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					                                                                   Laboratory 1: Prokaryotes and Eukaryotes
                                                                                        BE 209 Spring 2007


                                                                      Laboratory 1
                                                        Prokaryotes and Eukaryotes
Purpose
In this lab you will study the major structural similarities and differences between prokaryotes and
eukaryotes, the two primary categories of living organisms. This examination will introduce you to the
microscope and visualization procedures of cells.

Introduction
Scientists currently agree that there are three major groupings of organisms on Earth called domains: the
Bacteria, the Archaea and the Eukarya. Bacteria are believed to be direct descendents of the most
ancient organisms on Earth, later giving rise to Archaea and Eukarya (Fig. 1). Bacteria and Archaea are
called prokaryotes because they lack a true nucleus (pro=before, karyon=nucleus) and do not have
organelles, highly ordered structures found in eukaryotic cells that are dedicated to functions like
respiration, photosynthesis or protein packaging.

                     Figure 1 Three domains of life: Bacteria, Archaea and Eukarya




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                                                                    Laboratory 1: Prokaryotes and Eukaryotes
                                                                                         BE 209 Spring 2007

It is easy to believe that prokaryotes are the most simple of the existing organisms. They are essentially
invisible to the naked eye, they are capable of functioning as solitary cells and they lack organelles.
However, prokaryotes are the most widely distributed organisms, inhabiting the most benign and the most
extreme environments, from hot sulfur vents in the sea floor that reach over 100 °C to arid deserts, frigid
Arctic icebergs and highly acidic human stomachs. All modes of metabolism are represented by the
prokaryotes: respiration, fermentation, photosynthesis and anaerobic respiration. This wide range of
metabolic ability has allowed for bioremediation of radiation and toxic chemical spills.

Eukarya are the eukaryotes, organisms whose cells have a nucleus and organelles. Plants, animals,
fungi and protists are all eukaryotes. While many prokaryotes and eukaryotes can exist in a multi-cellular
state, only the eukaryotes have demonstrated a division of labor among the cells, the development of
tissues. In tissues, an aggregation of cells performs very specific functions, leaving other functions to
other tissues.

Today, you will compare the cell structures of Bacteria and Eukarya.

Technique
Proper use of the compound microscope
Microscopes use visible light and lenses to magnify an object. There are two lenses on a microscope, the
ocular lens, which magnifies things by 10 times (10X), and the objective lens (Figure 1). There are
usually four types of objective lenses to choose from on a typical microscope: 4X, 10X, 40X, and 100X.
The combination of these two lenses allows for a magnification between 40 (10 X 4) and 1000 (10 X 100)
times. Familiarize yourself with the parts of a microscope using Figure 1.

                                   Figure 1 A compound microscope




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                                                                     Laboratory 1: Prokaryotes and Eukaryotes
                                                                                          BE 209 Spring 2007

Preparing the microscope

   1. Carry the microscope to your bench top with two hands, one under the microscope and one
      holding the microscope arm.
   2. Set the objective magnification to the lowest power, usually 4X.
   3. Slowly crank the stage down using the course adjustment knob.
   4. Place your slide of interest on the stage.
   5. Turn on the light source with the on/off switch and set the brightness using the rheostat. Mid-level
      light will be best at this magnification.

Viewing an object

   1. Look through the eyepieces with both eyes. The eyepieces are designed so that your eyes
      should be about 1-2cm away from the lenses. The distance between the eyepieces is adjustable.
   2. Slowly crank the stage up using the course adjustment knob. Once the object on the slide comes
      into focus, use the fine focus knob to make the image clearer.
   3. You can now change the objective lens to a higher magnification. Do not change the height of
      the stage when doing this.
   4. When you view the image at higher magnification, you should only need to adjust the fine focus,
      not the course focus. If the image was in focus at a lower magnification, it should generally be in
      focus at higher magnification.
   5. When using the 100X objective, always place a drop of oil on the slide. The 100X objective lens
      will sit in this oil droplet to limit any light refraction. Be sure to wipe off the oil with lens paper
      (NOT a Kim-wipe) when you are done with the 100X objective.

