Dear Meggie, just came across your reply finding the question mentioned in the new rectangled overview "Questions and discussions matching your expertise").

Thank you for the details you gave.... I really hope I did not offend you with the request on the ingredients of your PHEM-buffer. But - usually - when I reply to methodological questions or problems of / during specimen preparation I think it is necessary to clear all pivotal parameters (at least as much as possible) - and - naturally - I think that there are a lot of "students" and young researchers out there which can profit from some explanation about the matter in question.

The fact that - for such critical specimen prep-work - you don't have an osmometer at hand is deplorable and reminds me on my years of study in zoology (in the 1970ies) when there (for appprox.40 PhD-students) only ONE osmometer was available (only by chance and "good will" of another working group leader who was able to get money for an osmometer from their research project funds ).

I do hope that most of what I wrote in my reply fits the request you wanted to be answered, and it is sad that you perhaps will have to learn about the alterations induced by 10x higher concentration of HEPES by evaluating these changes after a lot of manual work....(I know what you are talking about, since I was LM-/TEM-&specimen processing benchworker all the time)....

Just for convenience of any interested reader, I provide here also the coordinates (bibliographic data and URL's) for the really interesting article by MONTANARO et al, 2016 (as mentioned above by Meggie). It is open access (at least for me) and -especially also with regard to data of measurements (" Detailed overview of the different fixation and washing buffer formulations and their osmolarity.") and the consequences /effects of using the diverse formulations on (poor -better) stainability of structural components in resin sections should be incorporated in any (TEM-)specimen preparation protocol-collection:

PeerJ. 2016; 4: e1860.

Montanaro J, Gruber D, Leisch N. (2016)

Improved ultrastructure of marine invertebrates using non-toxic buffers.

PeerJ 4:e1860 https://doi.org/10.7717/peerj.1860

https://peerj.com/articles/1860/

Published online 2016 Mar 31. doi: 10.7717/peerj.1860

PMCID: PMC4824901 PMID: 27069800

Improved ultrastructure of marine invertebrates using non-toxic buffers

Jacqueline Montanaro, Daniela Gruber, and Nikolaus Leisch

(Vienna, Austria & Bremen, Germany)

PDF (open access, 28 MB): https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4824901/pdf/peerj-04-1860.pdf

[https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4824901/]

Dear Meggie, I wish you all the best with your specimen preparations and hope that the alterations might not be that dramatic as they c(w)ould be expected.

Last but not least I would like to find your kind reply on what you were able to find out after examination of your incorrectly treated specimens. And, for sure, the outcome should be considered to be documented and published anyhow....

Best regards, Wolfgang

Hi Meggie,

Are you working with animal or plant tissue?

Why did you choose these fixative methods?

Regards

João

@Joao: ...according to the data in Meggies question: "...200 sponge tissue samples have been fixed and ....[processed...] .... " I guess it is generally a zoological [PORIFERA] tissue matter, hence like "animalcules"....with very different but specialized cell (types), namely pinacocytes, choanocytes, and amoebocytes [am(o)eboid cells], respectively. These cells constitute the "living part" of sponges, but there are also skeletal elements (‚crystallites‘, ‚spicules/spiculae‘,composed of either calcite=calcium carbonate or silicic acid=silicon dioxide - produced by special cells, namly sclerocytes;) which constitute the more solid part of sponges. [according to Meggies information such (?? which ones) sponges were processed for long storage in ?? ethanol?%EtOH?? before further processing and embedding - & finally sectioning & staining for examination by means of Transmission Electron Microscopy(TEM).

@Meggie: The "PHEM-buffer" [most likely - we do not know exactly unless Meggie will disclose that secret(:-)) ] similar to a 'recipe' with the following ingredients: PHEM [500 mls; 2x]. 18.14 g Pipes. 6.5 g Hepes. 3.8 g EGTA. 0.99 g MgSO4. Adjusted to pH 7.0 w/ KOH] might be used in this special case (sponges usually saltwater inhabitants) since a zwitterionic buffer might overcome precipitation or tonicity problems / issues occuring with the use of anorganic buffers like PBS, or phosphate (Na-Na-, or K-PO4-) or cacodylate buffer. The added EGTA as of my knowledge acts as mild decalcifying agent -see "skeletal elements" above . I assume that the PHEM buffer was used for rinsing & equilibrating and secondly diluting the fixative (?? which one?). As a pragmatic solution, one could calculate the effect of 10x the concentration of HEPES on raising the tonicity of the buffer and then assume that a ionic tonicity or osmolarity way too high? for preservation of ultrastructure (with or without either shrinkage or bursting) or changing the pH to an inappropriate value for the 'surviving' of sponge cells.

General calculatory considerations regarding tonicity/osmolarity (serious personal recommendation: you should perform measurements of the P- 1x H vs 10x H – EM-solution by use of a suited osmometer device):

Assuming Hepes [HEPES Molweight= 238,3012 g/mol] is the monosodium salt, 10 mM is about 20 mOsm, 10x concentration=100 mM increases osmolarity approximately about / to 200 mosmol.

Meggie informed about the solution parameters she made: "instead 1 x PHEM (‚is 25mM‘) but I made 250mM".

