I need to study the flagella and external structures. I have some bacteria isolated and need to do SEM analysis of the same. I would like to know if any preparation protocol exists to condition my bacterial cells for SEM. Please help.
I think you can take a look on these papers. You can have an idea about the different protocols.
"A survey to assess the ultrastructural preservation of fixed biological samples after air-drying from tetramethylsilane"
"SEM sample preparation for cells on 3D scaffolds by freeze-drying and HMDS"
Good luck !!!!!!!
Dear Sasidharan,
The standard procedure for all SEM analysis of biological samples consists in dehydration followed by coating of the samples with gold, carbon, or platinum films (conductive materials). First, you can dehydrate the samples by using ethanol baths with different concentrations (30, 50, 70, 80, 90, 100 %, for instance). Currently, the last dehydration step can be done with Hexamethyldisilazane (HMDS) instead of critical-point drying (standard procedure). It is important to know that sometimes the dehydration may change the conformation of your bacteria and what you will see in the SEM analysis is not the real conformation. So, take care to do not dehydrate in excess.
PS: You can find several protocols online. Just search for SEM preparation of biological samples, dehydration of SEM samples, etc.
In my case, staphylococcus aureus adhered on textured surfaces of titanium were prepared for SEM analysis by dehydration (70, 80, 90, and 100% ethanol for 30 minutes in each solution) followed by air drying and coating with a carbon thin film (sputter coating machine).
I hope it is useful for you. Good luck !!!!!
Best regards,
@Alexandre Cunha
You forgot about very important part of protocol: fixation.
And what is to "not dehydrate in excess"? I am afraid it is a wrong advice.
Your problem is very close to SEM observation of suspended cell cultures. I have never done it myself (did it only for TEM), so you’d better wait for more qualified advice, of google something like “images sem bacteria flagella” and try to find a protocol.
As I see it, you may proceed in two ways (but again, it is “theoretical” advice, I have not tried it):
1. Collect bacteria on a filter and work with a filter as with standard biological specimen: fix, dehydrate, coat.
2. Instead of a filter, use gentle centrifuging in between steps, and again fix and dehydrade (skipping CPD part) in suspention. Then put a drop of final suspension in alcohol on a glass coverslip, let it dry and coat.
Thank you Alexandre and Vladimir sir. Can TMS be used to dehydrate the samples and as part of the dehydration, can lyophilisation be done? If not why? Is there any other cost effective method of preparing bacterial samples for SEM analysis?
First you must fix cells, specially for cells with flagella.
Before any dehydration you can fix with Osmium tetroxide under a "fume hood" because it is very dangerous, but the results are incredibly beautiful.
You can find procedure on web.
Good luck !
Dehydration is usually performed in graded alcohol solutions. There are a lot of protocols on the web, for example this one:
http://www.huck.psu.edu/facilities/microscopy-cytometry-up/faq/other/sample-preparation
If you do not have access to critical point dryer, you can replace it with final wash in HMDS or TMS.
Also if OsO4 is a problem for you, you (possibly) can skip its application.
I think you can take a look on these papers. You can have an idea about the different protocols.
"A survey to assess the ultrastructural preservation of fixed biological samples after air-drying from tetramethylsilane"
"SEM sample preparation for cells on 3D scaffolds by freeze-drying and HMDS"
Good luck !!!!!!!
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