1
$\begingroup$

I have bought a hobbyist kit which involves growing E. coli. The steps said to grow the E. coli on an LB agar Petri dish overnight. No incubation devices were included in the kit.

I let the E. coli grow overnight at room temperature (65-75°F), but there's pretty minimal growth, if any. I found this question which seems to imply that it's hard to grow E. coli without having temperatures near 37°C, which is much warmer than I can reasonably make my house.

Is it possible for me to do this experiment without purchasing an incubator of some sort? If so, does the lack of growth indicate that I've made a mistake somewhere?

$\endgroup$
  • 2
    $\begingroup$ You can always put the plates close to the heating source for your house - forced hot air vents, baseboard electric or hot water heaters, radiators, etc. (I'm assuming you're in the Northern hemisphere and you have the heat turned on). It will be several to many degrees warmer there than in the rest of the room. Make sure you place a good thermometer at exactly the distance away from the heat source that you'll be putting the plate(s) so you don't cook them accidentally - steam radiators, for example, get really hot. $\endgroup$ – MattDMo Dec 19 '16 at 0:16
4
$\begingroup$

The higher the temperature the faster E. coli will grow, with a maximum growth rate around 37 Celsius.

If you grow E coli at lower temperature, the ecoli will grow slower.

At 37°C you will see colonies overnight (16hr).

At 30°C you will see colonies in about 1 and a half days.

At 25°C you will see colonies in about 3-4 days.

You can do experiments at room temperature, but you will be waiting for days for what you will normally see after a 37°C overnight incubation.

As for satellite colonies.. yes ampicilin does break down with time and water. You can solve that problem by increasing the amount of Amp you are using from 25ug/ml to 100ug/ml. Or use 75ug/ml Carbenicilin. Or change your selection markers and use a more stable antibiotic like Chloroamphenicol.

$\endgroup$
  • $\begingroup$ Pedantic stuff: Overnight is typically 8-9 hours and the colony growth rate also depends on the concentration of the inoculum. $\endgroup$ – WYSIWYG Dec 19 '16 at 14:29
  • $\begingroup$ @WYSIWYG Your overnight is 8-9 hours? What are your lab hours? Mine is at least 16h. $\endgroup$ – MattDMo Dec 19 '16 at 15:28
  • $\begingroup$ @MattDMo 16h is your lab hours?!! I guess not, unless you are a robot :D I guess you meant "overnight". My lab hours are variable (as less as 3h to max 15h) :P Recently I was doing only computational; so I could fire commands from anywhere. When I was doing experimental then lab hours were on an average ~10h; 10 AM to 8 PM. All my "overnight" inoculations are done at ~9 PM (but later the better) and I harvest the cells in the morning ASAP. For maxipreps I inoculate late and harvest in 9-10h. Overgrown cells give bad preps. $\endgroup$ – WYSIWYG Dec 19 '16 at 16:50
  • $\begingroup$ @WYSIWYG sorry, I meant my "overnight" is about 16-ish hours. I work in industry, so 9AM-5PM is the norm :) It's been a few years since I've done plasmid preps, but I can't recall having any issues with the longer incubation time. YMMV, of course. $\endgroup$ – MattDMo Dec 19 '16 at 19:02
  • $\begingroup$ @MattDMo There are no big issues but the cell lysis doesn't happen properly when the cells are overgrown. The lysate is very sticky and viscous. The yield goes down. $\endgroup$ – WYSIWYG Dec 19 '16 at 19:06

Your Answer

By clicking “Post Your Answer”, you agree to our terms of service, privacy policy and cookie policy

Not the answer you're looking for? Browse other questions tagged or ask your own question.