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Generating a Dry Cell Weight Curve

Objective

Author(s): Pattarin Blanchard

This protocol will produce a curve that quantifies cell biomass at a given OD.

Materials

Procedure

OD measurements

  1. For ease of working and to avoid contamination, aliquot just over 5 mL of liquid culture into a 15 mL falcon tube.

  2. Under a flame, make the following dilutions of liquid culture in PBS in appropriately labelled microcentrifuge tubes. Use the aliquoted liquid culture.

    Tube12345678910
    Liquid culture (µL)05010020030050060075010001500
    PBS (µL)1500145014001300120010009007505000
  3. Blank the spectrophotometer with Milli-Q water at the appropriate wavelength for the organism.

    1. E. coli, CB2A: 600 nm
    2. UTEX: 750 nm
  4. Measure the OD of each tube starting from tube 1, wiping the cuvette with a kimwipe before each measurement. After each measurement, discard the contents in a waste beaker, rinse 3 times with water, then leave face-down on a paper towel soaking with ethanol or disinfectant if not reusing right away.

Dry cell weight

  1. Aliquot 25 mL of liquid culture into a 50 mL centrifuge tube and centrifuge at 10000G for 5 mins.

  2. In the meantime, weigh the drying dish in the analytical balance.

  3. Remove supernatant and resuspend with 10 mL PBS and centrifuge again at 10000G for 5 mins.

  4. Repeat step 3 once.

  5. Remove supernatant and resuspend with 3-5 mL water, try to recover all of the pellet. Pour into the drying dish.

  6. Place the drying dish in an oven and come back the next day to weigh the dish.

  7. Calculate the dry cell “weight” (per mL) from the obtained measurements:

    ρ=mdrymempty25 mL\rho = \frac{m_{dry}-m_{empty}}{25 \text{ mL}}

Dry cell weight curve

  1. Use the value from the previous step to calculate the dry cell weight at each dilution:

    DCW=ρVculture1.5 mLDCW=\frac{\rho \cdot V_{culture}}{1.5 \text{ mL}}
  2. Calculate true OD by subtracting each value with the OD of PBS only (tube 1).

  3. Plot true OD vs. DCW, it should be approximately linear between OD 0.1-1.

  4. Perform a linear regression on the data points in the linear region. The reciprocal of the slope is the desired quantity and has the units g/mLAU\text{g/mL}\cdot \text{AU}.