Good morning, I have to make a few experiments with the AFM, using a triangular tip, mounted on a cell fluid. On this tip I have to attack the yeast cells.
How could I do?
I suggested to use the poly-lysine, but carrying out some tests on a coverslip, do not notice the difference between the negative and positive, on both the yeast adheres perfectly.
Protocol for the coating:
- Wash the coverslips with etanole and water and let dry
- Coating with 0,5ul of poly-lysine (creating of the drops) and allowed to incubate for 15min
- Washing with water and left to dry for 2 hours
- Incubation with yeast for 30 min (S.Cerevisiae: 0.05g dissolved in 10ml of PBS)
- Washing with PBS (3 times)
Do You try to immobilize yeast on the D- and L- poly-lysine? If yes, this is obvious results that there is now differences between yeast adhesion. But if You try to immobilise yeast, which is resuspended in different pH buffers, maybe you will found changes, especially between pH 7 and pH 10 or higher. But actually, It is hard to understand what is the main goul? The protocol seems logic, but it is unclear also the poly-lysine concentration You used
thanks you!!!
I use L-poly-lysine.
STOCK 20ug/ul
I used 0.01% poly-lysine and PBS is 1X , ph 7.4
the concentration of poly-lysine seems to be enough for slips modification. Are You going to perform AFM experiments in the solution or dry sample state?
in the solution, whit a fluid cell.
When I observe the negative (without PLL coating) I always observe yeast cell! I don't understand!
Just another question: You think is possible to use any antibiotics to kill S.Cerevisiae? Like tetracycline, ampicillin?
Yeast should adhere on the glass, because of cell envelope structure and physiological properties. You can kill yeast in different way :) But I am not sure if tetracycline and ampicillin will afect yeast because they are addressed to bacteria. There are a lot antibiotics for fungy. You can also try different detergents to prevent yeast adhesion.
Thanks a lot for your help!
I believed that I also that antibiotics do not work, in literature I found very frequently use is acetic acid, lactic acid, salt and hypochlorite
Maybe this article will be helpful for you "Baker's yeast as a tool for the development of antifungal drugs which target cell integrity – an update" (DOI: 10.1517/17460441.3.8.931)
Different antibiotics against yeast are already published and you can easely find them using either spocus.com or pubmed. But the most simple compuonds, which can help you to monitor changes in yeast activities are cyanides (e.g. potassium cyanide - it irreversibly inhibits respiratory chain, potassium ferricyanide - it is alternative electron acceptor of respiratory chain elements). Additionally you can try sodium dodecyl sulphate, which provokes yeast envelop destabilisation and cell death.
Hi! thank you for your help!
A lab member suggested me to use the glutaraldeide instead the polylysine to improve the adhesion of yeast on the AFM tips. What do you think?
Hi, It's a hardest immobilisation method, because adhesion of yeast will be irreversible. And the most of methabolic activity of yeast will be lost. But this method is perfect, if you wont analyse only cell as a "shape". By fact, it is possible to find right concentration of glutarealdehide (GA) for smooth yeast immobilisation. It means, that yeast can be kept in active form for some time. My suggerstion is first to try direct immobilisation of yeast with GA, if something will be wrong with activity, mix the yeast cell suspension with albumine and then try to immobilise by GA. Good luck!
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