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Motility

Some bacterial species have flagella which allow the cells to travel from one place to another. These are referred to as motile species. For the purposes of this lab, we will not deal with other modes of bacterial motility. Those species in our lab devoid of flagella are non-motile. In this lab, we will only consider the motility (qualitatively, i.e. motile or non-motile) of Gram negative rods. Examples of clinically significant non-motile Gram negative rods that will be used in our lab include Klebsiella, Shigella and Acinetobacter. Although Alcaligenes is extremely motile, it only grows very near the top of the agar due to it’s requirement for a lot of oxygen. For this reason, it can be hard to detect Alcaligenes motility. Several methods exist for determining motility, and a few are discussed below. We will use the stabbed soft agar deep method in our lab.

Stab a soft agar deep

Motility can be determined by monitoring turbidity (cloudiness) following incubation in a stabbed soft agar deep medium such as TSA with 4 - 5gm agar/liter or SIM media (which we will cover in a subsequent lab). Normal hard agar, such as that used for plates or slants, contains 15gm agar/liter. This elevated agar concentration will not allow the movement of any motile cells trapped within the agar matrix. This is not the case with soft agar. To conduct the procedure, cells on the end of a straight needle are stabbed on a straight line into the center of a soft agar deep. Non-motile cells will remain on the stab line as they divide resulting in a distinct line in the agar with no surrounding turbidity. Since soft agar media is slushy motile cells can travel in it. These cells will not remain at the stab line but will radiate outward resulting in turbidity away from a stab line that is visibly less distinct, or even dissipated to invisibility. See image “Motility: left non-motile, right motile.” Highly motile cells can radiate quickly resulting in equal turbidity throughout the media. Less motile cells will radiate, but greater cell density will remain nearer to the stab line. To inoculate a soft agar deep first check your needle to make sure it is straight. Using aseptic technique, collect cells with your needle. Carefully stab the needle through the center of the deep to the bottom of the tube and withdraw along the same path. Do not mix or shake. Incubate at 37oC for 24 to 48 hours, however you cannot incubate motility deeps too long – see point number 2 below.

Results from the stabbed soft agar deep method can be misinterpreted. Consider the following points.

1. Soft agar is supposed to be sufficiently oxygen permeable to allow the growth of obligately aerobic species throughout the agar deep. This is not always the case, which can cause confusion when working with obligately aerobic motile cells that have an unusually high oxygen requirement. In this case, look for motility toward the top of the tube.

2. Keep in mind that ANY growth diffusing away from the stab line indicates motility. If not much cell division has occurred then any turbidity present will be difficult to distinguish from the pre-existing translucence of the media. This is perhaps the most common reason for misinterpretation of results using this method. For this reason, eventhough manuals often suggest 24-48 hours of incubation, I tell my students that “when in doubt, incubate longer.” You cannot over-incubate stabbed soft agar deeps. Always use an uninoculated soft agar deep as a control when checking your results.

3. Rarely will a needle follow a laser-straight line to the bottom of the agar deep. The needle will usually follow a crooked path, cutting the agar in one plane or more. Consider a non-motile organism growing along this flat cut path. When viewed perpendicular to the cut plane the growth can easily be mistaken as radiating turbidity. Simply rotate the tube 90 degrees to see if the growth is 3 dimensional (which would indicate actual motility), or if the growth is only following the cut path of the needle.

Wet mount / hanging drop method

Another way to determine motility is to examine living cells in a wet mount using the hanging drop method. To do this, place a small drop of water in the center of a cover slip using your loop. Collect a small amount of cells with your loop (as if you were making a smear) and gently mix them into the drop of water trying not to spread the drop out. Put small spots of petroleum jelly on the 4 corners of the cover slip. Place a small Sharpie dot next to the drop of water. Lay a depression slide down on the cover slip such that the drop is enclosed in the depression. Press lightly to secure the slip to the slide. Quickly invert the slide so that the drop is hanging from the bottom of the cover slip. View under oil immersion quickly before the heat inactivates the cells or evaporates the drop of water under the slip. The cells will have little contrast and will not be easy to see. Stop down the light with your condenser diaphragm. If everything works correctly you should see motile cells moving around. At first you may confuse the random jerky motion of Brownian motion (caused by random collision of atoms with the cells) for motility. This method is often used to view motile eukaryotic microbes such as protozoa and Euglena, but can also be used to examine the “twiddle and run” motility of bacteria.

Cells in the genus Proteus have peritrichous flagella meaning flagella all around the cell, thus they are extremely motile. These cells can even swim on top of agar, a characteristic called "swarming”. For this reason, Proteus can prove very difficult to isolate in a mixed culture, thus causing much frustration to microbiology students. Cells in the genus Pseudomonas have lophotrichous flagella, which is a tuft of flagella on one end of the cell. Lophotrichously flagellated cells are theoretically second only to peritrichously flagellated cells in regard to degree of motility. The remaining "enteric" bacterial genera (Gram negative, oxidasae negative rods) have polar monotrichous flagella, which is a single flagellum on one end of the cell. These are generally considered the least motile cells of the list above, however some strains of polar monotrichously flagellated genera are highly motile.

NOTES:

* We use SOFT agar deeps for motility tests, NOT tempered hard agar deeps.

* Inoculate motility deeps using a needle, NOT a loop. Before inoculating, make needle as straight as

possible to limit the diagonal cutting of the agar.

* It is hard to over-incubate a motility deep. If in doubt about your results, incubate another day.

* Motility results are more easily interpreted while the media is warm (directly out of the incubator).

Refrigeration causes the agar to become cloudy. This is a problem.

* We will only utilize motility testing no Gram negative organisms this semester. Motility will only be

useful in distinguishing the non-motile Gram negative rods (Klebsiella, Shigella and Acinetobacter)

from the other Gram negatives.

* It is easy to misinterpret a motility result on Pseudomonas due to its obligately aerobic nature.

Pseudomonas is motile, but will only show the spreading turbidity toward the top of the tube.

* Motility tests should NOT be used to distinguish degrees of motility (quantitatively), but should only

be used to distinguish motile from non-motile organisms (qualitatively).