Guide to Protozoan Identification PDF

Benson: Microbiological Applications Lab Manual, Eighth Edition II. Survey of Microorganisms 6 6. Protozoa, Algae, and Cyanobacteria Protozoa, Algae, and Cyanobacteria In this exercise a study will be made of protozoans, algae, and cyanobacteria that are found in pond water. Bottles that contai...

Benson: Microbiological Applications Lab Manual, Eighth Edition II. Survey of Microorganisms 6 6. Protozoa, Algae, and Cyanobacteria Protozoa, Algae, and Cyanobacteria 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 exercise will be used to assist you in an attempt to identify the various types that are encountered. Unpigmented, moving microorganisms will probably be protozoans. Greenish or golden-brown organisms are usually algae. Organisms that appear blue-green will be cyanobacteria. Supplementary books on the laboratory bookshelf will also be available for assistance in identifying organisms that are not described in the short text of this exercise. If you encounter invertebrates and are curious as to their identification, you may refer to Exercise 7; however, keep in mind that our prime concern here is only with protozoans, algae, and cyanobacteria. The purpose of this exercise is, simply, to provide you with an opportunity to become familiar with the differences between the three groups by comparing their characteristics. The extent to which you will be held accountable for the names of various organisms will be determined by your instructor. The amount of time available for this laboratory exercise will determine the depth of scope to be pursued. 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. All that is necessary is to place a drop of suspended organisms on a microscope slide and cover it with a cover glass. If several different cultures are available, the number of the bottle should be recorded on the slide with a china marking pencil. As you prepare and study your slides, observe the following guidelines: Materials: bottles of pond-water samples microscope slides and cover glasses rubber-bulbed pipettes and forceps china marking pencil reference books 1. Clean the slide and cover glass with soap and water, rinse thoroughly, and dry. Do not attempt to study a slide that lacks a cover glass. 26 © The McGraw−Hill Companies, 2001 2. When using a pipette, insert it into the bottom of the bottle to get a maximum number of organisms. Very few organisms will be found swimming around in middepth of the bottle. 3. To remove filamentous algae from a specimen bottle, use forceps. Avoid putting too much material on the slides. 4. Explore the slide first with the low-power objective. Reduce the lighting with the iris diaphragm. Keep the condenser at its highest point. 5. When you find an organism of interest, swing the high-dry objective into position and adjust the lighting to get optimum contrast. If your microscope has phase-contrast elements, use them. 6. Refer to Figures 6.1 through 6.6 and the text on these pages to identify the various organisms that you encounter. 7. Record your observations on the Laboratory Reports. THE PROTISTS Single-celled eukaryons that lack tissue specialization are called protists. Protozoologists group all protists in Kingdom Protista. Those protists that are animallike are put in Subkingdom Protozoa and the protists that are plantlike fall into Subkingdom Algae. This system of classification includes all colonial species as well as the single-celled types. SUBKINGDOM PROTOZOA Externally, protozoan 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, cytostomes, mitochondria, ribosomes, flagella, and cilia, may also be present. All protozoa produce cysts, which are resistant dormant stages that enable them to survive drought, heat, and freezing. They reproduce asexually by cell division and exhibit various degrees of sexual reproduction. The Subkingdom Protozoa is divided into three phyla: Sarcomastigophora, Ciliophora, and Apicomplexa. Type of locomotion plays an important role in classification here. A brief description of each phylum follows: