We have been advised to fix cyanobacterial samples before coating and SEM microscope analysis. I can't find a protocol for cyanobacterial cells fixation method to follow step by step. Do you have any literature or experience on that process?
We have done fixation for SEM in green microalgae. Please find the protocol in this paper, hope it may be helpful Article Mechanisms of detoxification of high copper concentrations b...
Best regards,
Jelena
Hi Eleni Keliri
You can use poly-L-lysine, PBS (phosphate buffered saline) method for this SEM analysis (JSM-IT300HR, JEOL)
Regards
I've never worked with cyanobacterial samples, but it should be Ok to use a standard SEM preparation. For example:
1. Cut small pieces (about 5x5 mm) of glass slide, silicon wafer, coverslip, etc.
2. Coat them with about 20 nm of gold using a sputter coater (very common tool, which can be found almost in every EM facility). Mark them scratching the gold layer with a needle;
3. Prepare a bacterial suspension in a buffer (usually PBS is Ok), concentration should be about 10^6 cells per 1 ml. It should be as pure, as possible (wash a few times with centrifugation);
4. Put a droplet (about 20 ul) onto a gold-coated piece of glass and incubate for a few min;
5. Wash with buffer, then put it into fixative (3 % glutaraldehyde in the same buffer) for 15 min;
6. Wash with buffer, then dehydrate with 30%, 50%, 70%, 90%, 96%, 100%, 100% ethanol, every step - 5 min;
7. Remove the ethanol residue with chemical drying (HMDS) or using a critical point dryer (I prefer this method, usually EM facilities have such a machine);
8. Glue the wafers to standard SEM stubs (usually it is possible to glue three or even four for one) with a conductive glue (carbon or silver paste, or something similar, usually can be found in an EM facility). Make an electrical connection between the surface of the sample and the stub using this conductive glue;
9. Coat with about 5 nm of gold or platinum/iridium;
10. Examine with an SEM. For modern FE-SEMs the best quality of imaging usually can be reached when the acceleration voltage is about 2 kV.
This protocol is described here (for sperm):
Article New Insights Into Sperm Ultrastructure Through Enhanced Scan...
and here (for bacteria):
Article Bacteriophage-resistant Acinetobacter baumannii are resensit...
Good luck!
Hi Eleni,
I would recommend that you use osmium tetroxide in a osmolarity adjusted phosphate buffer. See the protocol in this paper: Truby, E.W. (1997), Preparation of single-celled marine dinoflagellates for electron microscopy. Microsc. Res. Tech., 36: 337-340.
Cheers, Jennifer
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