Killing bacteria without denaturing proteins - (Mar/06/2008 )
Hi all,
This is a very naive question, and as a microbiologist I feel I should know the answer, but unfortunately I don't, so here it is : what would be the best way to kill bacteria without denaturing proteins? I'm trying to figure out if the bacteria that I'm studying need to be alive to cause damages to epithelial cells, so I want to try with dead bacteria. But if a proteinous factor is required for this process, I'm afraid that heat-killing bacteria won't really answer the question, because I will obtain completely denatured proteins as well as dead bacteria...
Thank you so much for your help!
I'd be inclined to radiation. It will damage some of the proteins, but most will be ok. A french press would kill most of the bacteria, but some would likely survive, at least in the threads. Toxic chemicals would be toxic to your cells, but otherwise sodium azide might be a good choice.
I'm not a microbiologist, but what about a highly concentrated solution (NaCl) killing the MOs by osmotic shock or vice versa dest. water to let them burst?
Unfortunately, bacteria are quite resistant to osmotic stress. I know for sure that mine is able to survive in water, even if I'm not sure about the salt... From what I read, people usually use osmotic choc to sensitise bacteria to increase the efficiency of further treatments.
Thought it depends on type of bacteria, some are some not?
So alternative: enzymes to lyse the cell wall, something like lysozyme, Achromopeptidase or whatever. The remains then open by some shear strain.
This is a very naive question, and as a microbiologist I feel I should know the answer, but unfortunately I don't, so here it is : what would be the best way to kill bacteria without denaturing proteins? I'm trying to figure out if the bacteria that I'm studying need to be alive to cause damages to epithelial cells, so I want to try with dead bacteria. But if a proteinous factor is required for this process, I'm afraid that heat-killing bacteria won't really answer the question, because I will obtain completely denatured proteins as well as dead bacteria...
Thank you so much for your help!
use appropriate prokaryotic antibiotic!
Why not fix the bacteria with PFA? or kill them with azide, pellet and resuspend in cell media or just use good old fashion antibiotics?
Many thanks for all your suggestions!
I tried azide and lysozyme treatments, and neither worked : I used a final concentration of 0.5M for the azide treatment, but I could not see any decrease in the cfu numbers... Same with lysozyme!
Unfortunately, "my" bacterium seems to be a bit of a pain to work with : it is known as resistant to all sorts of antibiotics, but I will give that a try, as well as the formalin fixation.
But if anyone as any other idea, I'd be glad to hear it!
I tried azide and lysozyme treatments, and neither worked : I used a final concentration of 0.5M for the azide treatment, but I could not see any decrease in the cfu numbers... Same with lysozyme!
Unfortunately, "my" bacterium seems to be a bit of a pain to work with : it is known as resistant to all sorts of antibiotics, but I will give that a try, as well as the formalin fixation.
But if anyone as any other idea, I'd be glad to hear it!
Hello,
It is not surprising azide is a cytochrome oxidase inhibitor it guarantee killing of all your eukaryoutic cells but not prokaryotic. many prokaryotic cells are anaerobic and others can switch from aerobic respiration to anaerobic respiration.
using formaldehyde will guarantee killing any living organism including bacteria, including viruses but won't preserve native form of proteins. formaldehyde covalently attaches to proteins and guarantee their denaturation.
If your bacteria is resistance to antibiotics you can isolate some of them that are sensitive to antibiotics by microbiological subculture techniques.
cheers,
DG
what about detergents such as NP-40 or Triton X-100 which are used to lyse eukaryotic cells in immunoprecipitations?