HandsOn 18 - Bacterial Growth

I. Bacterial Growth Preparation

II. Streaking and Inoculating Plates

III. Growth of Bacteria Under Starvation Conditions

IV. Staining Bacterial Colonies

III. Growth of Bacteria Under Starvation Conditions

As mentioned above, B. Subtilis, E. Coli, and E. Aerogenes are all commonly-occurring bacteria. In fact, we are surrounded by so many different bacteria in our environment that to grow a single strain requires careful attention to avoid contamination (infecting the bacteria you are growing with another strain). Always take standard laboratory precautions and wear gloves when handling the bacteria and agar plates.

1. Follow the instructions in HandsOn 5.1 to make your agar plates.


2. Follow the instructions in HandsOn 5.1 to inoculate and incubate your agar plates.


3. In addition to the single point inoculation experiments described in HandsOn 5.1, do an experiment in which two colonies compete on a single plate. On each of two plates, inoculate at two points, separated by 0.5 centimeter on one plate and by 1 centimeter on the other plate.


4. Measure the diameter of each bacterial colony daily and record it on a chart in your lab book.

Data Analysis

1. Record a digital image of the colony with a digital camera or scanner. It may look something like Figure .
Figure 5.8: Sample E. Coli bacterial colonies grown under starvation conditions.
2. Using the Fractal Dimension program, find the dimension of the colony. How does the dimension vary as nutrient concentration varies in the plates? Measure both the global dimension of the aggregate (the dimension of the entire colony including the interior), and the surface fractal dimension (the fractal dimension of the edge of the colony).


How does the global dimension compare with that of a diffusion-limited aggregate (DLA)?


How does the surface dimension compare with that expected from a random walk? Compare the dimension obtained with that found in HandsOn 5.1. If the dimension differs from that of a random walker model, does it behave as you would expect with bacteria that are socially cooperative? Write a short paragraph explaining why, or why not.


3. Plot the radius of the aggregate versus time and find the growth velocity. Is this also a function of nutrient level?


4. Is the growth of the colony limited by diffusion of nutrients? Suppose that a nutrient molecule moves an average distance r in a time t according to the diffusion law: r² = 4Dt where D, called the diffusion constant, has the approximate value 10^-6 cm²/sec. On average how far can a nutrient molecule diffuse in one hour? One day? How does that compare to the initial and final growth velocities you observed for the bacterial colony?


5. How do you interpret your results for the plate you inoculated at two points 0.5 centimeter apart? Is it consistent with a model in which access to nutrients is controlled by diffusion? What about the growth pattern on the plate inoculated at two points 1 centimeter apart? Can you simulate this experiment using the Aggregation program?