25 mM is about 50 mOsm, 10x concentration=500 mM increases osmolarity therefore approximately about / to 500 mosmol. This disproportional increase of the buffer‘s / fixative vehicle‘s osmolarity indeed though might have harsh consequences regarding disturbances in achieving a correct ‚morphological equivalent image‘ of the animal cells on ultrastructural examination.

NB: Admitting I'd never had such sponge specimen preps in "my professional life" and therefore have not made and/or measured 1x PHEM vs. 1x P-1x/10xH-EM-buffer solution.

Wolfgang H. Muss Thank you for your response. The PHEM buffer I use is no secret! Just a typical recipe found online (and originally found in a paper by Montanaro et al 2016, who compared it to PBS and cacodylic acid buffers). PHEM is used as the buffer for my fixative (2.5% glutaraldehyde, 1% PFA) and also in the proceeding dehydration series (up to 70% EtoH, where it is stored for a maximum of 2 weeks, and then my samples are embedded). I do not have a osmometer device unfortunately, and now I just have to wait until I actually section my samples and look at them under the microscope to see the damage done. I use TEM and then nanoSIMS so good preservation of ultrastructure is important.

Dear Meggie, just came across your reply finding the question mentioned in the new rectangled overview "Questions and discussions matching your expertise").

Thank you for the details you gave.... I really hope I did not offend you with the request on the ingredients of your PHEM-buffer. But - usually - when I reply to methodological questions or problems of / during specimen preparation I think it is necessary to clear all pivotal parameters (at least as much as possible) - and - naturally - I think that there are a lot of "students" and young researchers out there which can profit from some explanation about the matter in question.

The fact that - for such critical specimen prep-work - you don't have an osmometer at hand is deplorable and reminds me on my years of study in zoology (in the 1970ies) when there (for appprox.40 PhD-students) only ONE osmometer was available (only by chance and "good will" of another working group leader who was able to get money for an osmometer from their research project funds ).

I do hope that most of what I wrote in my reply fits the request you wanted to be answered, and it is sad that you perhaps will have to learn about the alterations induced by 10x higher concentration of HEPES by evaluating these changes after a lot of manual work....(I know what you are talking about, since I was LM-/TEM-&specimen processing benchworker all the time)....

Just for convenience of any interested reader, I provide here also the coordinates (bibliographic data and URL's) for the really interesting article by MONTANARO et al, 2016 (as mentioned above by Meggie). It is open access (at least for me) and -especially also with regard to data of measurements (" Detailed overview of the different fixation and washing buffer formulations and their osmolarity.") and the consequences /effects of using the diverse formulations on (poor -better) stainability of structural components in resin sections should be incorporated in any (TEM-)specimen preparation protocol-collection:

PeerJ. 2016; 4: e1860.

Montanaro J, Gruber D, Leisch N. (2016)

Improved ultrastructure of marine invertebrates using non-toxic buffers.

PeerJ 4:e1860 https://doi.org/10.7717/peerj.1860

https://peerj.com/articles/1860/

Published online 2016 Mar 31. doi: 10.7717/peerj.1860

PMCID: PMC4824901 PMID: 27069800

Improved ultrastructure of marine invertebrates using non-toxic buffers

Jacqueline Montanaro, Daniela Gruber, and Nikolaus Leisch

(Vienna, Austria & Bremen, Germany)

PDF (open access, 28 MB): https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4824901/pdf/peerj-04-1860.pdf

[https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4824901/]

Dear Meggie, I wish you all the best with your specimen preparations and hope that the alterations might not be that dramatic as they c(w)ould be expected.

Last but not least I would like to find your kind reply on what you were able to find out after examination of your incorrectly treated specimens. And, for sure, the outcome should be considered to be documented and published anyhow....

Best regards, Wolfgang

Wolfgang H. Muss Thanks for your detailed reply, and I was not offended at all with your PHEM question :) I understand clarity is key in these matters. Yes, an osmometer would definitely come in handy and I will look in to how much they cost. Although I am guessing that if you only had one between 40 PhD students they could be expensive! I will let you know how I fare with my fixation when I come to section the samples and look at them with light and electron microscopy (could be at the end of the year). Yes it was a lot of manual work to prepare and embed all these samples but (somewhat) luckily I discovered my mistake before starting my next large experiment, so hopefully at least one 'batch' of samples will turn out well.

Thanks again, Meggie

Dear Maggie and Wolfgang,

I read all the discussion above and I clear recognize the impressive efforts of Dr. Wolfgang. He shed light on the topic and really discuss in depth the subject. If you have further question about it please contact me.

Regards and thanks,

João

Wolfgang H. Muss Well I thought I should follow up with the results from above now that I have conducted SEM on the histology samples mentioned above. I could directly compare the sponge tissue from the correct PHEM recipe and those with the incorrect recipe (250 mM HEPES instead of 25mM), and can say that there was little difference between the two in terms of fixation. The only difference was that the cells fixed with the 'incorrect' PHEM buffer were slightly darker, and it was more difficult to see the vacuoles within the sponge cells (with SEM, with TEM the vacuoloes ay be clear I don't knowm). There was no tissue distortion though and my intercellular bacteria were nicely fixed and visible. Hope this helps!