Standard Marine Fixation

Chemicals Needed:

10 ml ampoules 16% Formaldehyde -EMS Sciences Cat #15710 Sodium Cacodylate Trihydrate bioxtra10 g - Sigma Cat #C4945 Na2CO3 Epofix

To Make Up Solutions:

22 g/L Na2CO3 in water filtered 0.2um

0.05M Na Cacodylate Buffer in 22g/L Na2CO3 Add 10.7 gms Cacodylic acid (to 1000 ml 22g/L Na2CO3)

To Make Up Fixative:

2% Formaldehyde in Sodium Cacodylate Buffered Sea-Water Fixative Add 10 ml ampoule of 16% Formaldehyde to 70 ml of the made up buffer

Fixation Steps

1. Place samples in fresh Formaldehyde fixative and keep it at RT 0.5 hrs
2. Rinse with buffer (2 changes at 5 mins each)
3. Dehydrate in 50,60,70, in 1g/L of Na2CO3 in 0.002M Na Cacodylate Buffer, 15 mins for each step
4. 80,90, in 0.5 g/L Na2CO3 in 0.002M Na Cacodylate Buffer and 100% x2 Ethanol
5. Vacuum the sample for 3 min to evaporate the ethanol.
6. Usually we make up the resin (follow kit directions) when the samples are in 90% ethanol.
7. Transfer into embedding resin (3 changes at 5 mins vacuum)

Embedding Steps

1. Measure amount of Epofix resin needed in a Dixie paper cup. Usually 8-10 grams is enough for one sample.
2. Calculate 3/25 amount of the resin to be the amount of hardener to add. Zero the balance and add the hardener into the cup. Take the cup and go upstairs.
3. Stir the mixture with a glass rod vigorously and make lots of small bubbles to ensure proper mixing. It usually takes 2 minutes
4. Place the cup in the desiccator (take the rod out) and pump it down for 5 minutes, then vent, and repeat 3 times. When venting, open the valve to ~2/3 of the full valve. There should be some bubbles only the surface by the end of this stage.
5. Take the epoxy out of the desiccator. Use a plastic pipet to drop the epoxy into the mold with the sample in it. Avoid bubbles as much as possible. Put enough epoxy to cover the entire sample. You can use the plastic pipet to remove some bubbles if needed.
6. Take the mold with epoxy filled and put it in the desiccator and repeat step 4 (pump 5 mins each for 3 times). After 3 times of pumping, leave the sample inside the desiccator for 12-14 hrs to cure.

Polishing Steps

1. Start with the coarse sandpaper. Rinse with 22g/L Na2CO3 solution. Grind the back of the sample to make it flat first. Then grind the sample side until the desired surface is exposed. Add 22g/L Na2CO3 solution during the grinding to keep the sandpaper wet. Press the sample with suitable force but keep in mind that the coarse sandpaper can remove materials very fast.
2. After the desired surface is approximately exposed, change to the fine sandpaper. Same as the previous step, add 22g/L Na2CO3 solution during the grinding. This step should remove all the large scratches from the last step, and the sample surface should appear foggy at the end of this step.
3. After grinding, move on to polishing with aluminum oxide particles. Change the sandpaper to the felt pad and add 0.3 µm Al2O3 suspension with Na2CO3 solution. Use hand to spread it across the felt and start polishing. Add 22g/L Na2CO3 solution during the grinding. Moderate force is important: if the force is too small, it will be difficult to remove the scratches from grinding; if the force is too large, alumina particles can get stuck in the sample and create pits on the surface. Polish until a shiny mirror surface is observed and no obvious scratches can be found.
4. Move on to the final polishing with 0.05 µm Al2O3 suspension. Same procedure as the previous step, but remember to change the felt to the one for fine polishing and don’t mix up with the previous pad. It’s difficult to judge if this step is done by naked eye, so you can use the microscope to help. Usually if there are no scratches under 20× it should be good enough.
5. After polishing, clean the sample with a series of wipes. Prepare 4 wipes with the following order: wet with Na2CO3 solution, dry, wet with ethanol, dry. After polishing with 0.05 µm Al2O3, clean the sample through these wipes as the order above. Gently wipe once on each wipe. If there are watermarks or weird patterns on the surface, it means that the cleaning was not done properly so you need to repeat this step.