For TEM sample preparation of bacteria, you may go through the following process;-

1.      First, I prepared the pellets of bacteria obtained by centrifugation and washed carefully with 0.1 M phosphate buffer (PBS).

2.      Afterwards, pellets were fixed with 0.5 mL of 2.5 % gluteraldehyde solution, and then subjected to a series of alcohol dehydration process (30%, 50%, 70%) followed by PBS washing.

3.       Finally, the pellets were dispersed 1-2 mL of pure ethanol (100%) and drop cast on TEM grids.

Your image [ultrathin section, ?nm thickness?, staining?] shows bacteria (E.coli) with an artificial morphology after "classical" fixation and perhaps processing (the latter we have no information so far, therefore one only can guess...about the causes of artifacts)  as this is displayed mostly in bacterial specs. processed without some special treatment*).

Special treatment could be: PLT-embedding (progressive lowering temperature embedding in lowicryls HM20 or K4M), Use of microwave fixation and embedding, additives to your primary fixative, Processing by initial HPF-technol, and others. IMHO there are two major parameters influencing "ultrastructural preservation" after >>>chemical fixation:

i) elution of bacterial core components (e.g. also due to damage of bacterial wall) by fixative and/or processing chemicals

ii) non-preservation of bacterial core components due to bacterial wall permeation hindrance to fixative(s) and processing chemicals.

Also one should not forget the temperature maintained during all processing.

There are a lot of methods/articles on bacteria ultrastructure preservation out in the wild waiting to be found by eager young researchers... 

(to  name only some of some hundred articles  cf: e.g. 

http://onlinelibrary.wiley.com/doi/10.1042/BC20070106/full, http://jb.asm.org/content/174/20/6508.full.pdf;  or also  www.researchgate.net/.../09e4150ab035d7de05000000.pdf,  && )

Kim,

If you could provide a little more detail on your protocol, it may help with trouble-shooting.  Otherwise I will re-post my earlier response to a very similar question:

A proper fixative in the proper buffer (osmolarity) is recommended.  Our EM specialist, Barbara Smith found that certain strains of E.coli will show severe membrane shrinkage, unless you fix with a  low osmolarity (20 mM cacodylate) buffer in glutaraldehyde only, followed by non-reduced osmium, and no UA en-bloc staining.  After dehydration we either critical point dry in liquid CO2 or use HMDS as the drying agent. good luck,

Michael Delannoy

Thank you for your responses!!  

Muss, you mean, not only the protocol but also the condition of E. coli can be influence on the results, right?

Delannoy, Here I add the protocol that I used, from http://www.ncbi.nlm.nih.gov/pubmed/22998466. 

Is there any additional recommendations?

Kim,

Two points:

1.  I would fix live suspensions with equal volumes of 2x fix to sample (fix live suspensions for 10 min, then centrifuge to a pellet, change  to fresh 1x fix and continue to gently rock the pellet). 

2.  I would use maleate buffer (0.1M) after osmium (since you not reducing the osmium with KFe(CN)6), and do the en-bloc staining in UA/maleate.

Do the wt samples (non treated before fixation) look the same?

sincerely,

Michael Delannoy

Kim/Hyung-eun (I am really sorry not to know exactly what your first/family name is),

From the article you cited to have adopted the protocol schedule above (= http://www.ncbi.nlm.nih.gov/pubmed/22998466. ) one can see ( a) from PubMed citation find the original article in ACS Journal (cf. http://pubs.acs.org/doi/abs/10.1021/es302379q, the article unfortunately only accessible as PPV=PAY-PER-VIEW) in the below "ABSTRACT" displayed "graphics"-figure ) that specific treatment will render altered bacteria (morphology WITH empty areas in bacterial core matrix). 

This in turn means b) that CONTROL bacteria specimen preparation has to follow the same processing schedule as the previously treated bacteria:  this is not only valid for the cited article (the content I unfortunately am NOT able to check due to blocked access) but also if you are in a process of preparing/processing your "own bacteria", treated or untreated ("wt "as designated by Michael).

