BEGINNING BEGINNER'S MICROSCOPY

    As always, one learns techniques much faster if there is a goal in mind. So today we shall see if we can answer a question by applying microscopic techniques, which we shall learn as we go along. Although this project certainly does not deal with the "warm and fuzzy" realm, it should introduce us to the relative size that bacteria are relative to familiar eukaryotic cells. This will allow us to adjust our mindset to later looking for and at bacteria.

Varigation and Chlorosis

    In the front of the lab are some plants with various shades of green on their leaves. We want to find out why some parts of the leaves are lighter green and other parts are darker green. Specifically, are the portions darker because those cells have chloroplasts with more chlorophyll (greener) than those in the less green areas, or are the chloroplasts all the same, but there are just more per volume of leaf in the darker portions? (We shall leave chlorosis for a later time. Chlorosis is when a plant has been taken away from sunlight for too long and the leaves get gangly and yellow.)

    How does this help us in microbiology? Chloroplasts are in the realm of prokaryotes and are about the size of large bacteria. You should be keeping your eyes open for the smaller mitochondria also as they are the size of smaller bacteria - in fact, they are in the realm of Archebacteria. So if you can find both mitochondria and chloroplasts in your leaf cells, then you are acquiring the microbiologist's version of size.

What To Do

    You probably cannot merely plunk a leaf down under a microscope and see very much other than a field of green. Your leaf is many cell-layers thick. But try looking at the leaf just in case!

    So you need to make some thin sections of the leaf. There is a really crude way to do that, and a less crude way.

Really Crude Way

    Take a razor blade and try to scrape off a very thin layer from the leaf. Immediately put that into NS so it won't dry out. Have a microscope slide ready with a puddle of NS on it. Once your thin slice is immersed in the NS, cover with a coverslip. What do you see under the scope? First, go to the lowest power and start by looking at an edge. This will help you to focus your microscope. Once that is in focus, swivel the nosepiece to the next higher power. DO NOT USE THE LENSES THAT HAVE RINGS AROUND THEM. (Those require the use of a special oil between them and the coverslips to go to very high power. We're not that advanced yet!)

    Next move the slide slightly so you are looking at the cells just inside the edge - the thinnest edge you think you have made.

A Merely Crude Way

    A Microtome is a device for slicing things in very thin slices and it works almost like a sausage slicer. We will be inventing our own crude microtome - merely a bolt and a nut. As the bolt turns, more of the sample will be exposed for our razor blade to slice away. Perhaps a ¾ turn will be enough, and if not then perhaps a ½ turn will do.

1. Insert the bolt just enough into the nut so that they don't fall apart. There should be a "cup" formed at the other side of the nut.

2. Fill this cup with paraffin wax. Really fill it! Add a little heat to the bolt so that the wax just barely melts. Add more wax to make the cup full.
   

   

3. Take a small segment of leaf - preferrably including an interface between two different shades of green. With a tweezers, or pin or needle, push this bit into the barely molten wax so that the bit is vertical in the wax.

4. Immediately put the head of the bolt into cold water. The wax will "freeze."
   

   

5. Run the razor blade across the face of the nut and shave off a portion of the wax. Advance the bolt a bit so that another shaving can be produced. Do you have any leaf in that shaving? If no, then advance the bolt some more until you do.

6. If you do have some leaf in that shaving, clean off the blade and advance the bolt perhaps ´ turn, and get another shaving.

7. Put that shaving onto a microscope slide. (Trick: slowly slip the blade into some water that is just hot enough to soften the wax. The wax-leaf shaving will float onto the surface of the water. Next, using a needle, tease the bit onto a microscope slide you have slipped into the water under the bit.)

8. To the unaided eye, the bit of leaf in the wax should be a mere speck, but that speck is many cells across, and perhaps only one cell thick - just what you want!

9. Add a cover slip and look at it under low power and higher powers. Compare the pale and the darker cells' chloroplast density and intensity of green-ness.

   

10. Can you find mitochondria. They usually have a lot of marginal crenelations:

    Footnote to this phase of microscopy: many of you are parents frequently in need of science projects for your school-age kids. Have them do the above with both these varigated leaves and with leaves that have been made chlorotic and leaves that are recovering from chlorosis. The equipment above is simple. Most high schools have microscopes, and if you have need of one, probably you can return with your child here to this lab to do that part of the work. You need merely be polite and nice to Dr. Rabiu and Mr. Patterson.

Looking at bacteria and yeast

    Next get some bacteria from plates or elsewhere and make some wet-mounts of those. Can you find the bacteria. If you are fortunate to have gotten a type of those in the pseudomonad family, they will be actively swimming - actually zooming around! No, you cannot see their flagella because they are narrower than visible light. But you can see their hotdog-shaped bodies twirling and speeding around. This twirling and then speeding off has a technical name: "twiddle and run!"

    If you don't have a pseudomonad, then you will see small hotdogs or golfballs jiggling around. Also try some bakers yeast cells (bigger or smaller than bacteria). Why are the bacteria and yeast jiggling? (Psst! It's due to Brownian motion. What's that?)