STUDENT HANDOUT:

• Motility Student Handout

LABORATORY REPORT FORM:

• Motility Laboratory Report Form

INTRODUCTION:

Various microorganisms have the ability to propel themselves.  This characteristic is termed motility.  Motility is not necessary for survival, but it can provide the microbe with advantages.  Motility may allow the microbe to move toward a more favorable environment, as in the search for nutrients.  It may also allow the microbe to flee from an adverse environment, such as a nearby predator or a toxic compound.

BACTERIA MOTILITY:

Bacteria are prokaryotic, unicellular organisms.  Motility in bacteria, if present, is accomplished by means of hair-like appendages called flagella (Figure 1).  These structures are composed of a single protein, flagellin.  Bacteria have four different arrangements of flagella based on the number and location of the flagella (Figure 2).  Bacteria with a single flagellum at one end of the cell are known as monotrichous (mono, meaning one, and trichous, meaning hair) bacteria. Amphitrichous bacteria have two flagella, one at each end of the cell.  Lophotrichous bacteria have a tuft, or group of flagella aggregated at one end of the cell, and peritrichous bacteria have flagella that cover the entire surface of their cell.  The number and location of the flagella can determine the degree of motility and directionality of a bacterium.

To visualize flagella under the light microscope is extremely difficult, if not impossible, unless special staining techniques are used.  These techniques often use hazardous chemicals and are environmentally unfriendly.  However, there are indirect means of demonstrating the presence of flagella that are less harmful.  The experiments in this laboratory exercise demonstrate indirect observation of the presence of flagella and motility.

Some bacteria exhibit “swarming” motility, where the growth of the bacteria appears as waves on the agar.  One species of bacteria appears as a wave on the agar. One of the most active bacteria that exhibits this type of motility is the rod-shaped organism, Proteus mirabilis. It is a gram-negative bacillus, similar to many other Enterobacterales. It is normally harmless but may be considered an opportunistic pathogen and is sometimes associated with urinary tract infections.

When this species of bacteria is grown in broth medium, it forms peritrichous flagella.  However, when the organisms are grown on a solid culture medium (solidified by agar), a change in cellular morphology takes place.  The P. mirabilis cells elongate to many times their original length with little change in width (Figure 3).  After some time, the elongated cells form septations and the cells divide, forming a microcolony.

Figure 3.  Elongation and swarming in Proteus mirabilis. 

These newly formed swimmer cells have more flagella than the original cells, enabling them to move over a solid medium by the process of swarming.  The swarming cannot be accomplished by individual cells.  The process requires a coordinated effort of differentiated cells.  It will occur when a group of the differentiated swarmer cells moves away from the central area of the medium.  The swarming continues until the number of cells in the swarm is reduced as a result of some cells being left behind on the agar’s surface.  At this point, the swarmer cells revert back to swimmer cells and the process is repeated.  Macroscopically (using the naked eye) the growth of P. mirabilis on agar will have a wavelike appearance (Figure 4). 

Figure 4.  (A) A Proteus culture view from above, (B) A cultured Proteus side view. 

Another indirect method for determining if bacteria have flagella is to use a “stab culture” technique (Figure 5). 

Figure 5.  Stab inoculation of an agar tube. 

This technique uses a semisolid agar medium that allows the bacteria to move within the agar rather than just on the surface.  The technique involves a straight-line inoculation of the microbe into the depths (3-4 cm) of a semisolid agar medium which makes up the stab.  After the tube is incubated for 24-48 hours at the bacterium’s optimum temperature for growth, the tube is examined to determine if the organism’s growth has spread away from the original line of inoculation. If the organism is motile, the agar in the inoculated tube close to the stab line will have a turbid or cloudy appearance (Figure 6).

Figure 6. (A) Schematic of non-motile growth following stab inoculation, (B/C) Schematic of motile growth following stab inoculation.  

                Figure (C) from Wikimedia Commons.

PROTIST MOTILITY:

Protists are eukaryotic microbes.  Protists can include both protozoa and algae.  Motility in the protists, if present, is accomplished by means of three different mechanisms.  These mechanisms use either cilia, pseudopods, or flagella (Figure 7). 

Figure 7.  Protist motility using (a) cilia, (b) pseudopods or (c) flagella.  Image from Wikimedia Commons.  

While both bacteria and protists can have flagella, the movement of the flagella varies.  The bacterial flagella “rotate,” while the protist flagella waves back and forth to make a cell move.  Cilia are short, hair-like structures that beat with many others to make a cell move.  Pseudopods are foot-like projections of the cell’s cytoplasm used to feed and move the cell.  

OBJECTIVES:

1. Learn the techniques for tests of bacterial and protist motility.

2. Observe various types of motility in bacteria and protists.

PRECAUTIONS:

1. Handle the bacterial cultures with care. Wash your hands with soap before and after handling cultures, and wash work surfaces with disinfectant.

2. Clean up spills using disposable plastic gloves, paper towels, and disinfectant. Dispose of all cleanup materials in a biohazard bag and autoclave before disposing.  

3. Do not put fingers or any objects near eyes or mouth while working.

4. Wear safety glasses and protective gloves through the entire experiment especially when handling any bacterial cultures or instruments that come in contact with bacterial cultures.

