Thread: Question about E.Coli culture rates.

Hi!
I just wonder is there a way to make a (very) rough estimation how many generations E.Coli creates on a agar plate, if its left in a incubator for a day. The temperature in the incubator was 37 Celsius and all I know about the agar is that it ``should be´´ ideal for growing E.Coli (so I quess that the bacterial culture has excess amount of nutrients).


I know that this would be quite easy if I could record the change of mass or e.g. test the chemical activity, but by relying on these bits of data I`m quite clueless.

Count the number of colony forming units produced in a 24h period? Best wishes,Tri

Originally Posted by tridimity

Count the number of colony forming units produced in a 24h period? Best wishes,Tri

Otherwise it would be possible, but I placed the bacteria by stroking the surface of the agar with a glass rod (covered with E.Coli) from multiple directions. So after incubating, there is more like layer of bacteria than circular units.

Well I found some literal values that the optimal temperature is 37 C and if the bacteria has excess amount of nutrients the generation time will be approx 20 minutes. So maybe I will just trust that information.

Yep, typical doubling time is 20 minutes. It depends on how accurate you want or need to be... one possibility is to establish a bacterial culture, rather than plating, and to indirectly determine the bacterial cell count by measuring the turbidity of the culture by spectrophotometry... then generate a growth curve with lag, log, stationary and death phases: http://faculty.sdmiramar.edu/dtrubovitz/micro/microhandouts/labs/growthcurve.pdf

Best wishes,

Tri

Another question relating in experiment of mine.

When I investigated the inhibition caused by antibiotics, I made one control group in which I used distilled water.
So I cut several holes in the agar plate and filled those with distilled water. The expectation was that the distilled water would not inhibit the bacteria growing on the plate, but it seemed to do that.

And well... I don`t have any idea to explain this phenomenon.
So what might make the inhibition? Does the water influence the nutrient levels on the agar or what ?

(I thought to ask this at the same post, so the question is not directly related to the topic mentioned in the post)

Hi Kikki,Distilled water is a good negative control to use but - why did you create several holes in the agar plate? Did you also create several holes in the antibiotic plates? If not, you will have been introducing needless variation between experimental and control plates. The holes may be what caused a lack of bacterial growth on the negative control plates.If it is possible to repeat the experiment, I would suggest doing the same again but - this time add distilled water to the negative control LB broth when making plates (as you would add antibiotic to the LB broth when making the experimental plates). Hopefully this should sort out the problem.Best wishes,Tridimity

Originally Posted by tridimity

Hi Kikki,Distilled water is a good negative control to use but - why did you create several holes in the agar plate? Did you also create several holes in the antibiotic plates? If not, you will have been introducing needless variation between experimental and control plates. The holes may be what caused a lack of bacterial growth on the negative control plates.If it is possible to repeat the experiment, I would suggest doing the same again but - this time add distilled water to the negative control LB broth when making plates (as you would add antibiotic to the LB broth when making the experimental plates). Hopefully this should sort out the problem.Best wishes,Tridimity

The reason why I cut several holes in the agar was that I pipetted the antibiotic solutions in the hole, from where it diffused to the agar (and to the surface also) causing the inhibition.
Bit like in Kirby-Bauer method, but instead of antibiotic disks, I had holes filled with antibiotic solution)

So the difference should be the percentage of water in the solution added (and of course the different concentration of the antibiotic in the other sample sets).
And reason for the 5 holes was to minimize the effect of errors (if I pipette some hole wrong, I would still have 4 other ones).

I tried also self made disks, but they didn`t work so well compared the other method.

I did another sample set with adding the distilled water directly on the agar, but (if I remember correctly ), it did have a negative effect on the growth of the bacteria.
The color of the area was lighter compared the other areas where there was no water added.

It might be partly the hole which causes the inhibition, but then in the other sample set (mentioned above) presence of water should not show any sign of inhibition.

(And by the way, thank you from the answers)

In that case, I don't know why you are seeing inhibition with water :SBest wishes,Tri

pick a colony, then use dillutions, 10*, 100*, 1000* etc. up to 10^15th, so... 15 tubes.. and see which tube shows no growth. Then you have a rough estimate of how many bacteria make up a colony, you can make smaller dillutions.

My guess, e.coli, colonies, after a day.. 25 billion..

Originally Posted by Zwolver

pick a colony, then use dillutions, 10*, 100*, 1000* etc. up to 10^15th, so... 15 tubes.. and see which tube shows no growth. Then you have a rough estimate of how many bacteria make up a colony, you can make smaller dillutions.

My guess, e.coli, colonies, after a day.. 25 billion..

Alright. I think that I will try to find out the estimations about the colonies and generations when I have the possibility to work in my school`s laboratory.

But I still wonder the inhibition effect that distilled water has on the bacterial growth .

(Thank you for the answers. I hope that I am able to illustrate the research well enough that you can get the main idea of the procedure and the problems)

distilled water kills bacteria..

use fysiologic salt..

Zwolver wrote:

distilled water kills bacteria..

Are you sure that distilled water is lethal to E. coli, and if so, please would you provide a reference?

I have looked (briefly) for proof of this in the literature, but to no avail...

I did however find an abstract (1) describing an experiment in which E. coli were present and viable in distilled water:

The effect of high pressure on the log reduction of six strains of Escherichia coli O157:H7... was investigated in tryptic soy broth, sterile distilled water... and apple cider... Generally, bacterial populations in fruit juices showed larger decreases than did populations in tryptic soy broth and distilled water.

Ref.

1. Whitney BM, Williams RC, Eifert J & Marcy J (2007) High-pressure resistance variation of Escherichia coli O157:H7 strains and Salmonella serovars in tryptic soy broth, distilled water, and fruit juice. J Food Prot 70 (9): 2078-83

pure distilled water, used at any time, provides an osmotic pressure in the bacteria, that makes them go pop like little balloons. I'd say that's pretty lethal.

But no, distilled water with salt/sugar to add up to the right osmotic pressure is not lethal..

pure distilled water, used at any time, provides an osmotic pressure in the bacteria, that makes them go pop like little balloons. I'd say that's pretty lethal.

But no, distilled water with salt/sugar to add up to the right osmotic pressure is not lethal..

I'm pretty sure that in the above-cited paper, pure distilled water was used, no spiking with salt or sugar.

Please will you provide a reference for your claim?

CHAPTER 6 MICROBIAL GROWTH

Not as sure now as i thought before. But it is stated it is harmfull to bacteria.

Although bacteria frequently find themselves in surroundings with a lower osmotic pressure than that inside the cell, the cell wall usually prevents excessive water from entering the cell. If osmotic pressure of a solution is extremely low, such as pure distilled water, some microbes might be damaged.

I usually find the concentration of bacteria to become extremely low or zero in practice if i dillute with distilled water. Though it is far more harmfull to eukaryotic cells, and in particular, red blood cells.

Fair enough, thanks