IGEM:IMPERIAL/2007/Projects/Cell by date/Implementation/Calibrating Absorbance

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Contents

Measuring Absorbance

Materials Required

Equipment

  1. Spectrophotometer set to wavelength 600nm
  2. Cuvettes

Reagent

  1. Experimental Sample

Protocol

  1. To remove interference that M9 medium has with culture readings, take a reading of the broth alone ('blank'). To do this, Add 4ml of M9 broth alone to cuvette and record down the readings at 600nm.
  2. Next, take absorbance readings at a wavelength of 600nm for each time point, and record the relative turbidity of the broth.
  3. Subtract the 'blank' to measure actual OD600 of culture.
  4. Convert readings to CFU with calibration curve and record in representative table below.

Treatment 1: M9, 4°C

Blank OD =

Time Point Visible Observation OD600 (abs units) Number of Cells(CFU/ml)
1
2
3
4
5


Calibrating Absorbance

Materials Required

Equipment

  1. Spectrophotometer set to wavelength 600nm
  2. Cuvettes
  3. Bunsen flame
  4. Vortex Mixer

Reagent

  1. Experimental Sample
  2. 9.9ml dilution tubes of sterile saline
  3. nutrient agar plates
  4. p1000 and p200 Gilson pipettes
  5. Glass spreader / Disposable plastic spreader
  6. 95% ethyl alcohol in glass beaker (WARNING: Keep alcohol away from flame!!)

Protocol

(b4 this, must culture bacteria o/n b4 placing in new media

  1. To remove interference that M9 medium has with culture readings, take a reading of the broth alone ('blank'). To do this, Add 4ml of M9 broth alone to cuvette and record down the readings at 600nm.
  2. Next, take absorbance readings at a wavelength of 600nm for each time point, and record the relative turbidity of the broth.
  3. Subtract the 'blank' to measure actual OD600 of culture.

Serial dilution is then carried out for each sample at every time point before plating onto the nutrient agar.

For Spread plate technique (dilution plate counting):

  1. Prepare serial dilutions of the broth culture below.
  2. Transfer 0.1ml to each of 9.9ml dilution tubes of sterile saline. Vortex mix.
  3. Repeat this x to obtain dilution factors of 10-2, 10-4 and 10-6. (This is because estimated bacterial count of OD600 0.1-1.0 is around 3x107 - 5x18.
  4. Use dilution factors 10-4 and 10-6 for further steps. Dilutions of such factors would allow the plating of 30-300 colonies. Anything outside this range is considered statistically unreliable.
  5. Position the beaker of alcohol containing the glass spreader away from the flame. Remove the spreader and very carefully pass it over the flame just once. This will ignite the excess alcohol on the spreader and effectively sterilize it.
  6. Spread 0.1 ml of the 10-4 inoculum evenly over the entire surface of one of the nutrient agar plates until the medium no longer appears moist. (Note: Increases dilution factor by another 10-2.
  7. Return the spreader to the alcohol.
  8. Repeat the flaming and spreading on plate for 10-6 inoculum.
  9. Invert the plates and incubate at 37°C for 24 hours.
  10. Repeat the whole procedure for each time point taken.

Results

Record results in the table as follow:

Time Point CFU count Dilution factor Number of Cells(CFU/ml)
1
2
3
4
5

With the results above, a calibration curve can be plotted with relation to the spectrophotometer data. Below is a sample from Endy Lab:

Image:MC4100_OD_vs_CFU_curve.jpg

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