The Cyanobacteria or blue-green algae CAUTION

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1 The Cyanobacteria or blue-green algae The cyanobacteria, also known as cyanophyta due to their photosynthetic properties, were once viewed as primitive algae, also referred to as blue-green algae. However, the absence of a nucleus and circular arrangement of the DNA throughout the cell place them firmly within the Subkingdom Prokaryota. The cyanobacteria are therefore closely related to the bacteria (Division Schizonta) but are differentiated from the Schizonta by their ability to photosynthesize and their utilization of chlorophyll a. These features link the cyanobacteria to both the bacteria and the algae. The occurrence of accessory pigments such as phycobilins (red) and carotenoids (orange, yellow) in addition to the green chlorophyll a generally impart a blue-green appearance on the cells. Pigment percentages and distributions may also in rare instances produce species that rang from dirty yellow to red, or dark brown to black. Whatever their color, cyanobacteria are photosynthetic, and so can manufacture their own food. Similar to other bacteria the cyanos are ubiquitous due to their small size and resistant prokaryotic cellular organization. Explosive growths, mats or blooms are frequently found in soil and aquatic environments and some cyanobacteria may be toxic (Anabaena, Lyngbya and Nostoc) while others are used in nutrient supplements (Spirulina). Unlike other bacteria or algae the cyanobacteria are one of very few groups of organisms that can convert inert atmospheric nitrogen into an organic form, such as nitrate or ammonia. It is these "fixed" forms of nitrogen that plants need for their growth, and must obtain from the environment. Nitrification cannot occur in the presence of oxygen, so nitrogen is fixed in specialized cells called heterocysts. These cells have an especially thickened wall that contains an anaerobic environment. Diversity of morphology may be seen among the 150 extant genera, though this diversity is reasonable subtle compared to other organisms. Cyanobacteria may be uni-cellular or colonial and both versions may show a covering of mucilage. Colonial cyanobacteria may be filamentous (simple, mixed and branched) and in rare occasions form irregular or cuboidal cell aggregates and in same cases plates or sheets. CAUTION Cyanobacteria are very sensitive to pollutants and environmental changes. Therefore, you will have to use water from the culture tube for all wet mounts. I. Cyanobacteria in gelatinous sheath 1. Gloeocapsa a few-celled colonial cyanobacterium occurring in either single (rarely) or clustered cells enclosed in concentric layers of mucilage. Typically, clusters of two to four are seen. These organisms are predominantly terrestrial though there are also aquatic species (Gloeocapsa crepidinum). 1

2 A. Prepared slide of Gloeocapsa observe on dissecting microscope first in order to find location of the cell aggregations (blue matrix due to stain utilized in slide preparation). Once you have found the cell aggregates general location transfer the slide to a compound microscope and observe at 4, 10 and 40x ocular magnification. Draw several aggregates and label the sheath and protoplasm. How are they distinguished from each other? (consider delineation, color and appearance) The chromoplasm contains the photosynthetic components, the centroplasm contains the genetic material. Base on this information can you discern the chromoplasm and centroplasm? If so, how? B. Pregare a Gloeocapsa wet mount using a drop of cultured water (containing the cyanobacteria) on a glass slide. Cover carefully with a coverslip to avoid air bubbles (presence of air bubbles will make it very hard to see these small organisms). Repeat the viewing sequence explained in A and note the differences between the live and preserved organisms. What color is the live Gloeocapsa? Why the difference in color between live and preserved materials? Can you still distinguish sheath from protoplasm? How? What is the reason for being able to differentiate the difference? Figure 1: Gloeocapsa (from left to right) live und comound microscope, schematic drawing showing the coats of mucilage and und the DIC microscope to distinguish firm, inner mucilaginous sheath surrounded by older sheath material from the parent cell, revealing the pattern of cell division. The sheaths are colorless or vivid shades of yellow, brown, red, orange, blue, or violet, and may be affected by changes in ph. 2. Chroococcus - Unicellular to colonial. Colonies usually microscopic, only few-celled, more or less spherical, rarely many-celled, forming macroscopic, gelatinous mats; colonial slime fine, diffluent, homogeneous and colorless or lamellated, limited and (rarely) colored; around gathered cells special own envelopes, usually copying the shape of cells, homogeneous or lamellated (up to rich lamellated) [subg. Chroococcus], or the old envelopes diffluent and the cells remain distant from one another [subg. Limnococcus]. Cells usually hemispherical to irregular (often in a form of a sector of a sphere), with homogeneous or granular content, sometimes with several prominent granules visible in light microscope. Widely distributed in freshwater and saline localities, mainly in metaphyton of varied bodies of water, also in 2

