UKC

If I seeded (if that's the right word) a surface with bacteria, effectively killed them all with biocide, then swabbed the surface and measured the amount of ATP present on the swab, would I expect to find any?

Effectively, it's a question about the presence over time of ATP in cells, where the cells (in this case bacteria) are not viable. I'm sure immediately after the cells die, there would be some ATP present, but does it remain for any appreciable time, or does it denature/change?

Thanks in advance.

In reply to Submit to Gravity: In meat it just breaks down via hydrolysis I think.. presume the same in bacteria. Depends on the biocide though, presumably it could fix the tissues. Not sure what happens to ATP when fixed though.

In reply to IainRUK: Thanks Iain. "Fixed" as in kept in its original form, unchanged/preserved? I'm aware of the term but not entirely sure what it means.

In reply to Submit to Gravity: well I think alcohol would precipitate it.. but an aldehyde should preserve the shape/bonds.. I think I'm not certain, but you'd use it it histology.

In reply to Submit to Gravity:

I don't think so, and I'm not entirely sure you could fix and view the structure of ATP using aldehyde fixation either. The latter takes quite a while and I would expect ATP to be pretty unstable, being hydrolysed in the usual ways.

I'm not a biochemist by any means but as far as I know, ATP presence is usually detected by colour changes in solution. So you would 'lyse', (break open), the bacteria on the given surface, using whatever lytic compound you have, then mount it all in some kind of buffer and add some kind of reactive dye in and measure the colour change intensity.

I think if you wanted to see the structure of ATP, you'd need to develop some kind of elaborate protein engineering protocol and use cryo-SEM or some similar technique.

In reply to Submit to Gravity:
Give us a clue what you are trying to achieve and it may help.
It depends on how you kill your bacteria. You can get bacterial strains that auto-lyse if chilled suddenly. Detecting the ATP if there could be done with a enzyme linked reaction requiring ATP. You add the enzyme and other cofactors and substrate then you could use a scanning confocal microscope for a detailed view or a plate scanner to get a general idea.

In reply to Submit to Gravity:
Quite a lot of commercial test kits for bacteria use ATP detection. I've never really trusted them and would think they were highly unreliable if you've used a biocide. I'd expect ATP to hydrolyse pretty quickly.

In reply to Submit to Gravity: I'm talking about understanding the results of ATP swabbing to test the efficiency of cleaning / disinfection, using a kit that measures the amount of light given off from the reaction of the ATP (which can be bacterial or other protein matter) and an enzyme that you mix with the swab.

In coarse terms, the less ATP present the better the cleaning, but I do wonder whether it's possible the disinfect a surface and leave it micro-biologically safe, but with ATP present in dead bacterial cells that would be picked up with swabbing.

In reply to Submit to Gravity: Thanks for the replies by the way.

In reply to Submit to Gravity: What surface? I'd have thought most industrially safe biocides, diluted bleach etc, would kill slow enough for ATP to be used up.

Even boiling takes time to kill bacteria (some sp).. they actually aren't that easy to kill.

In reply to IainRUK: A stainless steel worksurface, or a chopping board for example.

In reply to Submit to Gravity:
Seeded is the right word..stop me if i'm trying to tell you how to suck eggs, but: ATP will be in all cells, not just bacterial ones. The ATP/luminescence kits do have some tricks to reduce this.

working on a cleaned, sterilsed surface most of the result will be bacterial ATP, but you would need to have accounted for/removed all traces of the growth media. or you could accept inaccuracy. Centrifuging, washing and resuspending cells will stress them, of course. (I can't think of any bacteria that i'd be deliberately growing in the presence of live cells, though. there would be a few nasty ones that need them, though)

Soy lysate/tryptone soy agar is used for damaged bacterial recovery and has some neutralising effect on disinfectants. And there's some purple liquid that i forget the name of that you can use with swabs. Try searching for biocide testing protocols or maybe even 'Rideal walker reagents' as a start.

difference between cleaned, sterilised, disinfected and viable but not culturable (vbnc).

In reply to Submit to Gravity:
i might be talking rubbish about TSA..

Cultural methods: another thing to consider is that your seed mixture will contain clumps of bacteria. some of these will break up, some won't. A clump of 100 cells growing as a blob will appear almost the same as a single cell that has grown.

If you're not doing a cultural comparison, ignore my ramblings...it's been a while...

In reply to Submit to Gravity:
Compare your results with a live/dead stain if you have access to a fluorescence microscope.