Benson: Microbiological Applications Lab Manual, Eighth Edition II. Survey of Microorganisms 6. Protozoa, Algae, and Cyanobacteria © The McGraw−Hill Companies, 2001 Protozoa, Algae, and Cyanobacteria Figure 6.1 • Exercise 6 Protozoans 27 Benson: Microbiological Applications Lab Manual, Eighth Edition Exercise 6 • II. Survey of Microorganisms 6. Protozoa, Algae, and Cyanobacteria Protozoa, Algae, and Cyanobacteria Phylum Sarcomastigophora Members of 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 is commonly referred to as amoeboid movement. Illustrations 5 through 8 in figure 6.1 are representative amoebae. Mastigophora (Zooflagellates) These protozoans possess whiplike structures called flagella. There is considerable diversity among the members of this group. Only a few representatives (illustrations 1 through 4) are seen in figure 6.1. 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 short hairlike structures called cilia. Illustrations 9 through 24 are typical ciliates. Phylum Apicomplexa This phylum has only one class, the Sporozoa. Members of this phylum lack locomotor organelles and all are internal parasites. As indicated by their class name, their life cycles include spore-forming stages. Plasmodium, the malarial parasite, is a significant pathogenic sporozoan of humans. SUBKINGDOM ALGAE The Subkingdom Algae includes all the photosynthetic eukaryotic organisms in Kingdom Protista. Being true protists, they differ from the plants (Plantae) in that tissue differentiation is lacking. The algae may be unicellular, as those shown in the top row of figure 6.2; colonial, like the four in the lower right-hand corner of figure 6.2; or filamentous, as those in figure 6.3. 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 on the surfaces of rocks. Algae have distinct, visible nuclei and chloroplasts. Chloroplasts are organelles that contain chlorophyll a and other pigments. Photosynthesis takes place within these bodies. The size, shape, distribution, and number of chloroplasts vary considerably from species to species. In some instances a single chloroplast may occupy most of the cell space. 28 © The McGraw−Hill Companies, 2001 Although there are seven divisions of algae, only five will be listed here. Since two groups, the cryptomonads and red algae, are not usually encountered in freshwater ponds, they have not been included here. Division 1 Euglenophycophyta (Euglenoids) Illustrations 1 through 6 in figure 6.2 are typical euglenoids, representing four different genera within this relatively small group. All of them are flagellated and appear to be intermediate between the algae and protozoa. Protozoanlike characteristics seen in the euglenoids are (1) the absence of a cell wall, (2) the presence of a gullet, (3) the ability to ingest food but 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 semirigid 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. The photosynthetic euglenoids can be bleached experimentally by various means in the laboratory. The colorless forms that develop, however, cannot be induced to revert back to phototrophy. Division 2 Chlorophycophyta (Green Algae) The majority of algae observed in ponds belong to this group. They are grass-green in color, resembling the euglenoids in having chlorophylls a and b. They differ from euglenoids in that they sythesize 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 6.2) appears to be the archetype of the entire group and has been extensively studied. Many colonial forms, such as Pandorina, Eudorina, Gonium, and Volvox (illustrations 14, 15, 19, and 20, figure 6.2), 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 and Tribonema, all of the filamentous forms in figure 6.3 are Chlorophycophyta. All of the nonfilamentous, nonflagellated algae in figure 6.4 also are green algae. Benson: Microbiological Applications Lab Manual, Eighth Edition II. Survey of Microorganisms © The McGraw−Hill Companies, 2001 6. Protozoa, Algae, and Cyanobacteria Protozoa, Algae, and Cyanobacteria 4 • Exercise 6 5 6 3 2 1 8 7 9 10 11 14 12 15 13 17 19 18 20 16 Courtesy of the U.S. Environmental Protection Agency, Office of Research & Development, Cincinnati, Ohio 45268. 