In Re No 2 (by Michael) I guess he wanted to propose an end preparation / processing for SEM- instead as for TEM-observation.         (Michael, am I right?) 

And, Kim/Hyung-eun,

YES: IMHO, there are influences on ultrastructural morphology of bacteria (being either "CONTROL=wt=wild type" or, after a specific treatment, respectively)

i) in terms of culture/culturing (depending on several parameters like nutritive media consumption, duration, culture conditions)

ii) "wt" vs. "treated" bacteria.

Unfortunately I have to leave now.....   will come back to your Q tomorrow...

If you have in posession the cited article [=

Environ Sci Technol. 2012 Oct 16;46(20):11299-304. doi: 10.1021/es302379q. Epub 2012 Oct 5. "Role of reactive oxygen species in Escherichia coli inactivation by cupric ion." by : Park HJ1, Nguyen TT, Yoon J, Lee C.] 

as a pdf at hand, would you mind to send it to me/my e-mail account   [email protected]??

Thanking you in advance, Wolfgang

Kim, Wolfgang,

The protocol I listed above can be used for either TEM or SEM, samples can be run concurrently until the last ethanol step.  At that point the TEM samples would go into transition solvent (propylene oxide) then spurs parts resin embedding.  The SEM samples would then either be critical point dryed (liquid CO2) or HMDS dryed (from ethanol).

I will point out that certain samples do not do well with en-bloc staining for SEM.  We have found membrane shrinkage in them.

I hope this clarifies my response.

sincerely,

Michael Delannoy

Michael,

thank you really much for mentioning a disparate behaviour of some bacteria strains (membrane shrinkage) if using "classical" fixation protocols and the technical hint on using low osmolar buffer (as of your EM specialist, Ms/Mrs Barbara Smith). I always understand / understood your pointing also to the problems with en-bloc UO2Ac-incubation as a valuable information - not only for the EM-novice -. I remember the discussion about "adequate fixation of bacteria(and other critters)" between those using frequently only chemical fixation and those using most frequently if not ONLY CRYO-Fixation methodology.  I shall go through the literature I saved on this ....and if I find something worth to present in this forum, I shall do...

For now:  best wishes and regards, Wolfgang

Wofgang,

Seems like we are always in sync.

cheers,

Michael Delannoy

For TEM sample preparation of bacteria, you may go through the following process;-

1.      First, I prepared the pellets of bacteria obtained by centrifugation and washed carefully with 0.1 M phosphate buffer (PBS).

2.      Afterwards, pellets were fixed with 0.5 mL of 2.5 % gluteraldehyde solution, and then subjected to a series of alcohol dehydration process (30%, 50%, 70%) followed by PBS washing.

3.       Finally, the pellets were dispersed 1-2 mL of pure ethanol (100%) and drop cast on TEM grids.

@ Shahram Nazari :

Honestly: I found your recent answer (unfortunately as late as TODAY and only because my answer was recommended

by Paavo Bergmann (thank You!)

most recently [e-mail-notification was of today: 'Fr 28.06.2019 12:35'].

So - with my apology - I would like to ask nevertheless:

i) would you mind to upload an (a representative) image of the successful bacteria-TEM-preparation you did, and

at least

ii) if I got it right that you are washing your already dehydrated 'bacteria /cells' AFTER dehydration with a buffer*): (quote) " 2......subjected to a series of alcohol dehydration process (30%, 50%, 70%) followed by PBS washing. " and further on:

(quote) " 3.       Finally, the pellets were dispersed 1-2 mL of pure ethanol (100%) ..."

*) if that is correct, would you mind to disclose the type of bacteria as well as the composition (molarity, pH, etc., or at least the brand) of the PBS you used for that task?

Thank you in advance for clarification.

Best wishes WHM.