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Science in Motion Wilkes University Revised 2010 Pond Water Analysis: Survey of Microorganisms Standards: PA ST & E Standards: 3.1.10.A1. Explain the characteristics of life common to all organisms. 3.1.10.A5. Relate life processes to sub-cellular and cellular structures to their functions. 3.1.B.A5. Relate the structure of cell organelles to their function. 3.1.10.A8. Investigate the spatial relationships of organisms’ anatomical features using specimens, models, or computer programs. 3.1.B.A8. Recognize that systems within cells and multi-cellular organisms interact to maintain homeostasis. 3.1.10.A9. (Science as inquiry) Know that both direct and indirect observations are used by scientists to study the natural world and universe. PA E & E Standards: 4.2.7.C. Use appropriate tools to analyze a freshwater environment. 4.2.10.C. Explain the relationship between water quality and the diversity of life in a freshwater ecosystem. Safety Notes: 1. Do not drink the pond water; organisms can be present that can cause dysentery(diarrhea) or intestinal infections. 2. Use dropper carefully, do not squirt water into a person’s eyes as that could cause an eye infection. 3. Wash hands at end of lab. 4. Take care when using glass slides; if a slide breaks notify your teacher about clean up and disposal. Vocabulary: 1. Algae are any of various chiefly aquatic, eukaryotic, photosynthetic organisms, ranging in size from single-celled forms to the giant kelp. Algae were once considered to be plants but are now classified separately because they lack true roots, stems, leaves, and embryos. Dictionary.com · The American Heritage® Dictionary 2. Cyanobacteria are photosynthetic bacteria usually blue-green in color which are important in certain wet ecosystems. 3. Eukaryotes are organisms whose cells have nuclei including protists, plants, fungi, and animals. 4. Prokaryotes are unicellular organisms lacking a nucleus. 5. The kingdom Protista is composed of eukaryotes that cannot be classified as animals, plants, or fungi. They display the greatest variety; most are unicellular, but occasionally they can be multicellular. 6. Protozoans are “animal-like protists.” The four phyla are distinguished from one another by their means of movement. 1 Introduction: In this exercise a study will be made of protozoans, algae, and cyanobacteria that are found in pond water. Bottles that contain water and bottom debris from various ponds will be available for study. Illustrations and text provided in this lab will be used to assist you in an attempt to identify the various types of organisms encountered. Unpigmented, moving organisms will probably be protozoans; greenish or golden brown organisms are usually algae; and the blue-green organisms will be cyanobacteria. To study the microorganisms of pond water it will be necessary to make wet mount slides. The procedure for making such slides is relatively simple. The procedure is outlined below. Remember to identify the various organisms that you encounter in the lab report. Protozoa The subkingdom Protozoa includes the entire animal like microorganisms of the Kingdom Protista. All of the representatives in this subkingdom are single-celled organisms; however, some of them do form colonial aggregates. Externally, the cells are covered with a cell membrane, or pellicle; cell walls are absent and distinct nuclei with nuclear membranes are present. Specialized organelles such as contractile vacuoles, mitochondria, ribosomes, flagella, and cilia may also be present. All protozoa reproduce asexually by cell division. Some exhibit various degrees of sexual reproduction, called conjugation. Their ability to form cysts, which are dormant stages, enables them to survive drought, heat and freezing. The Subkingdom Protozoa is divided into three phyla: Sarcomastigophora, Ciliophora and Sporozoa. The type of locomotion plays an important role in classification. Phylum Sarcomastigophora Members within this phylum have been -subdivided into two subphyla: Sarcodina and Mastigophora. Sarcodina (Amoebae) Members of this subphylum move about by the formation of flowing protoplasmic projections called pseudopodia. The formation of pseudopodia as commonly referred to as amoeboid movement. Illustrations 5,6, 7 and 8 in Figure 1 are representative amoebae. 