LABORATORY EXERCISE # 2

MICROSCOPES AND MICROBES

LABORATORY OBJECTIVES

1.  Practice good microscope care and maintenance.

2.  Know the parts of the microscope and their functions.

3.  Know how to locate, focus, change magnification and view microscopic objects on the slide.

4.  Make a wet mount.

5.  View various microbes using the microscope.

6.  Classify selected microbes.

REFERENCE

Textbook: chapters 16 and 17

Photo Atlas: chapter 1, page 2; chapter 3; and chapter 4, pages 33-49

INTRODUCTION TO MICROSCOPES

Until late in the sixteenth century, much of the body of knowledge termed "biology" was unknown to science.  This is due to the miniscule size of some life forms, and the fact that a basic understanding of larger organisms requires the ability to see objects not detectable to the unaided human eye.  The development of precision lens grinding, and the construction of an instrument consisting of several lenses functioning together, revolutionized biology.  This instrument, the microscope, has become a major tool of biologists and few laboratories are without several.  With the passing of time and the development of new technologies, the microscope has proliferated into a variety of models, each designed for particular tasks.  Among these designs are:

A.  The light microscope - The type we will use in our laboratory. These use visible light which is magnified and concentrated by glass lenses.  There are two models which we will use.

1. The dissecting microscope is designed to study thick or opaque objects at fairly low magnification.

2. The compound microscope is used to observe small and/or thin objects at higher magnification.  Specimens must be thin enough for light to pass through from below.  This is the "workhorse" of biology and the one you will most often use.

B. The electron microscope uses a beam of electrons rather than light.  The electron beam is focused onto a photographic plate by means of electromagnetic "lenses".  There are two types of electron microscopes, both of which are large and expensive and are consequently confined to research labs.

1. The transmission electron microscope is used for specially-prepared transparent specimens.  It is capable of magnifying by thousands of diameters with great resolution.  Your professor will point our some photomicrographs in your Photo Atlas and/or in your textbook that were made using this kind of microscope.

2. The scanning electron microscope is not capable of extreme magnification, but can be used on opaque objects and shows remarkable surface details.  Your professor will point out some of examples of scanning electron microscopy from the textbook or Photo Atlas.

C. The dark-field microscope has the light entering from the side, where it is reflected off the object being observed. This shows a brightly illuminated object on a dark background. Your professor may show you some of these photomicrographs in your text or atlas.

D. Phase-contrast microscopes are useful for examining live, transparent objects.  Differences in thickness and composition are converted into visible differences which would be less apparent on another instrument.

PRELIMINARY INFORMATION - Please read before proceeding!

Microscopes are very expensive to replace; therefore, be particularly careful when handling and using them.  Always carry them with both hands, holding the instrument upright, so that the eyepiece does not slip out.  Use only clean dry lens paper to clean the lenses.  Do NOT use Kleenex, paper towels, sleeves, or the front of you T-shirt, as these can scratch the soft optical glass used in these lenses.  If there is a problem with your microscope, report this to your instructor immediately.  You will not be penalized in any way.

Activity

 I.  The Microscope and its Parts

Select one of the compound microscopes from the cabinet at the front of the room, carry it to your lab station and plug the power cord into a convenient outlet.  Using the figure in your Photo Atlas of a microscope similar to yours, locate all the parts shown.

     a. The ocular or eyepiece is the lens through which you view the object.  This lens magnifies the image passing through it by 10 times.  It also has a pointer which may be used to indicate objects being viewed.

     b. The revolving nosepiece holds three objective lenses.  Grasp the silver ring and note that it may be rotated, bringing each objective lens into place.  Feel the distinctive "click" as each objective is in proper position for viewing.

     c. The objective lens performs the initial magnification. In general, the lower the magnification, the shorter the objective lens and the farther it is positioned from the specimen.  Your scope has four of these, located on the revolving nosepiece.  They are as follows:

       - The scanning lens is a short lens which magnifies by 4 times. It as the broadest field of all your lenses and is used for initial location of objects.

       - The low-power objective has yellow color bands and magnifies by 10 diameters.  You will probably find this the objective most often used.

