Exercise 8: Bacterial Motility (Wet Mount Slide)

Introduction

Motility is the independent movement of a bacterial cell and is a function of bacterial flagella. Flagella are thin protein filaments capable of rotating in a clockwise or counterclockwise direction. The rotation of the flagella lead to one of two typical movements: tumbles and runs. Tumbling motility is non-directional and resembles spinning like a propeller. Runs are a more linear movement as the bacterium moves in a particular direction. Bacteria combine tumbles and runs to move toward or away from chemical stimuli such as nutrient concentration). This is known as chemotaxis.

The presence of flagella on the cell cannot be determined with standard staining techniques such as the Gram stain. Flagella are simply too thin to bind enough stain to be visible. Special flagella stains may be used to observe the number and arrangements but they are difficult and tedious. If done properly, the technologist can identify if the cell is monotrichous (one flagella), lophotrichous (several at either end), peritrichous (covering the entire surface) or non-motile (no flagella). Due to the fragile nature of the flagella they can be damaged or broken off during preparation leading to an incorrect result of non-motile.

Instead of flagella staining most labs simply prepare a wet mount slide to view the movement of the bacterial cells. This technique uses an unstained suspension of the bacterial culture covered with thin coverslip. Suspensions are made by applying a loopful of broth culture directly to the slide or a small amount of culture to a loopful of water. The hanging drop slide is another option for viewing the specimen in a suspension. The suspension is place on the coverslip for this preparation. Then an inverted depression slide coated with petroleum jelly is used to pick up the cover slip. The suspension then “hangs” from the underside of the coverslip while viewing with the microscope.

Observations should be done as quickly as possible because the specimen will typically dry out within 15 minutes. Unstained cells are very challenging to view with a light microscope. It helps to reduce the light intensity with the iris diaphragm and slightly lower the condenser. Once the specimen is in focus the technologist must distinguish between true motility (tumbles/runs) or Brownian motion. Brownian motion is random movement caused by the movement of water molecules in the suspension. It resembles shaking or vibration and should not be confused with independent movement of the cell.

Course Intended Outcomes

Perform and understand the basis of different types of microbiological staining techniques (e.g., simple stain and gram stain) to characterize and identify bacteria.

Materials Used

Students will work in small groups. Each group will need the following materials for this exercise.

• Bunsen burner
• Inoculation loop
• Glass slides, cleaned
• Glass coverslips
• Test tube rack

Additional materials and cultures will be assigned by your instructor

Procedure

Wet Mount Technique:

1. Refer to Exercise 2 for proper use of instruments and aseptic technique.
2. Add one loopful of water to the center of a slide. Then add a very small amount of the assigned bacterial culture. Mix gently but do NOT create a smear. The suspension should remain in a small and distinct drop.
3. Alternatively: If you are using a broth culture, you can add 1 drop of culture/suspension directly to the center of the slide.
4. Place a coverslip over the suspension. Hold the coverslip at an angle slowly lowering onto the slide to avoid air bubbles.
5. Place on microscope for observation.

Hanging Drop Slide Technique:

1. Place a clean, dry coverslip on a paper towel.
2. Add one loopful of water to the center of the coverslip. Then add a very small amount of the assigned bacterial culture. Mix gently but do NOT create a smear. The suspension should remain in a small and distinct drop.
3. Alternatively: If you are using a broth culture, you can add 1 loopful of culture/suspension directly to the center of the coverslip.
4. Obtain a clean depression slide and place depression-side up next to the coverslip.
5. Using a toothpick place a small dab of petroleum jelly on either side of the depression.
6. Invert the slide and gently press until it holds onto the coverslip.
7. Flip the slide so that it is upright again. Now the bacterial suspension should be “hanging” on the underside of the coverslip.
8. Place on microscope for observation.

Sampling/Inoculation:

1. Your instructor will assign the cultures to be used for staining by your group.
2. Collect the required materials for the lab.
   • Place all tubes in a tube rack.
   • Never hold a culture tube by its cap.
   • Bacterial culture tubes are always to be shared with your table group.
3. Use a wax pencil to label the slides with the initials of the cultures used.

Lab Clean-up

Aseptically remove coverslips and dispose in the SHARPS BOX.

Apply bleach to slides for 1-2 minutes to disinfect prior to cleaning.

Wash slides with detergent, rinse and dry. Return slides to your slide box and store in the bin labeled for your section.

Return depression slides to the front desk.

Return bacteria cultures to the front bench after inoculations are complete.

Thoroughly clean your microscope and return it to the lab prep room.

Disinfect your benchtop and wash your hands before leaving the lab.

Results and Interpretation

Record your results in your lab notebook. Create a table similar to the table below.

Critical Thinking Questions

Describe some of the complications that may occur with the wet mount staining techniques. What effect would they have on your interpretation of motility?

Does Brownian motion increase or decrease the longer that you view the slide? Why?