2 Mastigophora (Zooflagellates) These protozoans possess whiplike structures called flagella. There is considerable diversity among the members of this group. Only a few representatives 1,2,3 and 4 are seen in Figure 1. Phylum Ciliophora These microorganisms are undoubtedly the most advanced and structurally complex of all protozoans. Evidence seems to indicate that they have evolved from the zooflagellates. Movement and food- getting is accomplished with shot hairlike structures called cilia. Illustrations 9 through 24 in Figure 1 are typical ciliates. Phylum Sporozoa Members of this phylum lack locomotor organelles. All are internal parasites. As indicated by their group name, their life cycles include spore-forming stages. Plasmodium, the malarial parasite, is the most significant pathogenic sporozoan of humans. You will probably not usually observe this group in NE PA pond water. Algae Algae includes simple photosynthetic eukaryotic organisms which are either in the Plant or Protist Kingdoms. The algae may be unicellular, as those shown in the top row of Figure 2; colonial, like the four in the lower right-hand comer of Figure 2; or multicellular, as those in Figure 3. The undifferentiated algal structure is often referred to as a thallus. It lacks the stem, root, and leaf structures that result from tissue specialization. These microorganisms are universally present where ample moisture, favorable temperature, and sufficient sunlight exist. Although a great majority of them live submerged in water, some grow on soil; others grow on the bark of trees or the surfaces of rocks. Algae have distinct, visible nuclei and chloroplasts. Chloroplasts are packets that contain chlorophyll A and other pigments. Photosynthesis takes place within these bodies. The size, shape, distribution, and numbers of chloroplasts vary considerably from species to species; in some instances a single chloroplast may occupy most of the cell space. Although there are seven divisions of algae, only five will be listed here. 3 Division(Phylum)1 Euglenophyta (Euglenoids) Illustrations 1-6 in Figure 2 are typical euglenoids, representing four different genera within this relatively small group. All of them are flagellated and appear to be an intermediate between the algae and protozoa. Protozoa-like characteristics see in the euglenoids are (1) the absence of a cell wall, (2) the presence of a gullet, (3) the ability to ingest food (not through the gullet), (4) the ability to assimilate organic substances, and (5) the absence of chloroplasts in some species. In view of these facts, it becomes readily apparent why many zoologists often group the euglenoids with the zooflagellates. The absence of a cell wall makes these protists very flexible in movement. Instead of a cell wall they possess a semi rigid outer pellicle, which gives the organism a definite form. Photosynthetic types contain chlorophylls A and B, and they always have a red stigma (eyespot) which is light sensitive. Their characteristic food storage compound is a lipopolysaccharide, paramylum. Division (Phylum) 2 Chlorophyta (Green Algae) The majority of algae observed in ponds will belong to this group. They are grassgreen in color, resembling the euglenoids in that they synthesize starch instead of paramylum for food storage. The diversity of this group is too great to explore its subdivisions in this preliminary study; however, the small flagellated Chlamydomonas, Illustration 8 Figure 2, appears to be the archetype of the entire group. Many colonial forms, such as Pandorina, Eudorina. Gonium, and Volvox ( illustrations 14, 15, 19 and 20, Figure 2), consist of organisms similar to Chlamydomonas. It is the general consensus that from this flagellated form all the filamentous algae have evolved. Except for Vaucheria (5) and Tribonema (6) in Figure 3, all of these filamentous forms are Chlorophyta. All of the nonfilamentous, nonflagellated algae in Figure 4 also are green algae. A unique group of green algae are the desmids, see illustrations 16-20, Figure 4, With the exception of a few species, the cells of desmids consist of two similar halves, or semicells. The two halves usually are separated by a constriction, the isthmus. 