Re: Jellyfish for SEM
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|From:||Scott Whittaker <email@example.com>|
|Date:||Thu, 12 Aug 1999 08:34:10 +0100|
All sound good as well provided they have the equipment. Replica is a
quick and easy idea. Not too fond of the resin idea, but as I have never
tried it can't say it wouldn't work either. Wouldn't you just be imaging
the resin. SEM is monochromatic, imaging the sputtered heavy metal surface
and is seems the tissue and the resin would be indistinguishable. I just
based the reply on my work with the "goo" in tomatos. Standard fixation
and osmocation was the only thing which worked with a wet fracture from
ethanol/LN2. What a nightmare that was on the overripe samples though.
At 11:42 AM 8/12/1999 +1000, you wrote:
> Dear Scott,
> Although I cannot fault your method for routine tissue samples, I suspect
>there could be problems with a jellyfish. Now I have never worked with
>these organisms either, but I do know they are mainly water. Therefore any
>dehydration is bound to severely damage their structural morphology.
> For example I recently worked with chicken eyes. These organs are
>pressure filled spheres of mostly water. They do stand up to some rigorous
>treatment but only because the external layer of the eye is cartiledge.
>Inside the eye there are significant problems with shrinkage, seperation of
>tissue layers, & general morphological changes.
> The following is the advice I sent to Rena:
> "Tricky, I'm assuming its fixed & that you have more than 1 specimen to
>play with. My first thought would be to freeze it, coat it, & see what
> But this might require slightly more specialised equipment than you have
> Next choice is to attempt some sort of substitution, ie infiltrate with a
>hydrophilic resin (eg JB4, no dehydration required) to remove the water (&
>hopefully not change the gross or micro morphology of the thing), then coat
> Then there's always the possibility of making a replica, tricky for
>something so big (relatively speaking) but may give you the detail you ar
> Try contacting:
> Your next best bet is a marine sciences lab or a Uni that runs a
>specialised course in marine biology."
> Rob W.
>At 02:59 PM 8/11/99 +0100, you wrote:
>>Hi rena. I am new to the list, but primarily do SEM and TEM. Although I
>>have not actually worked with jellyfish, here is how I would proceed.
>>Fix in 2-2.5% glutaraldehyde in filtered seawater pH=7.6-8 (assuming
>>samples are marine) for 1hr. Otherwise use PBS buffer pH=7.2ish
>>Wash in seawater 3x 5 min.
>>Post-fix 1% buffered osmium tetroxide 1hr. This step is very optional
>>especially if you are not going to fracture the sample but the deposition
>>of heavy metal can help charging problems.
>>Wash dH2O 3x 5 min.
>>Graded Ethanol series 25-50-75-95-100-100% 5 min in each step.
>>>From here you need to either critical point dry or solvent dry with
>>HexaMethylDiSilizane (HMDS by far the fastest route but doesn't work for
>>mount, sputter, view.
>>If this doesn't work you may need to try some of the more exotic procedures
>>like OTOTO or a mercuric chloride/osmium fixation or even freezedrying from
>>LN2. Let me know if you need references or details on any of this. I will
>>be happy to pass them along.
>>I have also posted this message to the microscopy listserver dealing
>>primarily in SEM and TEM. Good luck
>>At 10:37 AM 8/11/1999 -0500, you wrote:
>>>I just received some small jellyfish (3/4 inch diameter) with attached
>>>Vibrio. Does anyone have any experience with processing such a
>>>for scanning electron microscopy? Your help is appreciated!
>> GO GATORS
>>Scott D. Whittaker 218 Carr Hall
>>EM Technician Gainesville, FL 32610
>>University Of Florida ph 352-392-1184
>>ICBR EM Core Lab fax 352-846-0251
>> The home of " Tips & Tricks "
>R. Wadley, B.App.Sc. M.L.S, Grad.Dip.Sc.MM
>Cellular Analysis Facility
>School of Microbiology & Immunology
>UNSW, New South Wales, Australia, 2052
>Ph (BH) +61 (2) 9385 3517
>Ph (AH) +61 (2) 9555 1239
>Fax +61 (2) 9385 1591
Scott D. Whittaker 218 Carr Hall
EM Technician Gainesville, FL 32610
University Of Florida ph 352-392-1184
ICBR EM Core Lab fax 352-846-0251
The home of " Tips & Tricks "
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