DIFFUSION

| Osmosis | Transpiration | VAST-2001 |

Diffusion is THE all-pervasive way nature moves molecules around. Sometimes, to help out, the solutions are stirred - the winds blow, the oceans have currents, our cells have internal flows. But diffusion is ALWAYS there! That is how molecules collide and react; that is how food molecules approach our cells, and how wastes exit away before the environmental stirrings take over.

1. BROWNIAN MOTION
   1. Take a drop of real "India Ink" (not just any black ink), and dilute it in half with distilled water.

   2. Place a drop of this "solution" onto a slide and cover it with cover slip.

   3. Under a light microscope, you should see small black specks that are jiggling around. THAT is "Brownian Motion." It is the basis of diffusion. Why do the specks bounce around?

   Ramification: Not only have you seen Brownian Motion, but you have also gained skill in the use of a microscope. That skill will come in very handy in future labs.

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2. DIFFUSION
   Moving to the sub-microscopic level of Brownian Motion, you are going to show how molecular size influences the rate at which things can diffuse away in an agar gel from a point of origin on the surface of the agar in a petri plate, which you will make.
   4. To make the agar plate (one plate per group) (Make a starch-agar plate unless the instructor is supplying them.)
      1. Into a 150 or 200 ml flask, weigh 0.5 gram of pure agar. (Thou shalt not confuse the substances "agar" and "agarose"!)
      2. Add 25 ml of 0.05% starch solution, which the instructor has already made.
      3. Add only two granules of NaN₃ (sodium azide; a very strong biocide). Do not lick your fingers unless you want to experience death by strangulation or drowning!
      4. Over a burner or on a hot plate, and using hotpads or mitts, continuously swirl the flask to boiling.
      5. Pour the contents into an empty petri plate (doesn't need to be sterile).
      6. Before the agar solidifies, waft a flame across the surface of the agar to burst any bubbles. (As you see them burst, think about the relationship between temperature and surface tension.)
      7. Allow the agar to solidify. You will call this a "starch-agar plate"

   5. Preparation or your diffusible sample (one sample per person)
      1. In the middle of a piece of aluminum foil, place a drop of red food coloring.
      2. From the instructor, obtain one of the vegetables, which may contain alpha-amylase, and squash it throughly upon a small portion of a filter paper.
      3. Immediately punch out three holes from that section of the filter paper. (Do not let the paper dry after squashing the vegetable.)
      4. Write the vegetable's first initial on the top of one "hole."
      5. Immediately add the "holes" to the drop of red food coloring. mix in a drop of red food coloring
      6. Stack your three "holes" up into a three-layer sandwich such that the vegetable's initial shows on the top one.
      7. Gently blot away excess liquid
   6. Using a tweezers, set your red disk sandwich in a triangle with the corners about 2 cm for the edge of the agar.

   7. Cover the plate, and store for 24 to 48 hours.

3. After this time has elapsed, you should see that the red has diffused out. (Red dye was used because you eyes are most sensitive to seeing that color.)

   

   MEASURE THE DIAMETERS OF THE RED RINGS AROUND YOUR INDIVIDUAL DISKS. (Or, using a marking pen, draw dotted circles on the bottom of the petri plate.) Also write the appropriate initials on the bottom of the plate under where the corresponding disks sit.

4. Next, pour about 10 ml of orange-colored iodine solution onto the surface of the agar. Allow to sit for no more than 2 minutes and then rinse away the iodine solution with running tap water. On the bottom of the plate make solid circles indicating the extents of the haloes have "eaten" into the starch by your samples of ginger root amylase. In the diagram to the right, Veggie "A" has a fast-diffusing amylase activity; "B" has no amylase activity (must have not dipped the disk properly in the beginning); and "C" has a slow-diffusing type of amylase activity. Consider how "fast" and "slow" might occur.

5. Ramifications: What all this gets for you: how to pour petri plates (for later labs); how to use diffusion disks on a gel (antibiotic testing); how the previously used iodine test can be used to test whether organisms are producing amylase, which is in itself an introduction to an upcoming lab unit; and the simple matter of diffusion of both a small molecule (red dye), and a large molecule (alpha-amylase).