1. Euglena (700X) 2. Euglena (700X) 3. Phacus (1000X) 4. Phacus (350X) 5. Lepocinclis (350X) Figure 6.2 6. Trachelomonas (1000X) 7. Phacotus (1500X) 8. Chlamydomonas (1000X) 9. Carteria (1500X) 10. Chlorogonium (1000X) 11. Pyrobotrys (1000X) 12. Chrysococcus (3000X) 13. Synura (350X) 14. Pandorina (350X) 15. Eudorina (175X) 16. Dinobyron (1000X) 17. Peridinium (350X) 18. Ceratium (175X) 19. Gonium (350X) 20. Volvox (100X) Flagellated algae 29 Benson: Microbiological Applications Lab Manual, Eighth Edition Exercise 6 • II. Survey of Microorganisms 6. Protozoa, Algae, and Cyanobacteria Protozoa, Algae, and Cyanobacteria A unique group of green algae is the desmids (illustrations 16 through 20, figure 6.4). With the exceptions 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. Division 3 Chrysophycophyta (Golden Brown Algae) This large diversified division consists of over 6,000 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 causes most of these algae to appear golden brown. Representatives of this division are seen in figures 6.2, 6.3, and 6.5. In figure 6.2, Chrysococcus, Synura, and Dinobyron are typical flagellated chrysophycophytes. Vaucheria and Tribonema are the only filamentous chrysophycophytes shown in figure 6.3. All of the organisms in figure 6.5 are chrysophycophytes and fall into a special category of algae called the diatoms. The diatoms are unique in that they have hard cell walls of pectin, cellulose, or silicon oxide that are constructed in two halves. The two halves fit together like lid and box. Skeletons of dead diatoms accumulate on the ocean bottom to form diatomite, or “diatomaceous earth,” which is commercially available as an excellent polishing compound. It is postulated by some that much of our petroleum reserves may have been formulated by the accumulation of oil from dead diatoms over millions of years. Division 4 Phaeophycophyta (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 phaeophycophytes in this laboratory experience. These algae have essentially the same pigments seen in the chrysophycophytes, but they appear brown because of the masking effect of the greater amount of fucoxanthin. Food storage in the brown algae is in the form of laminarin, a polysaccharide, and mannitol, a sugar alcohol. All species of brown algae are multicellular and sessile. Most seaweeds are brown algae. Division 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 6.2: Peridinium and Ceratium (illustrations 17 and 30 © The McGraw−Hill Companies, 2001 18). Most of these protists possess cellulose walls of interlocking armor plates, as in Ceratium. Two flagella are present: one is directed backward when swimming and the other moves within a transverse groove. Many marine dinoflagellates are bioluminescent. Some species of marine Gymnodinium, when present in large numbers, produce the red tides that cause water discoloration and unpleasant odors along our coastal shores. These algae have chlorophylls a and c and several xanthophylls. Foods are variously stored in the form of starch, fats, and oils. THE PROKARYOTES As indicated on the first page of this unit, the prokaryotes differ from the protists in that they are considerably smaller, lack distinct nuclei with nuclear membranes, and are enclosed in rigid cell walls. Since all members of this group are bacteria, the three-domain system of classification puts them in Domain Bacteria. Division Cyanobacteria Division Cyanobacteria in Domain Bacteria includes a large number of microorganisms that were at one time referred to as the blue-green algae. All these prokaryotes are photosynthetic, utilizing chlorophyll a for photosynthesis. They differ from the green sulfur and green nonsulfur photosynthetic bacteria in that the latter use bacteriochlorophyll instead of chlorophyll a for photosynthesis. Over 1,000 species 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 6.6 illustrates only a random few that are frequently seen. The