Question

5) As a brand new graduate student, you are required to design an experiment to test several che gene mutants, using capillary tube method described in Section 11.7 a) Provide a step-by-step experimental plarn b) What result would be observed using a bacteria that shows normal chemotaxis? What would you conclude?

Answer 1

5. a. Step – by –step method to detect several che gene mutants by capillary tube method

1. Cell culture of che gene mutants should be placed either on a parafilm strip or in a beaker.

2. Capillary tubes with control (eg.,phosphate buffered saline), capillary tubes with chemo-attractant, and capillary tubes with chemo-repellant should then be suspended in the culture.

3. The tubes should then be incubated for 60 min at room temperature.

4. After incubation, the capillary tubes should be removed from the culture and the contents should be sucked out of the capillary tube.

5. Serial dilutions of the contents should then be made and plated on agar plates, and the plates should be incubated at 37⁰C for 24-48 hours.

6. After incubation, the total number of colonies that are observed on the plate should be counted to calculate the number of colony forming units (CFU).

7. Chemotaxis index (CI) is then calculated using the formula

           CI = (CFU of test/ CFU of control) x 100.

b. If bacteria that show a normal chemotaxis are used, the following results would be observed:

In control: Few numbers of colonies will be observed. Bacteria during incubation with control containing capillary tubes will exhibit random movement with sporadic runs and tumbles. As a result few numbers of bacteria enter into the capillary tube during incubation that forms few numbers of colonies on agar plate.

In chemo-attractant: More numbers of colonies will be observed. Bacteria during incubation with chemoattractant containing capillary tubes will exhibit directed movement towards chemo-attractant with longer runs and less frequent tumbles. As a result more numbers of bacteria enter into the capillary tube during incubation that forms more numbers of colonies on agar plate.

In chemo-repellant: No colonies or little numbers of colonies will be observed. Bacteria during incubation with chemo-repellant containing capillary tubes will exhibit directed movement away from the chemo-repellant with longer runs and less frequent tumbles. As a result little numbers of bacteria enter into the capillary tube during incubation that forms no colonies or little numbers of colonies on agar plate.

From this we can conclude that bacteria move towards chemo-attractants and move away from chemo-repellants by exhibiting longer runs and less frequent tumbles. In controlled environment bacteria exhibit random movement with sporadic runs and tumbles.