Dear Namita, dear Frederick,

thank you for telling us about the task you would like to achieve (and thank you, dear Frederick, for your "background"-story about the vesicles on your daughter's arm and your suspicion of viral infection which always would be the differential to "bacteria".... ) as well as to point Namita to the possibility of purchasing or at least using the Beckman airfuge).

Ok, to be short (and this time I mean: really short):

IMHO, there is general acceptance that "assessment" of bacterial infection or localization of (single, or even some!) bacteria is no common diagnostic investigation or method used routinely. Therefore one has to figure out how bacteria (bacterial) contaminations or bacteria 'in fluids' or 'in tissue' can be/are to be

i) collected without the risk of being self-infected or to entrap contamination,

ii) fixed and

iii) processed for EM (now the question again arises: TEM or SEM?).

If these basic necessities are clarified one would search specific literature for collection - fixation - processing techniques/methods which were used in other scientific experiments or clinical applications, and have (already) been published in scientific papers or special Lab-techniques handbooks .

It is not easy to give you a (= one) recipe at hands since we only know for now that you would like to . Therefore I just wait for your, dear Namita,  kind reply telling us a bit more about the conditions you are faced with.  And, would you mind to comment on the possibility of your (EM-?) facility to get a Beckman airfuge? (which naturally would beof benefit and fortune!).  I have a lot of as well as sort of recipes and processing instructions concerning localization and diagnosis of Bacteria in general as well as B. spp....    but more detailed facts what you really want to do (i. e. which kind of clinical samples:  tissues, blood etc.SEM or TEM) for sure would be of benefit to answer not only correctly but seriously. Thank you for your understanding....best wishes and regards, Wolfgang

EM is not a diagnostic tool. In some cases (for example - differential diagnostics of smallpox) it is possible to use this method, but in a lot of cases it is very difficult or impossible.

The most important problem is an amount of particles (bacteria, viruses, etc.) in a sample. For example, it is necessary to have a concentration not less than 10^5 particles on 1 ml for classical negative staining TEM. The next problem is a purification of a sample (for negative staining TEM or SEM). The other method - preparation of ultrathin slides is very special technique with a lot of difficulties.

Not all clinical samples are suitable for EM study. Please describe your task more detail.

Good luck!

Actually I am writing one research proposal, thanks for your suggestion 

Hi dear,  I agree with you.  Try to read this article .. It may be helpful

Article Anti-methicillin Resistant Staphylococcus aureus Compound Is...

Dear Namita Ashish Singh, don't want to interfere with the posts of Denis and Hala. But you might see or at least imagine what is possible if you have a special situation where bacteria are abundant: cf. 

https://www.researchgate.net/publication/215824406_Purpura_fulminans_ultrastructural_findings_by_transmission_electron_microscopy_in_four_cases_with_special_reference_to_fixation_of_skin_biopsies?ev=prf_pub  and 

https://www.researchgate.net/publication/281120633_Oral_Lecture_PURPURA_FULMINANS_Ultrastructural_findings_by_Transmission_Electron_Microscopy_in_4_cases_with_special_reference_to_fixation_of_skin_biopsies?ev=prf_pub

If you want to discuss my findings (or know about the "TEM-processing protocol" please just come back with your specific question. Thank you and good luck

Poster Purpura fulminans: ultrastructural findings by transmission ...

Conference Paper Oral Lecture: PURPURA FULMINANS: Ultrastructural findings by...

Thanks Wolfgang H. Muss, Actually I am looking for a protocol of Bacillus spp. identification from clinical samples using Electron Microscopy

Years ago, when I was young and naieve, one of my daughters went thru a period in which vessicles appeared on her arms and hands.  It was diagnosed as bacterial and she was providede with an antibiotic.  I thought virus based on my limited experience.  I did not argue with my physician.  Instead, I brought home a fresh and sterilized carbon-coated TEM grid in a sterile container, a lance and a sterilized pair of #5 tweezers.  I lanced a vessicle and simply touched the carbon side of the grid to the brownish transparent drop of fluid and placed the grid back in its container at room temp.  Next morning, I applied my current negative stain and took the grid to the scope.  Virus!  Downside?  At that point in time, I was not equipped with an antibody against all.  However, there were many of them on the grid.  {When I was an undergraduate, I took a course in virology and followed it in graduate school with the lab.}    I'm certain that my effort did no good for my daughter.  She just got over it., Now my advice for your protocol.  I strongly advise you to check out a Beckman Airfuge that has an accessory that is designed to sediment suspended particles directly onto a grid in a closed environment.  Last time I saw a quote, the instrument was quoted at $12,000+.  [https://www.beckmancoulter.com/wsrportal/wsrportal.portal?_nfpb=true&_windowLabel=UCM_RENDERER&_urlType=render&wlpUCM_RENDERER_path=%252Fwsr%252Fresearch-and-discovery%252Fproducts-and-services%252Fcentrifugation%252Fairfuge-air-driven%252Findex.htm#2/10//0/25/1/0/asc/2/340400///0/1//0/%2Fwsrportal%2Fwsr%2Fresearch-and-discovery%2Fproducts-and-services%2Fcentrifugation%2Fairfuge-air-driven%2Findex.htm/]  118,000g.

