Exponential Growth of E. coli Cells

PC2267

Updated on 25 Sep, 2007

Aim

  • To investigate the exponential growth of E. coli cells
  • To understand the idea of doubling time.

Introduction

Restriction and modification enzymes made it possible to insert foreign DNA into bacterial cells that have the capacity to replicate it. A bacterial cell has a single chromosome, a circular DNA molecule with all genes that it requires to grow and reproduce. Many species of bacteria also may inherit plasmids. A plasmid is a small circle of extra DNA. It carries a few genes, and it gets replicated also. Some plasmid genes confer resistance to antibiotics.

Bacterial growth involves a process of doubling, and then doubling again and again. The time taken for the population to double is calledthe doubling time. A single cell E. coli would, under ideal circumstances, divide every thirty minutes. Before each division, each cell’s replication enzymes duplicate the bacterial chromosome and plasmids, so a cell can end of with many identical copies of foreign DNA.

A modified plasmid that can accept foreign DNA is called a cloning vector. It serves as a taxi for delivering foreign DNA into a bacterium, yeast, or some other cell that starts a “cloning factory” –a population of rapidly dividing descendants, all with identical copies of the foreign DNA. This experiment is to study the exponential growth of E. coli cells after the GFP gene for green fluorescent protein is transformed into the E.coli cells.

A colony is defined in bacteriology as a visible (to the naked eye) mass of cells which have arisen from a common source (usually a single cell).

Experiment procedure

Streaking

  1. Remove a single colony from the given plate with the sterile toothpick.
  1. Immediately streak the inoculating loop very gently a quarter of the plate using a zigzag motion. (See area 1 in Figure below.)

3. Take another toothpick .Going back to the edge of area 1, extend the streaks

into second quarter of the plate.(area 2)

  1. Take another toothpick. Go back to the area just streaked (area 2), extend the streaks

into the second quarter of the plate (area 3).

5. Take new toothpick. Go back to the area that was just streaked (area 3)

extend the streaks into the centre fourth of the plate (area 4.)

Imaging, counting and measurement of the cell size

SlidePreparation

1. Take4 colonies of pGFP transformed E. coli cells and suspend in 200 µl PBS.

2. Vortex for 20 seconds.

3. Take5 µl of the cell suspension and put on glass slide.

4. Put a glass cover slip over the slide (avoid air bubble formation).

Bright Field Microscope

1. Turn on power supply (both).

2. Place glass slide on the slide holder. Cover slip should face down.

3. Change object lens to 100 X, put a drop of oil on the lens.

4. Turn on power source for bright field.

5. Change the view to eye piece.

6. Adjust brightness and focus:

a) Innerlarge knob for coarse tuning

b) Outer small knob for fine tuning.

7. Adjust sample position by adjusting top and bottomknob.

Fluorescent Microscope:

  1. Turn off bright field power switch.
  2. Change filter to WB (No.3).
  3. Open the shutter.
  4. Change view to camera.

Software for taking image

  1. Click on DP controller.
  2. Click acquire button or press F7.
  3. Focus on sample.
  4. Add scale bar in scale tab. Click “Show Scale”. Resize scale bar to 10 or 5µm.
  5. Go back to capture tab.
  6. Change the scale objective to 100 X.
  7. Select exposure mode to manual. Change exposure time to 1 second.
  8. Click capture or press F8 to capture sample photo.
  9. Save the image.
  10. Move the sample slide, capture 5 pictures totally.

Calculating the doubling time of E.coli

  1. Calculate the average number of cells N0 in the 5 images.
  2. Measure the dimension of the image and calculate the image area S1 by resizing the scale bar to 100µm.
  3. The area of the cover slip is 22mmx22mm = S2
  4. The total number of cells on the glass slide is
  5. The doubling from one cell is after n-times doubling. ,  is the doubling time, T is the incubation time which will be given by the demo.
  6. Calculate the doubling time . Discuss the possible errors of your result.