       - The high-power objective, with its blue color bands and 40X magnification, is much longer than the first two.  It is the most powerful lens used in this class.

     d. The stage is the flat surface with the round hole or aperture.  The slide to be viewed is positioned on the stage over the aperture.  On your microscopes, the stage is equipped two slide clips that are used to secure the slide so that it can be smoothly moved about.

     e. The substage condenser is a package of lenses located under the stage and controlled by grasping the package and turning it.  Its function is to condense the light and focus it on the specimen.  You need not be concerned with this device.  SIMPLY RAISE IT TO ITS MAXIMUM HEIGHT AND LEAVE IT THERE.

     f.  The iris diaphragm is located on the condenser and functions to control the amount of light passing through the specimen.  A tiny lever projects from the front of this device and is moved from side to side to affect a change in adjustment.

     g. The substage illuminator is the source of light which illuminates the object being viewed.

     h. The adjustment knobs are located on both sides of the microscope, just above the base.  There are two of these, one located just above the other.

         -  The coarse adjustment knob is the largest of these and is above the fine adjustment  knob.  Its function is to focus the scanning and low-power objectives.  In our microscopes, the coarse adjustment may be tight and may require the use of both hands.  USE WITH THE SCANNING AND LOW-POWER OBJECTIVES, BUT NEVER WITH THE HIGH POWER OBJECTIVE.

         -  The fine adjustment knob is smaller and just below the coarse adjustment knob.  Its function is to focus the high-power and oil immersion objectives.  DO NOT USE WITH LOW-POWER OR SCANNING OBJECTIVES.

II.  Magnification

         The compound microscope combines the magnifying power of the eyepiece with that of the objective lens.  The magnifying power is marked on the housing of each lens.  Simply multiply the magnification value of the objective times that marked on the eyepiece to see how many times the specimen is enlarged.  Calculate the magnification of your microscope with each of the objectives in place.

III.  Resolving Power

         This is a measure of lens quality.  Quality lenses have high resolving power, the ability to deliver a clear image in fine detail.  If a lens has high magnification but low resolution, it is of little value.  Although such an image would be large, it would not be clear enough to show detail.  Resolution is also affected by the cleanliness of the lens.  It is a good idea to clean your lenses every lab.

IV.  Field of View

This is the size of the area that the lens views.  The higher the magnifying power of an objective lens, the smaller the area viewed.  When you switch to a higher-powered lens, you are actually looking at the central portion of what was visible under low power.  It is important to center the specimen before increasing magnification.

V.    Using the Microscope

          With your microscope sitting before you, grasp the monocular head and carefully rotate it.  Try turning the microscope so that the arm is away from you.  The rotating head allows you to position the instrument either way.  Which is most comfortable to you?  Now rotate the illuminator dial to the 7 position.  Carefully lean the microscope back in the direction of the arm.  Look under the stage and locate the iris diaphragm.  Move the control arm of the diaphragm and see how it affects the light passing through the condenser.

a.   From the slide tray on the counter at the side of the room, select a slide marked "Letter e”.  With the tip of your fingers lift the slide clips and position the sides of the slide under them.  This will hold the slide in place.  Using your thumbs and forefingers, move the slide about on the stage and finally center the tiny "e" over the stage aperture.

   b.   Turn the scanning objective so that it snaps into place directly over the aperture.  Now raise the stage so that the slide is as close to the objective as possible.  While viewing through the eyepiece, use the coarse adjustment knob to move the slide away from the objective and bring the letter "e" into focus.  Move the "e" around.  If you move the slide to the right, what is the apparent direction of movement of the "e"?  Look at the tiny "e" with your naked eye and sketch a picture of it.  Now look at it through the microscope and sketch that image.  Do you see a difference?  Carefully center the "e" in the field (the circle of light seen through the microscope) and proceed to the next activity.

           c. Without making any other adjustment to the microscope, turn the low-power objective into place.  Focus the image (using the coarse adjustment knobs) and compare it to the previous one.  Is it still in the center of the field?  If not, center it once more.

        d. Again without making other adjustments, rotate the high-power objective into place.  Viewing the objective from the side, note how close it is to the slide.  Look through the eyepiece and, using the fine adjustment knob, bring the "e" into focus.  If all is dark, move the slide slightly.  Now use the iris diaphragm to change the amount of light.  Did it make a difference?

             A feature of a good microscope (of which your's is one) is its parfocal capability.  This means that when a specimen is in focus under low-power magnification, you can switch to high-power magnification and have the specimen remain in fairly good focus.  Usually a slight adjustment with the fine focus knob is all that is required for a sharp image.

  e. When you have finished, turn the scanning objective back into place and remove the  slide.

  f. Select a couple of other slides from the tray indicated by your instructor and practice bringing them into focus.  Always use the same procedure shown above.  DO NOT TRY SHORTCUTS!

