Exercise 12.5 - Cell Mass by Measurement of Turbidity
- 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
- Obtain a trypticase soy broth culture of E. coli.
Trypticase broth is better than nutrient broth, since it is
inherently less light absorbent.
- Prepare trypticase soy broth dilutions of 10 and 10 of an appropriate bacteria culture, as
described in Exercise 12.4.
- 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.
- Use 1.0 ml of each of the twelve dilutions and plate on
nutrient agar plates to perform a colony count as in the
- Set a spectrophotometer for 686 nm wavelength and be sure it
is turned on and functioning properly. Adjust the dark current to
- 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.
- Transfer the remaining contents of the 12 dilutions to
spectrophotometer cuvettes and measure the %T for each. Compute
the absorbance for each sample. 5
- Count the number of colonies formed for each sample after 24
hours. of incubation.
- 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
to Table of Contents
Cell Biology Laboratory Manual
Dr. William H. Heidcamp, Biology Department, Gustavus Adolphus College,
St. Peter, MN 56082 -- firstname.lastname@example.org