Anthony checked the e.coli last night before he went home (after I went to class) and there wasn’t anything going on. He decided to leave the e.coli in the incubator shaker overnight at 150 RPM. This morning when I got into the lab, we checked the test tubes.

The P. Blue is cloudy which would indicate growth, yay!

P Blue e.coli... cloudy indicates that there was growth

The pALS is clear; probably no growth.

pALS e.coli - clear; probably no growth

Thermo Scientific Nanodrop 2000c SpectrophotometerTo accurately measure the number of cells we used a Thermo Scientific NanoDrop 2000c Spectrophotometer.  I’m new to spectroscopy, so for those of you who are also new:

“A spectrophotometer is a photometer (a device for measuring light intensity) that can measure intensity as a function of the light source wavelength. Important features of spectrophotometers are spectral bandwidth and linear range of absorption or reflectance measurement.” ~ Wikipedia – Spectrophotometry

Our method:

  1. Connect the nanodrop spectrophotometer to Anthony’s laptop and start the Nanodrop 2000 application.
  2. L to R: Growth Medium Only, GM & pALS, GM & P BluePrepare the three (3) sample cuvettes, with 1000 mL of LB Broth Growth medium.  The first cuvette has only the growth medium. This will serve as our blank reading (similar to the background reading we take for the Ft-IR).
  3. In the second cuvette we added 1000 mL of pALS to the growth medium.
  4. The third cuvette had an additional 1000 mL of P-Blue.

Now on to the scans:

  1. Click Cell Culture from the main screen of the nanodrop application.
  2. Starting with the “Blank” cuvette (the one with growth medium only), insert the cuvette into the cuvette holder of the nanodrop spectrometer.
    • Check (if not already) Add to Report and Use Cuvette. We used a 10 mm Path Length and no Stir Speed.
    • Click the Blank button (at top of screen).
    • Click the Measure button.
  3. Open the lid, remove the blank cuvette and insert the 2nd cuvette, pALS. Close the lid.
    • Enter a name (upper right of screen) for the new cuvette. We entered pALS.
    • Click the Measure button.
  4. Open the lid, remove the pALS cuvette and insert the 3rd cuvette, P Blue. Close the lid.
    • Enter a name for the new cuvette. We entered P_Blue.
    • Click the Measure button.
  5. Remove the last cuvette and dispose of all the cuvettes properly.

The nanodrop spectrometer software displayed a graph as we measured each sample. The A600 number for each sample was 0.001, -0.01, and 0.714 respectively.

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