Laboratory 1: Prokaryotes and Eukaryotes BE 209

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

It is easy to believe that prokaryotes are the most simple of the existing organisms. They are essentially invisible to the naked eye, 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

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Laboratory 1: Prokaryotes and Eukaryotes BE 209

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 membrane-bound 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 using compound microscope. Compound 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 (Fig. 2). 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 2. Figure 2 compound microscope

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.

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Laboratory 1: Prokaryotes and Eukaryotes BE 209

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.

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. Find the organisms under 10X objective lens, then switch to 40x objective lens. Fill in Table 1 of post-lab assignment with your observations about these cells. 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 of post-lab assignment: color, colony edge shape, colony elevation (look at a colony from the side), and colony surface texture. Use Figure 3 as a reference. 3. Make the same observations for Micrococcus luteus, Bacillus subtilis and Bacillus (Geobacillus) stearothermophilus. Figure 3 Examples of bacterial colony descriptions

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Laboratory 1: Prokaryotes and Eukaryotes BE 209

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 groups of bacteria (Fig. 4). One group has a thick layer of peptidoglycan in the cell wall. The other group however, has a thinner layer of peptidoglycan and a thick layer of lipopolysaccharide outside of it. During the Gram-Stain procedure, bacteria are stained with a purple dye first, washed with alcohol then stained with a pink dye. For the first group of bacteria, the thick layer of peptidoglycan allows the bacteria to retain most of the purple dye, resulting in the purple color at the end of the procedure. For the second group of bacteria, alcohol dissolves the outer lipopolysaccharide layer and removes all the purple dye. After counter stain with a pink dye, this group of bacteria appears to be pink at the end of the procedure. We define the first group of bacteria as Gram positive (purple) and the second group of bacteria Gram negative (pink). Figure 4 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! 1. Place a small drop of water on one end of a glass slide. 2. Put the loop end of a bacteriological loop into a colony of E. coli, and remove a small portion of the colony. Make sure not to touch the bacteriological loop to any other surfaces. 3. 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. 4. 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 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 bacterial. 5. Repeat steps 1-6 with M. luteus, B. subtilis and Bacillus (Geobacillus) stearothermophilus. 6. 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

7. 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. 8. 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. 9. 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. 10. Examine the cells under the microscope. 11. Record your observations in Table 3. Prepared Gram stained slides will be available if your slides do not come out well. 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. 5). Figure 5 Streaking procedures for inoculating quadrants on agar plates.

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Laboratory 1: Prokaryotes and Eukaryotes BE 209

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

Name: _______________________ BU ID: _______________________ Teaching Fellow name and section number: ______________________

Laboratory 1 Prokaryotes and Eukaryotes Post-lab Assignment Table 1 Comparison of Eukaryotic Cells: (20 pts) Magnification and Organism

approximate size relative to field

Cellular structures?

Drawing of Cell

Paramecium (a protist)

Amoeba (a protist)

Elodea (a plant)

Red blood cells

Cheek Cells

Question 1: What are the common eukaryotic cell structures you observed in the lab? (10 pts)

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Laboratory 1: Prokaryotes and Eukaryotes BE 209

Table 2: Observations of prokaryote colony morphology: (20 pts) Organism

Colony color

Colony edge shape

Colony elevation

Colony surface texture

E. coli M. luteus B. subtilis Geobacillus

Table 3: Observations of Gram-stained Bacteria: (20 pts) Organism

Color

Shape

Gram stain result (G+/G-)

E. coli M. luteus B. subtilis Geobacillus

Question 2: Gram stain result of a bacterium is based on the difference in which bacterial cell structure? (10 pts)

Question 3: According to the magnification of the objective lens and relative size you observed under the microscope, can you estimate the size range of the eukaryotic cells? How about the prokaryotic cells? Please explain how you come to your conclusions. (20 pts)

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