Pipetting, contamination....

peter via methods%40net.bio.net (by peter.ianakiev from gmail.com)
Thu Feb 22 21:57:09 EST 2007


On Feb 22, 9:09 pm, d... from no.email.thankstospam.net (DK) wrote:
> In article <1172192382.552109.275... from l53g2000cwa.googlegroups.com>, "peter" <peter.ianak... from gmail.com> wrote:
>
>
>
> >On Feb 22, 7:41 pm, d... from no.email.thankstospam.net (DK) wrote:
> >> In article <1172181256.088647.198... from a75g2000cwd.googlegroups.com>, "peter"
> > <peter.ianak... from gmail.com> wrote:
>
> >> >> Jeez, I've done this experiment yesterday:
> >> >> 1. Took a 1 ml Pipetman, set it to 1 ml and pipetted overnight E.coli
> >> >> culture up and down 20 times.
> >> >> 2. Immediately after that, changed a tip, took a bottle of sterile
> >> >> Terrific Broth, pipetted 20 times, set at 37C for 20 hours.
>
> >> >> The result: no signs of bacterial growth today ==> no aerosol
> >> >> contamination.
>
> >> >> DK
>
> >> >Seems like a good experiment, I just wonder what volume of the TB
> >> >media you used?
>
> >> The standard square bottle filled with 100 ml. I still have that bottle,
> >> so if you have your doubts, I can look at it very attentively by Monday.
> >> But, you know, E.coli can grow anaerobically so shaking is not
> >> strictly necessary...
>
> >> I pretty much knew the outcome based on the fact that I am still
> >> using the same ~1.5 years old bottle of SOC used for an umpteen
> >> (more like ~ 100 :-)) electroporations, each involving the same
> >> 1 ml Pipetman and no filter tips all along. Amazingly, still no visible
> >> growth in the bottle and, as recently as two days ago, 20 out of 20
> >> clones analyzed contained the expected plasmid.
>
> >> DK
>
> >> P.S. This topic reminded me of one curious observation:
>
> >> Everyone I know who does "microbiology" outside of sterile hood
> >> is engaged in the practice of flaming necks of every bottle and
> >> flask they use. When asked why they are doing it, the only answer
> >> ever given is "to keep it sterile". Problem is, for as far as I can tell,
> >> the way it is done, it's a completely useless exercise in at least
> >> 9 out of 10 times.
>
> >DK  ... People make night cultures for a reason before they seed 100
> >ml botles. wonder why? Can you repeat same experiment with like 2 ml
> >TB or SOC?
>
> I can but what's the difference from looking at the bottle 3 days later?
>
> DK

I guess we will find out.... sometimes bacteria will fail to grow if
you seed directly to >100 ml (even if you pick from a colony), that's
why people make small culture overnight.



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