Agar-Plate Diffusion

From DIFFUSION to the NOBEL PRIZE!

INTRODUCTION: When one thinks of winning high acclaim such as the Nobel Prize for making a major discovery or invention, most people erroneously envision very complicated and costly work. In truth, most Nobels were won literally on nickels and dimes - soup cans, paper towels, or even just a few simple notes jotted down on paper. This exercise you are about to begin is just one of those "simple minded" things that nobody previously "saw." But one scientist did, modified it ever so slightly and invented a cheap and extremely powerful tool for investigation in many fields of science and technology. In fact this scientist was _____ (you find out who!). It started as a duo - a wife and her husband who were tinkering around in their kitchen in the mid-1940's with ink and paper towels and paper napkins.

   (Here must be inserted a retrospective comment: intertwined with the notion of simple "diffusion," as done above and in all the procedures below, is the much more complex phenomenon of "partition" or "affinity" chromatography because in all these experimental cases the diffusion takes place in an immobile environment of paper fibers (above) or negatively charged agar matrices (below). Thus instructors must be very knowledgible about the chemistries of both their matrix molecules and their experimental solutes. Countless diffusion exercises on the "web" profess that larger methylene blue [cation] molecules do not diffuse as fast as smaller maroon permanganate [anions] do - purely on the basis of size. Yet the exercise below will show that amylase at 70,000 daltons will diffuse nearly as fast as will bromophenyl blue anions will. The reason is simply that methylene blue cations chromatography slowly because of their high affinity to the agar molecules. Non-ionic [but expensive!] agarose or cheaper starch gels would overcome this - except that amylase couldn't be used in a starch gel of course. / Another commonly used colored agent is potassium permanganate. Alas, although it starts out nicely colored, it is a strong oxidizing agent and soon becomes reduced to immobile solid particles of manganese oxide.)

    Thus doing diffusion in agar plates is really more chromatography than diffusion!

    Now let us modify what they were doing so we can get this working in our hands, and then go on to make big things of it. But first we need to know another fundamental - Brownian Motion.

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?

Now we are ready to begin "diffusion!"

  1. Each group: gather the necessary items together:
    • A clean and dry peti plate. This should be filled nearly to the brim with the following solution:
      1. In a 400 mL beaker put 50 mL of COLD water
      2. Add about 0.1 gm "soluble" starch (not what is purchased in a supermarket)
      3. Add 1 gm plain agar (not nutrient agar)
      4. Swirl to evenly disperse the two powders in the cold water.
      5. Heat to boiling. Frequent swirling is required. This process is very much like making gravy - dissolving starch in a liquid.
      6. Once the liquid becomes clear, pour it into the petri plate.
      7. Immediately rinse out the beaker for easy cleaning before the residual agar solidifies.
      8. Return to the plate and remove any surficial bubbles by either
        • using a pencil point to shepherd the bubbles to the edge, or
        • briefly wafting a flame across the surface
      9. Leave the cover off and allow the agar to gel (about 30 minutes).
      10. Now cover the plate.
    • A drinking straw.
    • A dropper for applying your experimental solutions
    • Experimental solutions (choose one from each list)

      List 1
      cationic      		List 2
      anionic      		List 3
      unknown      		List 4
      enzyme
      methylene
      blue      		bromo-
      phenyl
      blue      		food coloringα-amylase
      from saliva
      or ginger mash
      More dyes need to be searched.

  2. Preparation of the Plate (click this for an alternative disk method)
    1. Turn the plate over and place dots on the plastic indicating where you will locate your four sample "wells". These should be about 1 cm from the edge as shown to the right. In the small space near the edge write the samples' names. (Write nothing in the middle of the plate because that will become your viewing area. Write your [group's] name on the lid of the plate.)
    2. Flip the plate so that the gel side is up. Using your drinking straw, "drill" or bore down no more than 2/3 to the bottom at four locations as shown below. Pry out the agar plugs and blow them into the trash.
    3. Without missing the well, use the dropper to half fill each well with sample. Be sure to rinse the dropper between samples so as to prevent cross-contamination. Trick to prevent accidents: when loading a well, let the dropper's tip approach the well from the side of the plate. In this way, any spurious drips are most likely to fall on the tabletop. Directions for saliva and for ginger extract.
    4. Once all four wells are loaded, put the lid on the plate and place the plate in a refrigerator for at least 24 hrs (a week is also good).

