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LABORATORY NINE THE KINGDOMS OF MONERA AND PROTISTA OVERVIEW: In order to make it easier to study all the various organisms that have been identified and many more organisms that have not been identified, biologists have grouped the living world into five kingdoms. See Laboratory Five for the characteristics of the Five Kingdoms. These five kingdoms are separated by fundamental characteristics such as cellular qualities and modes of nutrition. In this laboratory we will be studying two kingdoms that are very diverse yet have two major characteristics in common — the organisms in these kingdoms are usually microscopic and many are solitary cells. To be totally accurate, we must admit that there are some filamentous or colonial groups in these kingdoms, and even some are multicellular. The Kingdom Monera contains prokaryotic life forms, such as the true bacteria and the blue-green bacteria or cyanobacteria, the latter formerly known as blue-green algae. Prokaryotic cells lack a nuclear envelope; that is, the genetic material is not doubly membrane-bound but loose in the cytoplasm. The Kingdom Protista is a diverse kingdom that contains the single-celled eukaryotic organisms, such as protozoa and some algae. Eukaryotic cells have a nuclear envelope that separates the genetic material from the rest of the cytoplasm. Many biologists place all of the seaweeds, for example, sea lettuce, a green alga; and kelp, a brown alga, in this diverse Kingdom.
OBJECTIVES OF LABORATORY: • To review the characteristics of the five kingdoms of life. • To learn the major characteristics of the Kingdoms Monera and Protista. • To review the steps of the Gram stain technique and to understand how it is used. • To use observed characteristics and a dichotomous key to identify organisms. • To be familiar with examples of the Kingdoms Monera and Protista that are available in the laboratory.
KINGDOM MONERA: Among the most commonly studied Monera are some of the bacteria that are medically significant. We will be looking at a technique today called the Gram stain that is used to separate disease-causing organisms into two large groups on the basis of the staining characteristics of the cells. Once the bacterial cells' particular Gram staining characteristic is known, medical personnel often can proceed with that information alone to prescribe an appropriate antibiotic. It turns out that the two large groups of bacteria have different cell wall qualities, and those qualities are what make the cells stain differently as well as cause the cells to respond to some antibiotics differently. Also, with the Gram stain, the clinician can describe the shape and possible grouping of the bacterial cells. Morphology and cell wall staining qualities are a great help in both identifying any pathogenic organism and then choosing the correct treatment for the condition. Choose one of the four kinds of cultures available on petri plates in the laboratory. Watch as your lab instructor demonstrates proper technique for using the inoculating loop to make a bacterial smear. Draw a dime-sized circle on the underside of a clean slide. Place a small drop of water in the circle on the topside of the slide. With the loop take a small, very small, amount of the culture from the plate and stir it into the drop of water. Allow the drop to dry without heating. When the drop is dry, heat fix the smear by passing the slide through the flame of the burner with the smear side up once or twice. Proceed with the following steps of the Gram stain. See the diagrams on the facing page.
You should see purple or pink (reddish) cells. You should be able to describe the morphology of the cells and perhaps how the cells are grouped. Those that are purple are said to be Gram-positive, and those cells that are pink are referred to as Gram-negative. Think back to the procedure that you performed. Cells that are purple, Gram-positive, retained the crystal violet stain throughout the whole procedure. Cells that are pink, Gram-negative, were decolorized by the alcohol wash even though they had been treated by the iodine, a mordant in stain technology terms. Those cells that retain the purple dye when treated with iodine have a thick cell wall of a material called peptidoglycan, polymerized sugar molecules linked with short amino acid chains. Those that are easily decolorized have only a thin layer of peptidoglycan but also an additional lipid bilayer in their cells walls; referred to as an “outer membrane” that does not hold the crystal violet dye. These latter cells are stained with the safranin in the last step so that we can better see their morphology and arrangement. In our work today you had four possibilities of cultures. Make a decision about the Gram reaction of the cells that you stained. Look at the smears of your neighbors to see other forms.
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