The goal of this methodology is to handle delicate tissues from plants, oomycetes, and fungi, for their optimal observation under a Scanning Electron Microscope, or SEM. This method can help us answering questions regarding practically any fungi, microbe, mor-phor-ity, and how they infect, colonize, and attach to their host. The technique in our improved methodology is for fixing samples, for where the mi-toh-mins in aquatic ecosystems.
The main advantage of this technique is that it allows us visualizing key aspects of infection using an expensive and easily available resources. Visual demonstration of this method is critical because the steps have been wildly modified from traditional protocols. And published methods describe general procedures without details.
To begin, prepare the formol and acetic alcohol, or FAA fixative in a fume cupboard fitted with an aldehyde filter. At 85 parts of 70%denatured ethanol, 10 parts of 60%formaldehyde solution, and five parts of glacial acetic acid. Under the fume cupboard, pour the stock of FAA into individual containers.
Select the floral or vegetative meristems to fix, ensuring that they are not damaged by insects, fungi, or extreme weather condition. Cut the branches, removing unwanted material, and deposit the sample immediately in the FAA solution. After 72 to 96 hours, pour the FAA into a plastic container for chemical disposal.
Immediately wash the samples three times with fresh 70%ethanol to remove any residual FAA. Fixed material can be stored indefinitely in 70%ethanol. Dissect the samples into a Petri dish covered with ethanol to prevent the tissues from drying.
Use a Petri dish with the base covered with a dry, black silicon to better see the contrasting white tissue. Transfer the tissue into separate containers and then immerse these into a Petri dish with 70%ethanol. Put the lids on the containers and deposit them in plastic centrifuge tube with plenty of 70%ethanol.
Transfer the dissected material through an ethanol series in hermetic jars or centrifuge tubes. Leave the samples in each solution for at least one hour. Then keep the samples overnight in a 100%ethanol solution.
Following the ethanol series, transfer the containers with material to the CPD. Write the sample identification number underneath the SEM sample holders. Then cover the top of the stubs with double-sided tape.
Place the stubs into a specimen holder. Under a stereo microscope, carefully open the containers carrying the young and delicate samples already dried in the CPD. Bear in mind that after the CPD treatment, the samples become lighter and sensitive to electrostatics.
Close the containers once the samples have been taken out to avoid dust or impurities. Put the samples on the sticky surface of the stubs planning ahead for the desired position. Once the samples touch the surface, it is very difficult to remove them.
Do not try to carry out a major dissection at this point. Just remove the unwanted tissue that is easy to pick up. For palynological studies, dissect the anthers and open them to expose the pollen on the stubs.
Keep the samples protected overnight in a hermetic container with silica gel to avoid re-hydration. Coat the samples using the spotter-coater and transfer them to the SEM as described in the text protocol. To test the differential growth of the spines of the cysts on separate Petri dishes, put 0.5 milliliters of the secondary cyst suspension onto different surfaces.
Surfaces can include carbon, gold, and copper transmission electron microscopy grids. Previously, bleached salmon and hake fish scales, and glass cover slips. Incubate the cysts at 20 degrees Celsius for 70 minutes, which favors the attachment of the cysts to the surface.
Remove the liquid and add 0.5 milliliters of 2%glutaraldehyde to each surface for the fixation of the cysts. Keep the samples at room temperature under a fume cupboard for one hour. Remove the glutaraldehyde and dehydrate the samples through an ethanol series, adding five milliliters of each of the ethanol solutions for 15 minutes.
Once in the last 100%ethanol solution, the sample can be stored for up to a month in a sealed Petri dish. Carefully transfer the grids and the scales from the Petri dish to a holder suitable for the CPD that keeps the samples separated from each other, such as a CPD grid holder, or a stacking specimen holder. Take the grid and scales with tweezers, keeping in mind that the cysts should be facing up on the grids at all times.
Proceed to dry the material using the CPD before performing SEM sample preparation of the oocytes to observe their behavior on different surfaces, as described in the text protocol. Wrap each sample carefully with filter paper forming approximately 0.5 to one square centimeter pencil labeled envelopes. Take care not to crush the samples.
Seal the filter paper with paper clips. Then transfer the packed samples to a Petri dish and immerse them in 10 milliliters of water to rehydrate the tissue around the spores. Immediately put the samples in a microwave.
Remove the material once the water starts to evaporate and allow it to cool down at room temperature. Then pass the sample through an ethanol series. Depending on the amount of sample, use a beaker or centrifuge tube for this step.
Leave the samples for 15 minutes in each solution. Next, place the samples into the CPD as described in the text protocol. To mount the spores for SEM observation, open the envelopes.
Then collect the spores with the sticky surface of the stubs, taking care not to crush them. Finally, perform gold coating of the samples and SEM observation as detailed in the text protocol. Shown here are floral buds of anacyclus clavatus treated with osmium tetroxide and prepared using the FAA CPD protocol demonstrated in this video.
Also shown are dried samples of phellorinia herculanea without any treatment, as well as fungal spores treated as demonstrated in this video. This figure illustrates early, mid, and late floral development captured under the SEM. This image is of the top view of a young reh-cim.
Shown here is a floral bud during gynesium differentiation and a flower at anthesis. Some structures can be difficult to fix and preserve for imaging under the SEM. Examples include those coming from wet environments, as well as those with ornamentations, and those with indumenta.
Shown here are differential growth patterns of the spines of saprolegnia parasitica on glass, carbon, copper, gold, hake scales, and salmon scales. This is a video showing the asexual life cycle of saprolegnia parasitica. The asexual lifecycle is important for the dispersal of these organisms in their aquatic or humid environments.
Moreover, zoo spoors produced in this stage represent the primary unit of infection in parasitic species of the order saprolegnials. While master this technique can be done in three to five days, it is as performed properly. We used money to complete the SEM therapies provide to external use for in this timeframe at the Royal Botanical Garden in Madrid.
After watching this video, researchers would know how to effectively collect and fix delicate biological material for SEM observation. Destruction of cells, organ distortion, or bad preservation of samples from wet environments can be easily overcome with this procedure. While attempting this procedure, it's important to remember that the observation through SEM is limited to high magnification study of surfaces.
Also before CPD treatment, the seals must be kept safe from shocking changes and direct contact with the air. After its development, this technique paved the way for researchers in the field of phytology to explore spore structure during the infectious process, particularly in species of interest to fisheries. Following this procedure, other methods like Transmission Microscopy or TM can be performed in order to answer additional questions, like how the internal structure of cell composition are in a specific organs or cells, or pollen.
Don't forget that manipulating formalin and glutaraldehyde can be extremely hazardous, and precautions such as working under a fume cupboard and using masks, lab coats, and gloves, should always be taken while performing this procedure.
