The overall goal of this procedure is to diagnose plant virus diseases within a few hours by transmission electron microscopy. This procedure begins with microwave assisted fixation and embedding of the infected plant material. The next step is to extract and stain virus particles from the plant samples by negative staining, followed by sectioning of the embedded plant samples.
The samples are then investigated in the transmission electron microscope and the size of virus particles is measured by image analysis. Ultimately, the results show typical virus induced ultra structural alterations and the size of virus particles through transmission electron microscopy methods. The main advantage of this technique over existing methods, such as conventional fixation and embedding at room temperature, which can take several days, is that sample preparation time for transmission electron microscopy can be reduced to about two hours, thus allowing a clear diagnosis of the viral disease in about half a day.
Demonstrating this procedure together with me will be Gerhard Kaba, a technician and Professor Zelnik, a colleague from our laboratory Before the start of sample preparation program, the automatic microwave tissue processor for electron microscopy LA A-E-M-A-M-W with the appropriate protocols. Prepare the solutions for the different steps described in the programmed protocol for fixation, dehydration, and infiltration, and fill them into the designated vials according to the protocol load the vials on the carousel. Insert the carousel into the microwave tissue processor, and then load the first vial into the mono mode chamber.
Next, cut out small sections of leaves from nicotiana tobacco infected with tobacco mosaic virus, or TMV with a razor blade on a modeling wax plate in a drop of glutaraldehyde in sorens and phosphate buffer at room temperature. During harvesting of the samples and loading of the samples into the baskets, it is important to make sure that they're always covered with fixative solution so that they do not try out Using fine tweezers. Transfer the sections immediately into the designated baskets with a mesh width of approximately 200 micrometers.
Stack the baskets on top of each other and insert them into the mono mode chamber. Start the previously programmed microwave assisted sample preparation protocol for fixation, dehydration, and infiltration. Just before the sample preparation protocol comes to an end fill freshly prepared agar 100 epoxy resin into the designated polymerization forms.
After the protocol is finished, release the stacked baskets containing the infiltrated samples from the mono mode chamber into the last vial of the carousel. Remove the carousel from the microwave device and take the vial containing the samples from the carousel. Then remove the baskets from the vial and unstack them using fine tweezers.
Load the samples into the designated polymerization forms. Next, stack the polymerization forms on top of each other and remove the previously used vials from the carousel of the microwave tissue processor. Load the carousel with the stacked baskets and insert the carousel into the microwave device.
Now start the previously programmed polymerization protocol. After the protocol is finished, remove the polymerization forms from the mono mode chamber and unstack them. Remove the polymerized blocks containing the samples, which are now ready for sectioning with a microtome to begin the procedure for sectioning the blocks containing the samples.
Insert one or more blocks into separate sample holders for ultra thin cutting. With the sample on top, sticking out about one centimeter of the holder. Trim the block with a specimen trimmer for TEM so that a block face of a maximum of one millimeter in length and 200 micrometers in width containing as much leaf material as possible is achieved.
Set the ultra microtome to cut sections of about 70 to 90 nanometers in thickness at a cutting speed of around one millimeter per second. Section the block using a diamond knife at a knife angle of 45 degrees. Pick up multiple sections with a form VAR coated copper or nickel 200 square mesh grid post stain the sections on the grid with lead citrate for five minutes in a Petri dish, partially filled with NAOH to create a CO2 free environment.
Wash the grids with distilled water for one minute. Finally, post stain for 15 minutes with 1%urinal acetate dissolved in distilled water at room temperature after washing the grids with distilled water for one minute, air dry in a grid box. Negative staining of the remaining plant material is carried out while sample preparation is being performed automatically by the microwave tissue processor.
First, harvest about 100 milligrams of TMV infected leaf material and prepare crude SAP by homogenizing the material for two minutes with a razor blade on a microscope. Slide in sorens and phosphate buffer. Transfer 20 microliters of the resulting homogenate onto the first well of a Teflon coated microscope.
Slide with four or more wells. Then place a form VAR coated grid on top of the homogenate with the form VAR coated side facing towards the drop incubate for five minutes. Wash the grid for two minutes by placing the grid on top of a drop of sorens and phosphate buffer.
Repeat the wash for another two minutes. Next, incubate the grid for one minute with a freshly prepared phospho tongue stick acid solution. After one minute, remove the grid and allow it to air dry.
In a grid box, load the negatively stained grid into the sample holder and examine it with a transmission electron microscope. While polymerization is being carried out automatically by the microwave tissue processor, take at least 10 images of randomly chosen negatively stained ion. At least 10 or more ion should be visible on each image.
With the transmission electron microscope at a primary magnification of 21, 000 x or higher care must be taken that all images have the same magnification. The sections containing the tissue samples that were automatically processed by the microwave tissue processor are also examined with a transmission electron microscope. Analysis of the images acquired by TEM can be performed using any image analysis.
Computer software with a particle analysis tool. Measure the length and size of at least 100 randomly chosen single virus particles on the micrographs taken from the negatively stained samples. Calculate the means and standard deviations in order to achieve an average length and width of the virus particles visualized by negative staining methods and TEM after microwave assisted sample preparation.
Typical TMV induced ultra structural alterations such as large areas containing ion's aligned in parallel form could be observed with the transmission electron microscope in the cytosol of infected nicotiana tobacco cells. In this transmission electron micrograph C represents chloroplast with starch. St.M is for mitochondrion and is for nucleus, and V is for vacuole.
Additionally, in the crude SAP of TMV infected leaves, TMV particles could be observed as ous rod-shaped structures after negative staining. Figure one B.This graph shows the relative distribution of length and width of 100 TMV particles as they appeared in the electron microscope. After negative staining, the sap of infected leaves image analysis of the virus particles revealed an average size for TMV of 280 nanometers in length and 17 nanometers in width After its development to rapidly diagnose brain virus diseases.
This technique may pave the way for researchers in the fields of animal and human pathology to rapidly diagnose animal and human diseases by transmission electron microscopy.B.
