Some bacteria are motile (able to move). Most motile bacteria are spirochetes or bacillus (rod-shaped) bacteria. These often have specialized organelles called flagella (or a flagellum, if singular) that is used to move away from harmful substances and to move towards nutrients. 

PART I  WET MOUNT

1. ​Prepare wet mount slides of both S. aureus and P. vulgaris using 5 loopfuls of each organism in the center of a slide
2. Cover the organism with a cover slip.
3. View under 400X magnification immediately, otherwise the culture will dry and you will not see anything move. Do not go to oil, you may crack the coverslip.

WET MOUNT PREPARATION

Organisms for Wet-Mount

​• Proteus vulgaris 
​Gram-negative, bacillus (rod-shaped) bacteria that reduces nitrate and produces hydrogen sulfide. It is found in the G.I. tract, soil, and fecal matter. Can cause wound infections and UTIs. 

​​• ​Staphylococcus aureus
Gram-positive, cocci (spherical) bacteria that reduces nitrogen and is considered the normal flora of the body. It is found in the nose, respiratory tract, and skin. 

​• Bacillus megaterium
Gram-positive, bacillus (rod-shaped), spore- forming bacterium. B. megaterium is amongst the largest bacteria we know of

MOTILITY STAB TEST
    For this test, a softer version of agar, called motility agar, will be used. Motility agar has a colorless dye (TTC or 2,3,5-Triphenyltetrazolium Chloride) that some bacteria have the ability to reduce. TTC becomes red when it is reduced by these bacteria. You may want to review your oxidation-reduction reactions (a.k.a. red-ox reactions from general chemistry). Bacteria that have the ability to reduce TTC, will reduce whatever TTC they may come into contact with. In this way, we will be able to visualize if the bacteria is motile or not, and if it is able to reduce TTC. 

PROCEDURE FOR MOTILITY STAB TEST

You will prepare 2 separate samples (1 using S. aureus and 1 using P. vulgaris) in exactly the same way, using sterile technique.

1. Flame inoculation needle, uncap agar tube of appropriate bacteria and stab 2-3 times in same spot.
2. Flame the mouth of the tube and recap tube., then remove cap from the motility agar and stab the motility agar ONE TIME ONLY. Flame mouth of motility agar tube and replace cap. Flame inoculation needle well (until visibly red) to sterilize.
3. Incubate this media at 25°C.

Reading the Results of a Motility Stab Test

	MOTILE	REDUCE TTC	Total ​Result
S. aureus	If there was either a red or a colorless blurry area radiating outward from stab-point = YES If not = NO	if red = YES ​if colorless = NO	??
P. vulgaris	If there was either a red or a colorless blurry area radiating outward from stab-point = YES If not = NO	​if red = YES ​if colorless = NO	??

• If the bacteria is motile , and is able to reduce TTC, you can visualize the motility by noticing a red area radiating outward from a red stab-point. 
  
• If the bacteria is NOT motile , and is able to reduce TTC, you can visualize the motility by noticing a red area radiating outward from a red stab-point. 
  
• If the bacteria is motile , but is NOT able to reduce TTC, you can still visualize the motility by noticing a "blurry" or "fuzzy" area radiating outward from a colorless stab-point.
  
• In contrast, if the bacteria is NOT motile and is NOT able to reduce TTC, then the motility agar will appear exactly as it did immediately after the stab (inoculation); as a colorless stab-point with no visible distortions radiating outward from it.  ​

HANGING DROP PREPARATION

MATERIALS

The organism you will observe for this motility test is B. megaterium.

You will be using a depression slide (also called a welled slide), a coverslip, a toothpick and some vaseline, for this test. 

coverslip

vaseline

depression slide (welled-slide)

toothpick

Procedure 

1. Using a toothpick, place a small amount of Vaseline on each corner of the cover slip. 
2. ​Add two loop-fulls of bacteria to the center of the coverslip. 
3. Invert the depression slide so that the welled portion is lined up over the coverslip and the bacteria, and gently press.
4. Quickly flip over the slide and view using the 40X objective (400X).

Step One

Step Two

Step Three

Flagella in bacteria cells are very small and thin. It is almost impossible to view these structures using a compound microscope without staining.