6.    Making a Wet Mount

While most of the slides you will use in this course are prepared slides; fixed, stained and mounted by professionals, from time to time you will be asked to prepare a temporary slide of some material.  These are termed wet mounts. You will now prepare a wet mount as directed below:

   a. Select a glass slide and a coverslip from those provided.  Carefully clean the slide as you would eye-glasses, using one of the KEMWIPE tissues provided.  The coverslips are unused and should be clean.  Always handle both slides and coverslips by the edges.

   b. Using a clean toothpick, carefully scrape some cheek cells from the inside of your mouth, as demonstrated by your instructor.  Touch the toothpick to the center of your slide, depositing a milky mixture of saliva and cheek cells.

   c. Place a drop of iodine stain on the cheek cells and carefully mix with the toothpick.  This will render the cells more visible.

   d. Now take a coverslip and carefully drop it into place on the drop of stained cells.  Set down one edge first, then slowly lower the coverslip into place.  Ideally, any air bubbles will be displaced by this maneuver.  Do not be distressed, however, if you get a few bubbles in this initial preparation.  If the fluid does not come to the edge of the coverslip, add a bit of water at one edge.

   e. Place the slide on your microscope and view the cheek cells.  Use all three objectives.  These are flat, tile-like cells with a prominent nucleus which should be stained golden brown by the iodine.  As there will probably be clumps, move the slide around until you find some isolated cells to view.

INTRODUCTION TO THE MICROBES

The term microbe is an ill-defined group of organisms that falls into more than one kingdom of life.  We will define a microbe to be any unicellular microorganism; especially if it is pathogenic (disease causing).  We will also include those microorganisms that are colonial or filamentous.  This definition includes all organisms classified in the kingdom Monera (bacteria and cyanobacteria); it includes all the primitive organisms classified in the kingdom Protista (protozoa and microscopic algae); and it includes the unicellular species in the kingdom Fungi.  All the organisms that are in the kingdoms Plantae and Animalia are multicellular and are too complex to be called microbes.

Kingdom Monera

This kingdom includes the most primitive organisms on earth, as well as those with the longest evolutionary history.  The fossil remains of creatures such as these have been recovered from rocks formed as much as 3.8 billion years ago.  The chief distinguishing characteristics of the monerans are derived form their prokaryotic form.  Unlike all other organisms, the monerans lack a true, membrane-bounded nucleus.  The term “prokaryotic” comes from pro (before) and karyos (nucleus).  The single cells making up these simple creatures, in fact, have no membrane bounded organelles at all!  Although colonies of monerans are quite visible to the human eye, the individual cells are microscopic.  The world’s smallest organisms belong to this kingdom.  While there is evidence of sexual reproduction in some monera taxa, most reproduce for simply dividing.

What is meant by the term “prokaryotic?”

What is the term used to describe cells that are more advanced than monerans and have a nucleus and other membrane-bound organelles?  (You may have to go to your textbook or ask your instructor.)

1. The bacteria – These are the most well-known monerans for obvious reasons – they are responsible for more human diseases than any other pathogen.  Their importance does not end there, however, for they function both for and against us in many less spectacular ways.  For example, they are important as decomposers in ecological systems – recycling living or once-living cells of other organisms by breaking them down into their smallest molecules and absorbing them.  Unfortunately this process also occurs when foods “go bad.”  Some types of bacteria play a role in flavoring cheeses, nitrogen-fixation, and genetic engineering.

The three basic shapes of bacteria include the spherical bacteria or cocci, the rod-shaped bacilli and the spiraled bacteria or spirilli.

What is a pathogen?

What is a decomposer?

What is nitrogen-fixation and what is its roll in ecosystems?

2. The cyanobacteria – Known previously as the blue-green algae, these prokaryotic organisms are much like the bacteria, with one major exception – they contain chlorophyll.  This allows them to form their own food reserves through the process of photosynthesis.  In all probability, these were the first autotrophic (self-feeding) organisms on earth.  Though essentially unicellular, the cyanobacteria may form clump-like colonies or linear filaments.  Like the bacteria, the cyanobacteria play the important role of nitrogen-fixation.  On the negative side, they are usually responsible for taste and odor problems in domestic water supplies.

What is meant by the term “autotrophic?”

What is meant by the term “heterotrophic?”

Activity

9. Shapes of Bacteria

Your instructor has set up three microscopes along the side counter.  Briefly examine the slide on each and notice the general shape of the bacteria on each slide.  Determine if they are bacilli, cocci, or spirilli.  If the bacteria form clumps they are designated by; the prefix “staph” (as in staphylococcus).  If they are arranged in chains of cells they are called “strep” colonies, as in streptobacilli.  Are any of these examples of “staph” or “strep” colonies?