4 Division 3 Chrysophyceae (Golden Brown Algae) This large diversified group consists of over 6000 species. They differ from the euglenoids and green algae in that (1) food storage is in the form of oils and leucosin, a polysaccharide, (2) chlorophylls A and C are present, and (3) fucoxanthin, a brownish pigment, is present. It is the combination of fucoxanthin, other yellow pigments, and the chlorophylls that cause most of these algae to appear golden brown. Representatives are seen in figures 2, 3, and 5, Chrysococcus (12), Synura (13), and Dinobyron(16) are typical flagellated chrysophycophytes. Vaucheria (5)and Tribonema (6) are the only filamentous chrysophycophytes shown in figure 3. All of the organisms in figure 5 are chysophycophytes and fall into a special category called the diatoms. The diatoms are unique in that they have hard cells of pectin, cellulose, or silicon that are constructed in two halves. The two halves fit together like lid and box. It is postulated by some that our petroleum reserves were formulated by the accumulation of oil from dead diatoms over millions of years. Division (Phylum) 4 Phaeophyta (Brown Algae) With the exception of three freshwater species, all algal protists of this division exist in salt water (marine); thus, it is unlikely that you will encounter any brown algae in this laboratory experience. All species of brown algae are multicellular and sessile. Most seaweeds are brown algae. Division(Phylum) 5 Pyrrophycophyta (Fire Algae) The principal members of this division are the dinoflagellates. Since the majority of these protists are marine, only two freshwater forms are shown in figure 2; Peridinium (17)and Ceratium (18). Most of these protists possess cellulose walls of interlocking armor plates, as in Ceratium. Two flagella are present: one is directed backward in swimming and the other moves within a transverse groove. Many marine dinoflagellates are bioluminescent. Cyanobacteria The blue green prokaryotic bacteria, or Cyanobacteria, comprise Division I of the Kingdom Monera. Although these microorganisms were formerly referred to as algae, their prokaryotic-type nucleus definitely sets them apart from the eukaryotic algae. Although some bacteria are phototrophic, the difference between phototrophic bacteria and cyanobacteria is that the cyanobacteria have chlorophyll a and the phototrophic bacteria do not. Bacteriochlorophyll is the photosynthetic pigment in the phototrophic bacteria. Over 1000 bacteria of cyanobacteria have been reported. They are present in almost all moist environments from the tropics to the poles, including both freshwater and marine. Figure 6 illustrates only a random few that are most commonly seen. 5 The designation of these bacteria as "blue-green" as somewhat misleading in that many cyanobacteria are actually black, purple, red and various shades of green instead of blue-green. The varying proportions of the numerous pigments present produce these different colors. These pigments are chlorophyll a, carotene, xanthophylls, blue cphycocyanin, and red c-phycoerythrin. The last two pigments are unique to the cyanobacteria. Cellular structure is considerably different from the eukaryotic algae. As stated earlier, nuclear membranes in cyanobacteria are absent. The nuclear material consists of DNA granules in a more or less colorless area in the center of the cell. Unlike the algae, the pigments of the cyanobacteria are not contained in chloroplasts; instead, they are located in granules (phycobilisomes) that are attached to membranes (thylakoids) that permeate the cytoplasm. Materials: bottles of pond water samples droppers 6 page key Forceps microscope slides cover slips Kim wipes miscroscope Procedure: 1. Use forceps to remove as much filamentous algae as possible from the bottled sample 2. Make a wet mount by placing a single piece of algae, a drop of pond water, and slowly lowering a cover slip at an angle to avoid air bubbles. 3. First observe using the lowest objective (4x), then switch to 10x or 40x to observe more details. Reduce the lighting with the iris diaphragm. Keep the condenser high. 4. Next use a dropper to extract water (and organisms) from the bottom of the sample bottle. Very few organisms will be found swimming around in mid-depth of the bottle. 5. Squirt water and organisms onto the middle of the slide. Avoid putting too much material on a slide. 6. Place a cover slip over the slide. 6 7. Explore the slide with a low power objective. Reduce the lighting with the iris diaphragm. Keep the condenser high. 8.When you find an organism of interest observe and sketch in notebook or data sheet. 9. Refer to Figures 1-6 to identify the various organisms you encounter. You may not be able to identify every organism, try your best to look through the key provided. 10. You may have more than one species on your slide. 11. You may make another slide with a new drop of pond water following the same Procedure until the end of the lab period. 12. Complete sketches and answer the questions on the data sheet. 7 8