Exercise 12.5 - Cell Mass by Measurement of Turbidity

LEVEL II

Materials

• Trypticase soy broth culture of E. coli
  
• Tubes of trypticase soy broth (9.9 ml each)
  
• Tubes of trypticase soy broth (5.0 ml each)
  
• Nutrient agar plates
  
• Sterile transfer pipettes
  
• Spectrophotometer and cuvettes 4

Procedure

1. Obtain a trypticase soy broth culture of E. coli. Trypticase broth is better than nutrient broth, since it is inherently less light absorbent.

2. Prepare trypticase soy broth dilutions of 10 and 10 of an appropriate bacteria culture, as described in Exercise 12.4.

3. Now, for each of the two dilutions, set up a second series of dilutions, this time diluting by 1/2 each time. That is, transfer 5.0 ml of the 10 culture to 5.0 ml of fresh broth and mix thoroughly. Use 5.0 ml of this and transfer to 5.0 ml of fresh broth to produce a 1/4 dilution. Repeat for 1/8, 1/16, and 1/32 dilutions. Repeat the entire 1/2 dilution series for the 10 dilution.

   You should now have twelve tubes, six for each of the 10 and the 10 dilutions. These are then diluted 1/2, 1/4, 1/8, 1/16, and 1/32.

4. Use 1.0 ml of each of the twelve dilutions and plate on nutrient agar plates to perform a colony count as in the preceeding section.

5. Set a spectrophotometer for 686 nm wavelength and be sure it is turned on and functioning properly. Adjust the dark current to 0% T.

6. Place a cuvette containing trypticase soy broth into the spectrophotometer and adjust the reading to 100% T. This is the blank for all subsequent measurements.

7. Transfer the remaining contents of the 12 dilutions to spectrophotometer cuvettes and measure the %T for each. Compute the absorbance for each sample. 5

8. Count the number of colonies formed for each sample after 24 hours. of incubation.

9. Plot the absorbance of each sample against the plate count for that sample.

   Once this has been accomplished, you will have a value for computing the cell population number directly by measuring the absorbance of a suspension. This can be more readily measured on a continuous basis. To do this, grow suspensions directly in cuvette tubes and measure the A at timed intervals. Record a growth curve. By connecting a recorder to a spectrophotometer and keeping the chamber at 37° C, a continuous growth curve can be automatically recorded (assuming care is taken to ensure proper suspension of the bacteria throughout the time period involved). Lacking such sophisticated equipment, the tube can be removed from an incubator at intervals, gently suspended, measured for absorbance and returned immediately to the incubator. Plot cell growth against time to produce a growth curve.

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