I have a problem for preparing the SEM specimen of Colepidae. The amour plates of the species appears to melt during the preparation procedure. I have attached my preparation and it was prepared as follows: fixed by HgCl2 (or glutaldehyde with osmium), washed in DW, then ethanol series, CPD (about 4 hours), coated, and FE-SEM. The preparations of live cells, air-dried, showed good quality, but the CPD preparations appears to meld. Thanks in advance.
For me it looks like precipitation, not melting.
Do not use mercury chloride. And what does it mean "glutaldehyde with osmium"? Do you mix then? You do not need Os for SEM. Try just 2.5% of glut (in a buffer). Carrying CPD for 4 hours is an overkill, but is should not contaminate specimen.
The attachment fixed by 2.5% glutaldehyde (final conc.) and carried CPD for 1.5 hr. But the amour plates still appears to melt.
Hi Jae-Ho,
Well, at first, I have to say that I do not know anything about Colepidae biology.
So, let's assume that CPD processing of your samples was done well. Is there any possibility that amour plates are etched before final CPD processing? The amour plates are calcified (J Eukaryot Microbiol. 2008 ; 55(3): 185–200. doi:10.1111/j.1550-7408.2008.00323.x.). Maybe that "amour plates melting" reflects the low pH effect that might happen during the fixation process.
I'm really curious If you solve the problem.
Best regards
Oldrich
Dear Jae-Ho,
As you know, cracks and holes in the specimens commonly point to the CPD protocol. It strikes me that the CPD procedure last 4 hours (I do not know which CPD apparatus you are using…), but I am not sure if this could affect the calcium carbonate of the armour, or if some pH issues during the fixation, as pointed by Oldrich, could be behind what you called "melt-appearance" of your specimens.
Regarding the fixatives you have used, I am not sure from your message whether you have tried the mixture of 2% saturated aqueous osmium tetroxide with saturated aqueous mercuric chloride (Parducz´ fixative), which would be my first choice.
As an alternative to CPD, in case you keep getting the same problem, have you considered a chemical drying using hexamethyldisilazane (HMDS)? I have never tried it with ciliates covered with calcium carbonate scales but generally it works fine (e.g. have a look to the paper about Microdiaphosoma arcuatum in J. Eukaryot. Microbiol., 58(2), 2011 pp. 114–119).
Kind regards,
Pablo
Dear Jae-Ho,
Could you post an image of your air dried sample for comparison please. Could you get someone to use ESEM on your sample?
Dear David,
Here the air-dried specimen, but different species in the same genus. Now I am trying to get air-dried SEM but it did not work very well because it is saline water species that means I should remove the salt before SEM.
Unfortunately, we do not have ESEM and have to search it from other institutes.
Dear David,
here the air-dried specimen from the original question.
Dear Pablo,
all solutions/combinations, Parducz's, glu, Os + glu, HgCl2, showed bad results. I have checked the paper of Foissner, Kusuoka, and Shimano (2008) in J. Euk. Mic. and they also took the SEM of Colepidae from air-dried specimens, not CPD. It is interesting and I want to know why. If someone know the reason, it will be very helpful for preparations of biomineralized species.
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