Putting away the microscope

   1.   Wipe off any oil on the 100X objective with lens paper (NOT a Kim-wipe).
   2.   Set the objective lens to the lowest magnification.
   3.   Crank the stage all the way down.
   4.   Turn off the light source. Wrap the cord loosely around the microscope. Place it back in the
        cabinet.




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                                                                     Laboratory 1: Prokaryotes and Eukaryotes
                                                                                          BE 209 Spring 2007


Procedures
A. Examination of Eukaryotic Cells

In this laboratory session, you will examine live and stained representatives of eukaryotic cells: protists,
plant cells and animal cells. You should note characteristics such as size, shape and cellular structures.
You should be able to identify key features of these three major groups by the end of the exercise. At
labeled stations around the room, you will find representatives of eukaryotes to view under compound
microscopes. Do not move the field or change the magnification. You should only need to adjust the fine
focus. Fill in Table 1 with your observations about these cells.

                               Table 1 Comparison of Eukaryotic Cells
                         Magnification and
                                                   Cellular
       Organism          approximate size                                        Drawing of Cell
                                                 structures?
                          relative to field
    Paramecium
    (a protist)




    Amoeba
    (a protist)




    Elodea
    (a plant)




    Red blood cells




    Cheek Cells




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                                                                      Laboratory 1: Prokaryotes and Eukaryotes
                                                                                           BE 209 Spring 2007



B. Examination of Prokaryotic cells: Colony observations
For our investigation of prokaryotic cell structure, we will first identify colony morphology, characteristics
of Bacteria on agar plates. Then, we will stain several types of Bacteria, allowing us to investigate cell
membrane structures. As you progress through this section, pay attention to similarities and differences
in structure and morphology between the different types of Bacteria.

    1. Obtain an agar plate containing the bacterium Escherichia coli. The agar in the plate is similar to
       Jello and contains nutrients allowing the bacterium to grow. Each dot on the plate represents one
       colony of cells. Each colony is probably no more than 1-2mm in diameter.
    2. Describe the following characteristics of the colonies in Table 2: color, colony edge shape, colony
       elevation (look at a colony from the side), and colony surface texture. Use Figure 2 as a
       reference.
    3. Make the same observations for Micrococcus luteus, Bacillus subtilis and Bacillus (Geobacillus)
       stearothermophilus.

                          Figure 2 Examples of bacterial colony descriptions




                        Table 2 Observations of prokaryote colony morphology
                               Colony              Colony               Colony          Colony surface
        Organism
                                color            edge shape            elevation            texture
    E. coli

    M. luteus

    B. subtilis

    Geobacillus




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                                                                        Laboratory 1: Prokaryotes and Eukaryotes
                                                                                             BE 209 Spring 2007

C. Examination of Prokaryotic cells: Microscopic Examination of Gram-Stained Cells
This staining procedure will allow you to infer basic differences in the cell wall structure between two
different kinds of bacteria (Fig. 3). After staining, the bacteria will appear as one of two colors, pink or
purple, based on these structural differences. Gram-positive bacteria have a thick layer of peptidoglycan
exterior to their plasma membrane. This layer retains the first stain in the procedure, resulting in the
purple color of gram-positive cells. Gram-negative cells have a thinner layer of peptidoglycan that does
not retain the purple dye well because it is surrounded by a thick layer of lipopolysaccharide. When
alcohol is applied during the staining process, the lipopolysaccharide layer is dissolved and all dyes are
removed from the cell. When you counterstain with a pink dye, you see the characteristic pink color of
gram-negative cells.

     Figure 3 Comparison of cell wall structures in Gram positive and Gram negative bacteria.




               NOTE: The timing of each step of the Gram stain is very important.
                     Do not allow a stain to remain on the slide too long!

   4. Place a drop of water on one end of a glass slide.
   5. Place the loop end of a bacteriological loop in the flame of a Bunsen burner until it turns red hot.
      Allow the loop to cool for about 15 seconds.
   6. While making sure not to touch the bacteriological loop to any surfaces, remove the lid from a
      plate of E. coli.
   7. Place the loop into the colony of E. coli, and remove a small portion of the colony.
   8. Place the loop in the droplet of water and swirl around gently to dislodge cells from the loop. The
      droplet should now be slightly cloudy.
   9. Grasp the end of the slide with a clothespin, and flame the slide from underneath the droplet
      GENTLY! Three or four quick passes over the flame should dry out the droplet. This affixes the
      bacteria to the slide.

