For most, probably not an issue, but I have teh rage against Ampicillin for being such an awful antibiotic in general. It's only bacteriostatic for about 48h at 30/37 C and beta-lactamase generates large zones of clearance within ~2 hrs, so contaminating bacteria can easily screw up an experiment.
Worse, in liquid culture, contaminants can actually build up serially from culture to culture, so that the proportion of contaminants to desired cells can grow and grow with each generation. With unstable plasmids, this can be a real killer. I've actually lost plasmid stocks thanks to this; hence my nerd rage.
For Kanamycin, I have suffered less trouble, something I attribute to it killing contaminants at the outset of culture, rather than just delaying their growth.
Honestly, I haven't used other antibiotics in years, so I don't know, but as you say; many are stored in Ethanol, which is pretty effective against vegetative or dormant cells, and may be bacteriocidal to boot. Chloramphenicol comes to mind. Incidentally, Chloramphenicol can be autoclaved, and can't be reliably heat-inactivated.
Agreed. I wasn't advocating shortcuts just telling my empirical experience. However, I have never worked or seen in an academic lab someone filter sterilize an antibiotic mixture. Considering the price of filters these days that seems like a big waste of money.
Antibiotics are stored at -20 and kill or slow growth of bacteria. Usually many also in 70 % ethanol. I hardly see a need for filter sterilization but each person needs to find their own level of comfort in how strict they need to be in their experimental protocol.On Jan 5, 2013 11:55 AM, "Cathal Garvey" <cathalgarvey@gmail.com> wrote:--Depending on the Antibiotic, this may be more or less of a good idea. If the antibiotic is bacteriostatic, then it'll only delay the growth of contaminants, so you should autoclave and add filter-sterilised antibiotic; this is the case with Ampicillin.
However, for Kanamycin, you can probably get away with adding directly, as it's bacteriocidal. However, it still makes sense to autoclave the medium prior to adding the antibiotic, or even simply boil it in the microwave briefly, to kill wild resistant bacteria. It's a nice shortcut, but adding directly may still allow wild colonies to grow.
As innocuous as it sounds, that can really mess up an experiment if you take it for a positive result. I've wasted weeks on broths I thought were sterile when pure chance yielded a few colonies on the experimental plates and not the controls.
On 5 January 2013 08:50, Andreas Sturm <masterstorm123@gmail.com> wrote:Never heard you can forgo autoclaving, but it does make sense.
Some hard-core antibiotics kill quite everey bug (be it eucaryotes or prokaryotes). But of course there's the danger of having one contaminant who is naturally resistant.
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