Dear Namita, dear Frederick,

thank you for telling us about the task you would like to achieve (and thank you, dear Frederick, for your "background"-story about the vesicles on your daughter's arm and your suspicion of viral infection which always would be the differential to "bacteria".... ) as well as to point Namita to the possibility of purchasing or at least using the Beckman airfuge).

Ok, to be short (and this time I mean: really short):

IMHO, there is general acceptance that "assessment" of bacterial infection or localization of (single, or even some!) bacteria is no common diagnostic investigation or method used routinely. Therefore one has to figure out how bacteria (bacterial) contaminations or bacteria 'in fluids' or 'in tissue' can be/are to be

i) collected without the risk of being self-infected or to entrap contamination,

ii) fixed and

iii) processed for EM (now the question again arises: TEM or SEM?).

If these basic necessities are clarified one would search specific literature for collection - fixation - processing techniques/methods which were used in other scientific experiments or clinical applications, and have (already) been published in scientific papers or special Lab-techniques handbooks .

It is not easy to give you a (= one) recipe at hands since we only know for now that you would like to . Therefore I just wait for your, dear Namita,  kind reply telling us a bit more about the conditions you are faced with.  And, would you mind to comment on the possibility of your (EM-?) facility to get a Beckman airfuge? (which naturally would beof benefit and fortune!).  I have a lot of as well as sort of recipes and processing instructions concerning localization and diagnosis of Bacteria in general as well as B. spp....    but more detailed facts what you really want to do (i. e. which kind of clinical samples:  tissues, blood etc.SEM or TEM) for sure would be of benefit to answer not only correctly but seriously. Thank you for your understanding....best wishes and regards, Wolfgang

Dear Wolfgang H. Muss,

Actually I am writing research proposal, so I have some queries.

Generally If we want to analyze Bacillus bacteria from blood, first of all we culture bacteria then staining followed by biochemicals and antimicrobial susceptibility test. Suppose if I don't follow this protocol and after culturing bacteria from blood on media directly go for SEM after sample processing,whether it is possible or which protocol I can follow?

Dear Namita, thank you.

Naturally you can go from bacteria culture directly to e.g. SEM (as well as to TEM)-observation. For both examination types the first steps of processing will be  (or perhaps nearly !) the same:

NB: CAUTION necessary (and perhaps a permit to work with unfixed bacterial cultures). Personal 'safety measures' apply.

Carefully and cautiously handling the culture media dishes (as usual), decanting perhaps the culture medium, followed by immediately overlaying the cultured bacterial lawn with the / a suited fixative (at RT).The level of fixative should be approx. 1 mm or better 2 mm above the bacterial lawn surface. (alternative:  add to the existing vol. of culture medium [if any] 1 : 1 vol. fixative [2fold concentration!] ). When I did some preparations for scientific reasons/(publication)work it wasn't really easy (being only a natural scientist!) to get permission for doing that work-step by myself in the microbiology Lab! 

Fixative as usual either suitably buffered (P)FA/GA (concentration e.g. 0.5 / 1%) followed by another buffered GA (4% ) fixation. Duration FIX I at least 2 h, FIX II at least 2.5 -4 hrs followed by fix II (fresh) over night at 4°C. washing, osmication, washing,dehydration, eventually adding 1%(w/v) PPD = paraphenylenediamine to the 70% Ethanol during dehydration... The only thing to think over would be eventully to add some other reagent to the initial fixativefor stabilizing membranes a bit better...for SEM you know the further steps from 70 or higher alcohol to CPD etc. If you are in need for more detailed recipe please let me know. NB: since you wanted to get info-data on SEM I found it unnecessary to mention an other technique, namely: negative staining.

Best regards, Wolfgang

NB: There is -at least - an old publication by Bozzola et al 1973 which eventually I could provide (copyright issues):  

Stain Technol. 1973 Nov;48(6):317-25.

In situ multiple sampling of attached bacteria for scanning and transmission electron microscopy.

Bozzola JJ, Johnson MC, Shechmeister IL.

PMID: 4129036 

as well as a more recent article (2002)

Bacterial adhesion to sulcular epithelium in periodontitis

Ljubomir Vitkov et al, 2002: FEMS Microbiology Letters 211 239-246

Dear Wolfgang H. Muss,

Thanks for your valuable suggestions, it was nice discussion with you.