II. Slide of Nostoc

Select a slide of the cyanobacteria Nostoc.  Examine it carefully on high power.  Locate the bead-like chains of cells.  Note the general size and shape of the individual cells.  Can you see anything in the cells?  Do they all look alike?  You should be able to see some larger, colorless cells.  These are termed heterocysts and may function in nitrogen fixation.  Can you distinguish between a colony and a filament?  Perhaps you can see the gelatinous sheath secreted by the cells.

Kingdom Protista

With the exception of members of Kingdom Monera, all organisms are said to be eukaryotic.  Most organisms are multicellular, but the vast majority of protists are unicellular.  These single-celled protists can be called microbes.  Some are pathogens.

Though small, the protists are often quite complex.  Several modes of locomotion are found among the members of Kingdom Protista and both autotrophic and heterotrophic forms abound.  For convenience we will divide the kingdom into two groups, based primarily on their mode of nutrition.

1. The Animal-like Protists, or Protozoa.  These organisms are all unicellular and they ingest their food in animal-like fashion.  The four phyla of this kingdom get their names from their method of locomotion.
1. The flagellates move by means of one or two thin, whip-like flagella (singular; flagellum).  Most flagellates are free-living (not parasitic), however, some are important human pathogens.  An example is trypanosoma.  Trypanosoma is responsible for the debilitating disease known as African Sleeping Sickness.  The trypanosomes inhabit the blood vessels and are spread by the little, biting tsetse fly.

2. The ciliates are the most complex of protozoans.  They move by the coordinated rowing motion of many cilia (singlular; cilium).  These are similar in construction to flagella, but are much shorter.  Most ciliates are free-living predators or scavengers, inhabiting ponds or temporary pools.  These organisms, unlike any others, have two different nuclei.  The tiny micronucleus guides the cell’s reproductive activities, while a much larger macronucleus contains information for all other functions.  There is more diversity in form and function in this group than in any other protozoan taxon.

3. The amoebae and their kin are distinguished by their pseudopodia (singular; pseudopodium).  These are temporary outgrowths of protoplasm with which creatures feed and/or move.  Some members of this group secrete tiny shells; most, however, are naked.  The best place to find these protozoa is in the bottom mud of shallow freshwater pools, but a few are important pathogens of animals, causing gum disease, encephalitis and dysentery.

4. The sporozoans have no mode of locomotion.  They don’t need one.  These are blood parasites that are passed from one host to another by an insect vector – usually a mosquito.  The disease malaria is caused by a sporozoan

Activity

I. Slides of Paramecium

Select a prepared slide of Paramecium.  Find a good specimen and examine it under high power.  Can you find the two nuclei?  Can you see any other details like vacuoles?  Can you see its cilia?  Why or why not?

To observe live Paramecium, prepare a wet mount by placing a drop of the Paramecium culture inside a thin ring of Protoslo as directed by your instructor.  This will slow these very active creatures enough to allow their observation.  Can you see any detail?  Under high power you might be able to see hints of movement of cilia, but not the cilia themselves.

II.   Slide of Amoeba

Examine a prepared slide of Amoeba proteus.  Can you identify the pseudopodia?  The nucleus?

2. The plant-like protists are all photosynetic.  Some are unicellular, some form loose colonies and some are multicellular.  We will consider two groups that are unicellular and can be referred to as microbes.

1. The euglenoids are often classified with the flagellates because of their mode of locomotion, yet they are photosynthetic organisms.  Not only that, but they store food in a decidedly plant-like form – starch.  Euglena, one of the most common laboratory organisms, is in this group.

2. The diatoms are a group that is found in both fresh and salt water.  Their delicate valves (shells) are constructed of silicon dioxide – glass.  These protists store their food reserves as oil and may be a major source of our natural petroleum.  More importantly, these organisms form the base of the oceanic food chain.  Due to the presence of certain accessory pigments in addition to the chlorophyll, the diatoms are a golden color, rather than green.

Activities

9. Slides of Euglena

Place a prepared slide of Euglena on your microscope and observe these organisms under high power.  Can you see the flagellum? 

Make a wet mount using Protoslo as directed in the Paramecium activity above.  Select a slow-moving specimen and look for the flagellum, the eyespot and the chloroplasts.

II. Slide of Diatoms

Next select a slide of Diatoms and examine it under high power.  You may have to close down your iris diaphragm to see these glass-shelled organisms.