The Prokaryotes & Viruses Lab Exercise Contents Objectives 1 Introduction 1 Activity.1 Prokaryotic Cell Structure 2 Activity.2 Blue-Green Algae 2 Activity.3 Viruses 3 Activity.4 Gram Staining of Bacteria 3 Resutls Section 6 Objectives - Know the principal characteristics of the Prokaryotes - Identify three basic shapes of bacteria - Understand the Gram staining technique - Identify Gram + and Gram bacteria Introduction The Prokaryotes include all organisms which lack a nucleus. This characteristic is termed prokaryotic. Many scientists now divide these organisms into two Domains: the Bacteria and the Archaea. The Archaea is a very ancient group which is thought to resemble the first organisms on earth. These organisms are frequently found in very harsh environments. There are several phylogenetic groups within the Bacteria. However, with one exception, we will not be concerned with the characteristics of these separate groups. Rather we will only be concerned with more general characteristics of these organisms. The one group which we will specifically identify are the blue-green algae, the Cyanobacteria. In the Diversity Section of the BiologyOne DVD, select The Prokaryotes simulation. 1
Activity.1 Prokaryotic Cell Structure Activity.2 Blue-Green Algae This simulation illustrates some of the basic features of prokaryotic organisms. Review these to be familiar with the characteristics of these organisms. For the Eubacteria, one of the more important features used to distinguish these organisms is their shape. Three general bacterial shapes are termed coccus, bacillus, and spirillus. There are also variations based on these shapes. Because of the small size of bacteria, these shapes can only be determined under high magnification with light microscopes. The Cyanobacteria are photosynthetic prokaryotes. The combination of photosynthetic pigments found in these organisms frequently makes them blue-green in appearance, thus the common name, the Blue-Green Algae. In The Prokaryotic simulation on the BiologyOne DVD, examine micrographs of the four species of blue-green algae shown. Make your own diagram of these species in the Results Section. Coccus (pl: cocci) - This is a spherical or round shape. Bacteria with this general shape may be found singly, in pairs (diplococcus), in long chains (streptococcus), or in clusters (staphylococcus). Bacillus (pl: bacilli) - This is a rod shape. Bacteria with the bacillus shape may also be found singly, in pairs (diplobacillus) or in long chains (streptobacillus). Spirillum (pl: spirilla) - These bacteria have a spiral or corkscrew shape. Turning of the corkscrew shaped cell allows motility. Bacterial Shapes In the Prokaryotic tutorial on the BiologyOne DVD, after reviewing the material concerning the general structure of bacteria, examine the micrographs of the cell shapes found in bacteria. Label the illustration of the bacterial cell in the Results Section. Coccus Bacillus Spirillum 2
Activity.3 Viruses Activity.4 Gram Staining of Bacteria While viruses are not prokaryotes, usually not considered to be living organisms at all, viruses are often discussed with the prokaryotes. Perhaps because of their small size and tendency to cause disease. This simulation follows this tradition and illustrates some of the characteristics of viral particles. After review these features, label the diagram of a viral particle in the Results Section. An important method to classify bacteria is by observing their ability to complex with various chemicals (stains). This represents differences in bacterial morphology and physiology. One of the most commonly used stains for bacterial classification is the Gram Stain. This staining procedure will distinguish between two morphological types of bacteria, those with a thick, uniform cell wall and those with a thinner, bilayered cell wall. In this simulation you will follow the Gram staining procedure to determine the Gram staining characteristics for several species of bacteria. When you start this simulation you will see a shelf with the supplies you will need to complete this simulation (see diagram below). For most of these, clicking on the item will pull it off the shelf so you can use it. Clicking on the item again will place it back on the shelf. Just as in an actual laboratory setting, it is important to follow these directions in order or you will not obtain the desired results. Simulation Screen for Gram Staining click on item to pull from shelf 1. To begin, click on the Bunsen burner to bring it off the shelf. Then click on the container of inoculating loops. click on stains to remove from shelf move loop into tube for sample to view, move slide to scope slide arrow to select bacteria 3
You can click on and drag the loop which appears. Clicking on the loop container again will put away the loop. Drag the loop over to the Bunsen burner so its end enters the flame. This sterilizes the end of the loop. You can put the burner away at this point. 2. Click on the rack of culture vials. You have ten different cultures to select from. In each vial, the bacteria are growing on media called agar. Move the arrow under the culture you would like to begin with. When you release the arrow, the culture vial will be removed from the rack and opened so you can collect a sample of the bacteria. Select a culture, then move the loop so that its tip slowly enters the vial and collects a sample of the bacteria. Slowly remove the loop. Click on the culture vials to put them back on the shelf. You now have the bacteria on the end of the loop. 3. Click on the container with the forceps. Once you have forceps on the screen, click on it and drag it up to the box of slides on the shelf. You will pick up a microscope slide on the end of the forceps. Move this to the middle area of the screen. 4. Move the loop so that its tip contacts the slide. This will transfer the bacteria from the loop to the surface of the slide. You can put the loop away now by clicking on its container. (In an actual lab you should flame the loop before storing the inoculating loop.) Solution 1 is crystal violet stain. Dip the slide into this jar. If you were doing this procedure live, you would leave the slide in the solution for one minute. You do not have to time any of the steps in the simulation. Dip the slide into solution 2. This is water to wash off excess crystal violet stain. Dip the slide into solution 3. This contains Gram s iodine. If the cell is a Gram + bacterium, the iodine will react with the crystal violet causing it to adhere to the cell wall. The slide would remain in the iodine for one minute in the actual procedure. Dip the slide into solution 4. This is water to wash off excess iodine. Dip the slide into solution 5. This is alcohol. This solution will remove any crystal violet stain which has not reacted to adhere to the cell wall. The slide should remain in the alcohol for 10 to 30 seconds. Dip the slide into solution 6. This is water to remove excess alcohol. Dip the slide into solution 7. This is safranin stain. It will stain any bacteria which did not retain the crystal violet. This allows you to see these bacteria. The slide should be stained in safranin for 30 seconds. 5. You need to fix the bacteria to the glass of the slide to prevent them from washing off during the staining process. To do this, pull down the Bunsen burner and pass the slide through the flame several times. When completed, put the burner away. 6. You are now ready to stain the bacteria. Click on the stain tray to bring the stains off the shelf. When you do this the forceps and slide will change orientation for staining. The Gram Stain procedure requires that you pass the slide and bacteria through eight different solutions in order. In the simulation you do this by dipping the slide slowly into the solution until the tip of the forceps is about at the level of the top of the stain jar. Dip the slide into solution 8. This is water to remove the excess safranin stain. Once you ve removed the slide from solution 8 you can put the staining jars away by clicking on them. The slide and forceps will return to a side view. 7. You are now ready to view your results. If you were using a real slide and microscope you would probably want to place a cover slip on the slide. The simulation skips this step. Move the slide over to the microscope image in the lower left corner. When the slide is placed on the microscope stage, the screen will change to view your results. 4
8. For the bacteria you selected, record the bacteria s name, identify its shape and whether the bacteria is Gram + or Gram - in the Results Section. Repeat this experiment for at least three different bacteria. Before you finish, be sure you have observed at least one Gram + and one Gram - bacteria. 5
Lab Exercise Name Results Section Activity.1 Prokaryotic Cell Structure 1. 3. 2. 6
Activity.2 Blue-Green Algae object object object object 7
Activity.3 Viruses surface proteins (found in some viruses) lipid envelope (found in some viruses) 1. 2. 8
Activity.4 Gram Staining of Bacteria 9