   Heating the slide for too long will cause the slide to break. You should still be
   able to touch the slide without burning yourself. You should not smell burning
   bacteria.

   10. Repeat steps 1-6 with M. luteus, B. subtilis and Bacillus (Geobacillus) stearothermophilus.
   11. Place a drop of crystal violet on each dried bacterial smear and allow the smear to rest for one
       minute. Gently rinse the stain off with water.

            Crystal violet stains all cells purple, regardless of the cell structure.



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                                                                  Laboratory 1: Prokaryotes and Eukaryotes
                                                                                       BE 209 Spring 2007



12. Stain the smears with Gram’s iodine solution for one minute. Gently rinse the stain off with water.

           Gram’s iodine causes large crystals to form in the peptidoglycan layer.
           Because the peptidoglycan layer is thicker in Gram+ cells, more iodine
           is retained in these cells.

13. Apply a couple of drops of 95% ethanol to the smears for approximately ten seconds, then gently
    rinse off the slide with water.

           Ethanol removes the lipid layer of Gram- cells, removing the dye from
           the peptidoglycan layer. Gram- cells will now be colorless, while
           Gram+ cells will still be purple.

14. Stain the smears with safranin for thirty seconds then gently rinse the slide with water and blot
    dry.

           Safranin stains all cells pink, but because Gram+ cells are already
           purple, only the Gram- cells display the pink color.

15. Examine the cells under the microscope. Directions on how to use a microscope are on the
    following page.
16. Record your observations in Table 3. Prepared Gram stained slides will be available if your slides
    do not come out well.

                           Table 3 Observations of Gram-stained Bacteria
          Organism                   Color                   Shape              Gram stain result
                                                                                      (G+/G-)
E. coli

M. luteus

B. subtilis

Geobacillus




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                                                                    Laboratory 1: Prokaryotes and Eukaryotes
                                                                                         BE 209 Spring 2007

D. Growing and characterizing your own bacterial isolate
You will have the opportunity to practice some of the observational skills you have acquired in today’s lab
by studying some local bacterial flora. Your job today is to sample some bacterial flora from various
sources.

   1. Obtain one Petri plate from your TF for your group. Mark the bottom of the Petri plate (the half
      containing agar) into four quadrants with a permanent pen. Label the bottom of the plate with
      your group name and the date. Keep the plate closed at all times, unless inoculating it.
   2. Obtain four sterile-wrapped swabs.
   3. Among your group, decide on four locations in and around the building that you would like to
      sample for microbes. These may include people, handles, sinks, fountains, rain puddles or soil.
      Each group member should choose one site.
   4. Take a tube of sterile water with you on your sampling field trip.
   5. Before you sample a site, get the swab wet with water. Then sample the site. Make sure that all
      surfaces of the swab have touched the sample.
   6. To the quadrant of interest, apply the sterile swab. You do not need to apply much pressure to
      the agar. Make sure to keep the swab only within that quadrant.
   7. Use a back and forth motion to spread the sample across the agar (Fig. 6).

              Figure 4 Streaking procedures for inoculating quadrants on agar plates.




   8. Use a new swab for each sample.
   9. When you have sampled four sites, be sure to label each quadrant with the location sampled.
   10. Give your plate to your TF, who will incubate it at 37 °C overnight to facilitate bacterial growth.
       They will then be kept in the cold room until the next lab.
   11. Discard contaminated swabs in the orange/red biohazard bag in your classroom.




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                                                                  Laboratory 1: Prokaryotes and Eukaryotes
                                                                                       BE 209 Spring 2007

Name: _______________________

BU ID: _______________________

Teaching Fellow name and section number: ______________________
                                                                     Laboratory 1
                                                       Prokaryotes and Eukaryotes
                                      Post-lab Assignment

   1. What is the difference in the average size of a prokaryote to a eukaryote?




   2. What eukaryotic cellular structures are visible under a microscope?




   3. What prokaryotic cellular structures are visible under a microscope?




   4. What does the gram stain tell you about the structure of a bacterium?




   5. If you were to look under a microscope and identify whether the cell was a prokaryote or a
      eukaryote, what characteristics would you look for?




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