Motility and Chemotaxis

1. Hardware.

Most motile bacteria possess flagella. Because flagella are made of protein subunits, they are not thick enough to be visible under the light microscope. However, there are a variety of methods for detecting the presence of flagella or the ability to move.

Flagella stain. pp 102-103

Wet Mount pp 74-76, 100. Simply by putting a drop of liquid on a slide and placing a cover slip over it, the evidence of motility can readily be seen.

bubbles good! Because of refractive index differences, the interface between air and water creates a sharp dark line which is easy to focus on. Bacteria should be just on the water side of that line. Under 100x total mag, they are very hard to see, but they are in focus.

Just because you don’t see the bacteria swimming doesn’t prove they can’t, but seeing them swim proves they can. Distinguish between swimming and streaming and Brownian motion.

Soft agar stab tube p 346. By inoculating a medium with a reduced agar concentration. Bacteria that are not motile grow only along the line of inoculation, but because of the reduced agar concentration, motile bacteria spread through the medium. Contrast sharp growth with cloudy, diffuse growth.

Determining the motility of wild bacteria can be valuable in identification, although motility depends somewhat on growth/environmental conditions. I don’t trust motility much for identification of routinely cultured bacteria because of mutations, strain variations, etc.

Different species vary in how "flagellated" they are. Mostly bacteria have a polar flagellum or have peritrichous flagella. With sufficient experience, it is possible to determine which simply by watching the movement of the cells.

in this lab we hope to see the motility of spiral shaped bacteria. There are two distinct types that vary with regard to their flagella. Spirilla are fairly large bacteria that typically have flagella at both ends (in tufts; lophotrichous). Spirochetes are different; much skinnier and have internal flagella in the cell wall which corkscrew the bacteria and allow rapid movement. Spirilla are often found in nature; spirochetes are often parasites and cause diseases such as Lyme disease and syphilis.

Gliding bacteria are those that move only across a solid surface. There appears to be 3 different mechanism by which gliding occurs. Because it has been difficult to visualize any structures that can be proven to be responsible, it has been difficult to figure it out. Gliding bacteria typically exist in groups and on surfaces where there are lots of polymers to degrade.

2. Software

Bacteria move for a reason. They swim away from harmful substances and they swim toward nutrients. Movement allows for dispersal so that bacteria can occupy more nutrient rich areas.

Chemotaxis: movement in response to a chemical such as glucose or amino acids. bacteria rapidly sample their environment; if they are going the right way, they keep going. if not, they randomly reorient and try again. Membrane Proteins with one end in the periplasm bind attractants and repellents and bring about chemical changes which result in continued swimming or tumbling. In one of the soft agar stab tubes for motility, the agar was overlain with an agar plug containing a high concentration of acetic acid which is supposed to be a repellent for the organism. We will see if it has any noticeable effect on the spreading of the cells.

Other types of taxis include aerotaxis, phototaxis, magnetotaxis

Bacteria respond in other ways to their environment. Bacteria are able to detect a solid surface and turn on different genes in response, The enteric bacterium Proteus mirabilis produces a different set of flagella for "swarming" instead of swimming. The bacteria can now move over a damp slid surface. Gliding bacteria require a surface for gliding and must be in contact with it in order to move. Some gliding bacteria perform elasticotaxis, movement in response to the deformation of the surface.

What you are doing:

1. You have five different cultures. You will use two methods to determine whether or not they are motile. Work in pairs. Place a loopful of culture on a slide. Look for air bubbles under 10x. Center the edge of an air bubble, then move to 40x. Be sure your lens is clean. Adjust the iris diaphragm!! You will see bacteria floating. Are they swimming? Check a second culture by placing a loopful next to it on the slide and adding a cover slip and repeat. Evaluate all 5 cultures.

Then look carefully at the soft agar stab tubes and determine which ones are motile. Do the results agree? We will discuss your findings before the end of class.

2. Do a wet mount (in pairs) of the hay infusion. This will have a variety of swimming organisms. a) look at and enjoy all the diversity. Remember that protozoa will be much bigger than the bacteria. View this on 40x and adjust the iris diaphragm as will. Find and watch any swimming spiral shaped bacteria and show your instructor.

3. View the plate with swarming Proteus mirabilis cells. Remember that they form a very thin layer, so hold the plate at different angles to be sure you see them. These bacteria have produced a different type of flagella so that they can spread over a moist surface.

4. Hope for the best:

View a Rhodospirillum centenum plate. Did the bacterial colony move across the agar in response to light?

View the chemotaxis tube; did acetic acid affect motility?

The instructor hopes to have a sample of gliding bacteria for you to view before the end of class. Either individual bacteria attached to glass will be moving slowly or the edge of a concentrated spot of cells will send out flares comprised of groups of cells.