My bacterium is Aeromonas dhakensis and I had observed very pretty lateral (peritrichous) flagella under oil immersion light microscope using traditional flagellar stain after growing my bacteria on swarming agar for 6 hr. However, when I used the same method to grow bacteria for TEM, I hardly viewed any flagella on my bacteria. The protocol I used for TEM sample preparation is that I used a sterile disposable inoculation loop to take some of the bacteria from the swarming edge very gently and resuspended in sterile distilled water. The suspension was slightly turbid, and I had checked under light microscopy to ensure flagella were still intact on the bacteria. Then, I proceeded with fixation using 4% glutaraldehyde in cacodylate buffer for 30 min at room temperature and 10 uL of the suspension was absorbed onto 300-mesh Formvar-coated copper grid for 10 min, blotted with filter paper, followed by negative staining with 10 uL of 2% PTA solution (pH=7) for 2 min and blotting again. The grid was dried in a dessicator for at least 3 days prior to TEM viewing (Libra 120, Carl Zeiss) at 120 kV. I would be grateful if anyone can share your knowledge with me in which step I might do wrongly in TEM sample preparation that causing my flagella to lose.
Interesting problem! I had a similar issue with another single cell organism. A single cell, motile alga. The organism sported two cilia used for locomotion. However, when stressed, the alga let go of their cilia, with considerable energy,
I purposely stressed the alga, on glass slide, under coverslip, with the camera running in movie mode. I started removing liquid, slowly by way of periodic wicking of the water with a strip of filter paper. When the alga felt stresses from lowl levels of water the cilia literely shot off of the alga with considerable force. They would shoot out several micrometers from the organism.
I do not know if your issue is like this one; under stress (perhaps with the introduction of fixative Being the stress input). But it is one possibility.
I hope this helps.
@ Var St. Jeor, thank you for the suggestion. Yes, I believe fixative can be one of the possibilities to lose flagella as they are very fragile and I have also came across a number of papers that skip the fixative step. I will try to omit fixative to see if it will work out. Thank you vy much!
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