Exercise 12.4 - Prokaryote Cell Number by Dilution Plating
Figure 12.5 Colony counter
- Broth culture of E. coli
- Tubes of nutrient broth (9.9 ml each)
- Nutrient agar plates
- Sterile transfer pipettes (1.0 ml)
- Quebec colony counter (Optional)
- Obtain a broth culture of E. coli and carefully mix the
contents to ensure equal suspension of the bacteria.
- Obtain four test tubes each containing 9.9 ml. of nutrient
broth. These will be used to produce a serial dilution of the
- Using sterile pipettes, remove 0.1 ml of well suspended
cells from the stock culture and transfer these aseptically to
one of the waiting test tubes of broth. This tube now contains a
dilution of 1/100 or 10. Gently but
thoroughtly mix the contents and label this tube at 10.
- Repeat this process, but now aseptically remove 0.1 ml of
culture from the 10 tube and place it into a
new broth tube, which now becomes a 1/10,000 or 10 dilution. Mix the contents and label as 10.
- Repeat this procedure twice more to produce respectively a
10 and a 10 dilution. Be
sure to mix thoroughly and label each tube.
- You should now have a serial dilution of the stock culture
with tubes at 10, 10, 10, and 10 . The original stock
culture will almost invariably be too high a population for the
next step, so you will only use the four dilutions that you have
Using four separate petri plates containing 15 ml. of nutrient
agar each and four separate sterile pipettes, transfer 1.0 ml of
each dilution broth suspension onto the surface of a petri plate.
Carefully label the plates and place them in an incubator for 24
hours at 37° C.
- At the conclusion of incubation, remove each of the four petri
plates and count the number of colonies formed on the plates. For
proper statistical analysis, the plate containing between 30 and
300 colonies will give the most accurate results.
The colonies can be more easily counted by using a Quebec Colony
Counter which allows proper illumination, a grid overlay and by
slight magnfication of the plate surface.
- Multiply the number of colonies counted by the dilution
factor to obtain the population density of the original broth
A growth curve can be established by repeating this procedure
every two hours. Since the number of bacteria can be large, it
will be necessary to plate cultures serially diluted. Count the
number of colonies for each dilution and average the results.
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Cell Biology Laboratory Manual
Dr. William H. Heidcamp, Biology Department, Gustavus Adolphus College,
St. Peter, MN 56082 -- firstname.lastname@example.org