Scientific Ltd. | R1213 | |
Formvar | Agar Scientific Ltd. | R1202 | |
Leica EM AMW | Leica Microsystems | ||
Leica (Reichert) Ultratrim | Leica Microsystems | Newer model is available | |
Leica (Reichert) Ultracut S | Leica Microsystems | Newer model is available | |
Diatome Ultra 45 | Gröpl | ||
Philips CM10 TEM | FEI (formerly Philips) | Newer model is available | |
Cell D | Olympus Inc. | ||
Optimas 6.5.1 | Media Cybernetics Inc. | Newer version is available |