designation of these bacteria as “blue-green” is somewhat misleading in that many cyanobacteria are actually black, purple, red, and various shades of green instead of blue-green. These different colors are produced by the varying proportions of the numerous pigments present. These pigments are chlorophyll a, carotene, xanthophylls, blue c-phycocyanin, and red c-phycoerythrin. The last two pigments are unique to the cyanobacteria and red algae. Cellular structure is considerably different from the eukaryotic algae. Although cells lack visible nuclei, nuclear material is present in the form of DNA granules in a 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. Benson: Microbiological Applications Lab Manual, Eighth Edition II. Survey of Microorganisms © The McGraw−Hill Companies, 2001 6. Protozoa, Algae, and Cyanobacteria Protozoa, Algae, and Cyanobacteria 1 4 9 7 12 Exercise 6 6 5 3 2 • 8 13 14 10 15 16 11 17 Courtesy of the U.S. Environmental Protection Agency, Office of Research & Development, Cincinnati, Ohio 45268. 1. Rhizoclonium (175X) 2. Cladophora (100X) 3. Bulbochaete (100X) 4. Oedogonium (350X) Figure 6.3 5. Vaucheria (100X) 6. Tribonema (300X) 7. Chara (3X) 8. Batrachospermum (2X) 9. Microspora (175X) 10. Ulothrix (175X) 11. Ulothrix (175X) 12. Desmidium (175X) 13. Mougeotia (175X) 14. Spirogyra (175X) 15. Zygnema (175X) 16. Stigeoclonium (300X) 17. Draparnaldia (100X) Filamentous algae 31 Benson: Microbiological Applications Lab Manual, Eighth Edition Exercise 6 • II. Survey of Microorganisms © The McGraw−Hill Companies, 2001 6. Protozoa, Algae, and Cyanobacteria Protozoa, Algae, and Cyanobacteria 2 3 1 4 6 7 5 8 9 10 15 13 12 11 14 17 18 16 17 19 20 Courtesy of the U.S. Environmental Protection Agency, Office of Research & Development, Cincinnati, Ohio 45268. 1. Chlorococcum (700X) 2. Oocystis (700X) 3. Coelastrum (350X) 4. Chlorella (350X) 5. Sphaerocystis (350X) Figure 6.4 32 6. Micractinium (700X) 7. Scendesmus (700X) 8. Actinastrum (700X) 9. Phytoconis (700X) 10. Ankistrodesmus (700X) Nonfilamentous and nonflagellated algae 11. Pamella (700X) 12. Botryococcus (700X) 13. Tetraedron (1000X) 14. Pediastrum (100X) 15. Tetraspora (100X) 16. Staurastrum (700X) 17. Staurastrum (350X) 18. Closterium (175X) 19. Euastrum (350X) 20. Micrasterias (175X) Benson: Microbiological Applications Lab Manual, Eighth Edition II. Survey of Microorganisms © The McGraw−Hill Companies, 2001 6. Protozoa, Algae, and Cyanobacteria Protozoa, Algae, and Cyanobacteria • Exercise 6 3 1 5 4 2 7 6 9 8 15 10 11 13 12 14 16 17 19 21 20 22 18 23 Courtesy of the U.S. Environmental Protection Agency, Office of Research & Development, Cincinnati, Ohio 45268. 1. Diatoma (1000X) 2. Gomphonema (175X) 3. Cymbella (175X) 4. Cymbella (1000X) 5. Gomphonema (2000X) 6. Cocconeis (750X) Figure 6.5 7. Nitschia (1500X) 8. Pinnularia (175X) 9. Cyclotella (1000X) 10. Tabellaria (175X) 11. Tabellaria (1000X) 12. Synedra (350X) 13. Synedra (175X) 14. Melosira (750X) 15. Surirella (350X) 16. Stauroneis (350X) 17. Fragillaria (750X) 18. Fragillaria (750X) 19. Asterionella (175X) 20. Asterionella (750X) 21. Navicula (750X) 22. Stephanodiscus (750X) 23. Meridion (750X) Diatoms 33 Benson: Microbiological Applications Lab Manual, Eighth Edition Exercise 6 • II. Survey of Microorganisms © The McGraw−Hill Companies, 2001 6. Protozoa, Algae, and Cyanobacteria Protozoa, Algae, and Cyanobacteria 2 1 3 6 4 5 7 13 9 8 10 11 12 14 15 17 16 21 18 19 22 20 Courtesy of the U.S. Environmental Protection Agency, Office of Research & Development, Cincinnati, Ohio 45268. 1. Anabaena (350X) 2. Anabaena (350X) 3. Anabaena (175X) 4. Nodularia (350X) 5. Cylindrospermum (175X) 6. Arthrospira (700X) Figure 6.6 34 Cyanobacteria 7. Microcoleus (350X) 8. Phormidium (350X) 9. Oscillatoria (175X) 10. Aphanizomenon (175X) 11. Lyngbya (700X) 12. Tolypothrix (350X) 13. Entophysalis (1000X) 14. Gomphosphaeria (1000X) 15. Gomphosphaeria (350X) 16. Agmenellum (700X) 17. Agmenellum (175X) 18. Calothrix (350X) 19. Rivularia (175X) 20. Anacystis (700X) 21. Anacystis (175X) 22. Anacystis (700X)