Dear Dr. Burns;

I suggest you to read an article below.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC379936/pdf/applmicro00035-0122.pdf

T.K.

Dear Josua,

I suppose to fix the culture adding the 1.5 per cent solution of glutaraldehyde in the 0.1M cacodylate buffer over the culture, 1 hour fixation in the closed plate at the room temperature. Thenafter washing several times in saline (10 minutes), dehydration in the ethanol series (70%, 96% and absolute), and drying and dissection of the area of interest. Before the observation coating by the carbon or gold.

Best regards

Pavel

Hi,

Thank you for the article.  I have heard that growing on cellophane/cellulose/dialysis tubing can sometimes give a different morphology - do you know if this is the case?

Otherwise, thank you!

Josh

Hi,

I have no informations in this field.

Pavel

If you have such information, please try another method to fix bacterias taken directory from plate. Good luch. T.K.

Use a very thin sterile needle for the purpose. First observe the specimen under ordinary microscope before going in for SEM sample preparation. 

Dear Joshua Burns,

Take the stub with cork and add the drop of glue on it. Then allow patch of cellophane from dialysis tubing (or piece of paper filtre) to fix over it. Press the cellophane (or piece of paper) against the lawn of your bacteria and dry the replica in sterile air flow. Then use cold 2,5% glutaraldehyde solution in buffer to fix the specimen. After that dehydrate it in acetone and make it finally dry in a ‘Critical point drier’. After coating with gold in a sputter coater you can examine your specimen in SEM.

Good Luck!

Dear Joshua:

I think that Dr. Sharga's comment must be more helpful for your SEM technology. I suggest you to read Kumar's paper published recently, 2011, if you could find it described below, on ResearchGate network. Good luck!

V. Kumar et al: Scanning 33(6):446-9 · November 2011

Tateki Kikuchi

Dear Joshua Burns

In définition: 

-Electron microscopy is one of a limited number of techniques suitable to show bacteria in great detail in their natural environment. There are two basic modes, scanning electron microscopy (SEM) and transmission electron microscopy (TEM), and each of them uses specially designed microscopes. SEM is relatively easy to perform and the results are easy to interpret, because the specimens are shown as three-dimensional objects. TEM produces different kinds of images based on the fact that the electron beam passes through the specimen and forms a shadow-like image on a fluorescent screen which is photographed.

-Scanning electron microscopy (SEM)  consists of different techniques. Conventional SEM is used to visualise bacteria which had been fixed, dehydrated, and dried. Rapidly frozen (cryofixed) bacteria may be examined at very low temperatures (below –120°C) by cryo-SEM. Both techniques are carried out with the specimen placed in a high vacuum chamber. There, the specimen must not release any volatile substances.

You can find more details in the following publication: Miloslav Kaláb, Ann-Fook Yang, Denise Chabot. 2008. Conventional Scanning Electron Microscopy of Bacteria

Abstract: 

Scanning electron microscopy (SEM) is one of the best suited out of a variety of procedures to visualise the external appearance of bacteria. Bacteria live in various environments and their preparation for SEM thus takes their nature into consideration. The basic principles of isolation, fixation, dehydration, drying, mounting, and photographing have many variations, some of which are discussed in this article.

Cordialy

Marius

Hi everyone,

Thanks for all your help, it's much appreciated.  I'll try the Kumar method and see how it works out.

Thanks again!