    5. You will visually be able to see the degree to which each of the dyes have been able to diffuse. Correlate their distances with both molecular weight and charge.
    6. Finally, place about 1 mL of tincture of iodine (aqueous solution of KI3) on the surface of the plate and swab it completely over the surface of the agar. Within a minute you should be able to see where the amylase has "grazed" its way through the starch that was imbedded in the agar.
    7. Now make final comparisons of molecular weight and charge.
    8. There should be few relationships with regard to molecular weight, but a vast difference between slow-moving cationic dyes and faster-moving anionic or neutral substances.

     

        Saliva Preparation. Using the inside of the lid of the plate, place a glob of saliva near the edge and then using a rinsed dropper pick up enough to half-fill a well. Again: do NOT miss the well. Mopping up misses merely spreads unwanted sample around. Wipe out the top. Let the saliva donor do all this. Later on after the diffusion has taken place, any pathogens will be inactivated upon application of tincture of iodine so as to visualize the effects the saliva has had on the starch. Return

        Ginger Preparation. Smash and crush and grind about a gram of ginger root in 2 mL of water. Squueze out the liquid, which you will draw up into your clean dropper for transfer to a well. Again: do NOT miss the well. Return

        Tincture of Iodine Preparation. This is in lieu of purchasing some tincture of iodine from a supermarket or pharmacy: place about 2 mL of water in a flask. Add about 2 gm of KI crystals; swirl to dissolve. You MUST have a highly concentrated solution of very soluble KI. Next add about 1 gm of black iodine crystals; swirl to dissolve. Dilute up to about 25-50 mL with water. This is good for testing for starch, and for use in the Gram Stain procedure. Return

    Disk Method

        Instead of "drilling" wells, consider laying filter paper disks on the surface of the agar. These disks are easily produced using a hole puncher. Using a tweezers, dip the disks into your samples, blot them slightly so they don't drip, and then place them on the surface of the agar in the same pattern as mentioned above for the wells. Be sure to rinse the tweezers between disks of different samples. When about to do the iodine test, remove all disks.

  3. NOBEL PRIZE QUESTIONS AND MODIFICATIONS
    1. Suppose you had two colorless disks in which one contained an agent that would react with and form a precipitate with what was in the second disk. Suppose you had put these two disks about an inch apart. Draw what you think your result would be. (This is the basis of immunodiffusion analysis - a major test of whether your have been properly immunized without subjecting you to the disease to see if you contract it or not. You will do something similar to this later in the course!)
    2. In your plates your water was "immobile": it was stationary and wasn't moving.Suppose you did something to make that water flow very, very slowly in one direction past the multicolored disks. (The paper becomes the stationary or immobile phase.) What would you see? Draw a picture of what you would expect on the basis of what you have seen in the diffusion results.
      • The water can be moved within the paper by letting the water soak across the paper. Usually this is done by letting the water soak UP the paper when the end is dipped in the solvent. Think about this in the context of your first experiment in which you studied some of the properties of water. This is called "paper chromatography", and won the Nobel Prize because it became the first major way to separate many things. Can you think of why some solutes might move faster than others. (Answer!)

        Suppose you chromatographed a mixture of monosaccharides, or amino acids. How would you "visualize" them (stain for them) on the paper? You will be doing this later this semester.

      • The water AND charged solute molecules can be moved by an electric field: if a negative electrode is inserted on one side of the plate, and a positive electrode is inserted on the other side of the plate, the molecules will move. This is called "electrophoresis" and won a Nobel Prize for Dr. Arrhenius. This procedure is even more powerful that paper chromatography in separating things. Why might one solute molecule move faster than another? (Answer!) If you are really good students, you will be doing this later in the semester - electrophoresing genes and our friends the dyes, which can be "visualized" directly.

    3. How might you employ agar or starch or polyacrylamide as the "immobile" phase?