Exercise 12.4 - Prokaryote Cell Number by Dilution Plating

LEVEL II

Figure 12.5 Colony counter

Materials

• 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)

Procedure

1. Obtain a broth culture of E. coli and carefully mix the contents to ensure equal suspension of the bacteria.

2. Obtain four test tubes each containing 9.9 ml. of nutrient broth. These will be used to produce a serial dilution of the stock culture.

3. 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.

4. 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.

5. Repeat this procedure twice more to produce respectively a 10 and a 10 dilution. Be sure to mix thoroughly and label each tube.

6. 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 produced.

   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.

7. 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.

8. Multiply the number of colonies counted by the dilution factor to obtain the population density of the original broth culture.

Notes

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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