I want to check the effect of nanoparticle on bacterial ultrastructure by TEM analysis. For this I will be washing cells with PBS and then fix it in 2% gluteraldehyde overnight at 4 degree centigrade . The subsequent step is fixation of the bacterial pellet using 1% osmium tetroxide (not available with us) is to be done by another institute after 2—4 days followed by dehydration using alcohol. In that case can i dispatch the sample after discarding the gluteraldehyde from the vial or shall I keep the sample back in PBS after gluteraldehyde fixation and then dispatch. Please give your valuable suggestions. Thank you.
Keep your samples in the glutaraldehyde fixative. The glutaraldehyde should be diluted in PBS. Keep the samples at 4 degrees C until you can ship the samples to your collaborators. Ship the samples on ice in a styrofoam shipper. Do NOT ship the samples with dry ice, if the samples freeze they are no longer of any use to you. Once your collaborators receive the samples they can continue with the processing; washing the glut out, osmification, dehydration etc.
I routinely get samples from other researchers this way, sometimes with delays of a couple of weeks. The morphological results are usually excellent.
As always: it depends.... Brent Gowen's answer principally accepted... but I 'd differentiate somehow:
PBS = (phosphate) buffer (appropriate pH and osmolarity)
If sending via parcel or even air mail (if you are allowed to do so) you can do it i) cooled by dry ice, which - IMHO - after a proper primary fixation (e.g. (1) Karnovsky (at RT at least 3 hrs), followed by (2) 3-5% buffered GA - since bacteria pellet (?) - over night at 4°C) isn't necessary any more So: ii) also without cooling (as long asduring the wole duration of transportation the temperature doesn't exceed 28° C). Sometimes transportation time is longer than expected...and dry ice evaporates ... Container box MUST be an 'open' construction during transport (i.e. there must be holes in the cardbox or sytrofoam container (otherwise: danger of explosive hazard may arise...)..and, last but not least,
Don't forget to add the notice on status of specimen prep... (:-))
Good luck!
In question, what will be the following steps in the protocol (you can ask your collaborators from the other institution). If Pb-ions are to be included at some steps in the protocol, these could react with some residual phosphate in your sample from the PBS, which can result in production of insoluble lead phosphate crystals and this will be a problem in the EM analysis. This can also happen with phosphate buffer and uranyl acetate. Other buffers may be preferable in electron microscopy, especially if you aim preparing samples by negative staining. You best discuss this with the people that will process your samples.
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