5. Keep all flammable material away from the flame of the Bunsen burner.

EXPERIMENT #1:  Swarming Motility

MATERIALS (Work in pairs):

1. 2 sterile semi-solid nutrient agar stab tubes (containing 0.7% agar)

2. Culture of Serratia marcescens

3. Culture of Micrococcus luteus

4. Inoculating needle/wire.  If a Bunsen burner is not available, use a disposable inoculating needle/wire.

5. Bunsen burner (if available)

6. Permanent marker

7. Incubator set at 37°C

8. Biohazard bag

9. Test tube rack

PROCEDURE:

1. Using a permanent marker, label your Nutrient agar plate with your name, the date, and the name of the bacteria.

2. Sterilize the inoculating loop by heating it to redness in the Bunsen burner.  Then let it cool for 15-30 secs, without blowing on it or waving it around. If a Bunsen burner is not available, use a plastic sterile disposable inoculation loop.

3. Aseptically pick up some of the P. mirabilis with the inoculation loop.

4. Open the agar plate like a clamshell, inoculate the center area with the bacteria.

5. Using two small pieces of tape, tape the plate closed and incubate upside-down at 37°C for 24-48 hours. 

6. If waves of growth are not seen after incubation, incubate for another 24 to 48 hours.

OBSERVATIONS/CONCLUSIONS:

1. On your Microbiology Laboratory Report Form, draw a picture of the growth of P. mirabilis on the agar plate.  Record your conclusions on the motility of P. mirabilis. 

QUESTIONS:

1. Is the swarming of the bacteria directional or random?

2. How does this swarming help to identify the bacteria?

EXPERIMENT #2:  Stab Technique

MATERIALS (Work in pairs):

1. 2 sterile semi-solid nutrient agar stab tubes (containing 0.7% agar)

2. Culture of Serratia marcescens

3. Culture of Micrococcus luteus

4. Inoculating needle/wire.  If a Bunsen burner is not available, use a disposable inoculating needle/wire.

5. Bunsen burner (if available)

6. Permanent marker

7. Incubator set at 37°C

8. Biohazard bag

9. Test tube rack

PROCEDURE:

1. Using a permanent marker, label each stab tube with your name, the date, and the name of one of the two test bacteria. Be sure to label the test tube itself, not the lid.

2. Flame-sterilize the inoculating needle by heating it to redness in the Bunsen burner.  Allow to cool for 15-30 seconds without blowing on it or waving it.  If a Bunsen burner is not available, use a disposable inoculating needle.

3. Using the needle, aseptically transfer S. marcescens to the corresponding uninoculated semisolid stab tube by passing the needle straight down into the agar and pulling it straight back out. (See Figure 5).

Figure 5.  Stab inoculation of an agar tube. 

4.  Flame-sterilize the needle by heating it to redness again, then allow it to cool for 15-30 secs without blowing on it or waving it, or acquire a new sterile inoculating needle.

5.  Repeat the procedure using M. luteus and the second stab tube.

6.  Place the tubes upright in a test tube rack.

7.  One half of the class should incubate their tubes for 24-48 hours at room temperature while the other half should incubate their tubes for 24-48 hours at 37°C.

8.  Your instructor will include semi-solid stabs that have been inoculated with a sterile needle to serve as a control.

OBSERVATIONS/CONCLUSIONS:

1. On your Microbiology Laboratory Report Form, complete Table 1.  Sketch the appearance of the cultures and the culture after inoculation.  Draw and label the line of inoculation and write the names of the test bacteria and the incubation temperature. Record your conclusions on the motility of both bacterial species. 

QUESTIONS:

1. Which of your cultures shows evidence of motility?

2. Compare your results with those from another student/group who incubated the cultures at a different temperature.  Is there any difference in the motility of the bacteria based on different temperatures?  Why?

3. What do you conclude about the relationship between motility and temperature?

EXPERIMENT #3:  Protist Motility

MATERIALS (Work in pairs):

1. Cultures of Amoeba, Euglena and Paramecium

2. 3% methylcellulose solution (or Protoslo)

3. Clean microscope slides and coverslips.

4. Compound light microscope.

5. Disposable pipets

PROCEDURE:

1. Using a disposable pipet, add a drop of the Amoeba culture to the center of a clean microscope slide.

2. Gently put a coverslip on the drop to prepare a wet mount. 

3. Put the slide onto the stage of the microscope.  Beginning at scanning power (4x objective, 40x magnification), focus on the slide.  When the slide is in focus, change the objective to low power (10x objective, 100x magnification).  When the slide is in focus, change the objective to high power (40x objective, 400x magnification). 

4. Look for Amoeba and take note of how the Amoeba moves using pseudopod motion.

5. Repeat steps 1 - 4 for Euglena and Paramecium.  

6. Since Euglena and Paramecium move quickly, prepare an additional two slides by first adding a drop of Protoslo to the slide before adding the culture.  The ProtosloⓇ will slow down the cells so you can see them move. Use a disposable pipet to gently mix the ProtosloⓇ into the culture.  

OBSERVATIONS/CONCLUSIONS:

1. On your Microbiology Laboratory Report Form, complete Table 2. Name the method of motility for each protist. 

2. Record your observations on the motility of the three different protists.

QUESTIONS:

1. Based on your observations:

(a)  Did each of the protists you observed move randomly or in a particular direction?  

(b)  Why do you think they do this?

KEY TERMS:

• algae

• amphitrichous

• bacillus

• cilia

• Enterobacterales

• eukaryotic

• flagella

• gram-negative 

• lophotrichous 

• microbe / microorganism

• monotrichous 

• motility 

• opportunistic pathogen

• optimum temperature

• peritrichous

• prokaryotic 

• protozoa

• pseudopod

• semi-solid agar 

• septation

• stab

• swarming 

• swimmer 

• turbid

SUGGESTED READINGS:

In this lab, you observed different types of protists by looking at how they move.  Scientists learn more and more about protists each day.  Click HERE to learn more about the protists.