3 aerophytic, thermal and soil biotopes; a group of species lives in plankton of reservoirs (few species facultatively with aerotopes). Many species were incorrectly identified and misinterpreted repeatedly. The ecology is important for the correct determination. A. Prepared slides of Chroococcus. Observe the color and the pigments of Chroococcus and compare them to Gloeocapsa. What are differences and similarities? What is the difference in the gelatin sheath and the overall structure between Chroococcus and compared to Gloeocapsa? How can you distinguish between these two cyanobacteria? Can you distinguish organelles? If so which organelles are discernible? If not, why? Figure 2: Chroococcus (from left to right) live and schematic drawing. Observe the differences between Gloeocapsa and Chroococcus. 3. Coelosphaerium - Coelosphaerium comprises free-living, spherical or oval mucilaginous colonies, in which the cells are arranged irregularly in one marginal layer (but sometimes slightly shifted to one another), or near the surface of the sphere. Colonies sometimes are composed of subcolonies. Mucilage is colorless and homogeneous. Cells are spherical, pale or bright bluegreen, and 1-7 µm in diameter. One species has visible aerotopes. Cell division occurs in two planes in successive generations, perpendicular to one another and to the colony surface. Reproduction is by disintegration of a colonv and bv liberation of subcolonies. Colonies microscopic, more or less spherical or oval, sometimes composed of subcolonies, rarely irregular when old, free-living, usually floating in plankton, enveloped by fine, colourless, usually not sharply defined, sometimes indistinct mucilage; mucilage limited to just around the peripheral layer of cells or forming a gelatinous layer (margin) around cells. Prepared slide of Coelosphaerium. Observe the cell shape of Coelosphaerium. Is the mucilage of Gloeocapsa or Chroococcus. Which cyanobacteria do the cells most closely resemble? Are aerotropes present (and what are they)? What may be the use of aerotopes and what does that mean for the environment these cyanobacteria live in? 3

4 Figure 3: Coelosphaerium live (see the cell layering by using the focus) and a schematic diagram. 4. Lyngbya - Filamentous; filaments thick, rarely solitary, usually connected into thick, layered, sometimes leathery strata (mats) surrounded by a thin layer of mucilage. Usually macroscopic, to several cm in diameter, with firm sheaths, which are sometimes layered or stratified and brownish colored, opened at the ends. Unbranched or slightly falsely branched with short branches. Trichomes straight or slightly waved, thick (5.5-60µm), uniseriate, composed of shortly cylindrical or barrel-shaped discoid cells, unconstricted or constricted at the crosswalls, not attenuated or attenuated to the ends, nonmotile or slightly motile (but with motile hormogonia). Cells very short, always shorter (usually several times) than long, without or rarely with facultative aerotopes. Prepared slide of Lyngbya. Observe the trichomes of Lyngbya. Can you determine the mucilaginous sheath? Are aerotropes present? Which other cyanobacterium does Lyngbya most closey resemble? What are the main differences to that cyanobacterium? Are heterocysts present? What does that indicate? Figure 4: Lyngbya live trichomes (can you observe the mucin sheath?), overall general cell structure (cell shape?) and formation of hormogonia. 5. Merismopedia - Unicellular-colonial; colonies free floating, microscopic, usually flat or slightly waved, with cells situated in one plane, in rows more or less perpendicular one to another (sometimes 2 or 4 cells more or less in dublets or quadruplets). Colonies with 4-16 cells, but in several species to several hundreds of cells (up to 4000). The mucilaginous envelope of colonies is colorless, usually homogeneous, fine, diffluent at the margin. Cells sometimes with own, colorless mucilaginous envelopes. Cells spherical or widely oval, sometimes finely 4

5 granular or with visible chromatoplasm. In several planktonic species one or more aerotopes per cell present. Free floating in water or in metaphyton, detritus, among aquatic plants, usually in submerged biotopes with numerous water vegetation. Several small species are planktonic, several live in halophilic biotopes (seashores, saline swamps) or in thermal springs. Distribution worldwide, but several species have clearly ecologically and geographically limited areas of distribution. Prepared slide of Merismopedia Observe the prepared slides of Merismopedia available. Determine what cells are present (you should by now know what the use of these cells is and what that means to the lifestyle of the cyanobacterium you are examining). How is the structure of Merismopedia different from other cyanobacteria? What are similarities to other cyanobacteria? Figure 5: Merismopedia live, cell shape and overall detailed cellular arrangement. 6. Microcystis - Gelatinous, free floating or attached, microscopic, spherical, discoid or irregular, colonies with numerous cells without own mucilaginous envelopes, irregularly distributed within the common slime, sometimes densely agglomerated. The colonial mucilage is colorless, usually homogeneous, fine, diffluent, rarely with a limited margin. Cells spherical or (after division) hemispherical with several up to numerous aerotopes; sometimes with visible chromatoplasm. Several strains toxic. Free floating in plankton of freshwater and marine waters in more or less eutrophic water reservoirs (sometimes forming heavy water blooms). Several species live benthic in moors, epiphytic or epilithic in different types of stagnant and running waters and on wet rocks. Figure 6: Microcystis colonies and toxic bloom. 5

6 Prepared slide of Microcystis. Observe the cell structure, cell aggregation and mucin sheath. How do these compare to other cyanobacteria you have observed. 7. Nostoc filamentous, thallous cyanobacterium. The thallus may be micro- or macroscopic, gelatinous, amorph or spherical, later irregularly spherical, lobate, smooth or warty on the surface, filamentous or forming flat gelatinous or "paper-like" (when dry) colonies, usually with distinct periderm on the colonial surface. Filaments within colony irregularly coiled and loosely or densely agglomerated, sometimes more gathered in peripheral layer; sheaths around trichomes present, but visible usually only in the periphery of colony or in young colonies, wide, fine mucilaginous, confluent with colonial mucilage, sometimes yellowish-brown. Trichomes isopolar, of the same width along the whole length, apical cells morphologically not different from other cells; cells cylindrical, barrel-shaped up to almost spherical (forming moniliform trichomes; variability of cell-size and -shape sometimes distinct in one and the same species). Heterocytes solitary, develop in trichomes terminally or intercalary. (their frequency or absence is dependent on nitrogen metabolism); trichomes in principle metameric. Akinetes arise apoheterocytic, oval, little larger than cells; almost all cells between heterocytes change successively in akinetes towards heterocytes. Nostoc has special life (vegetation) cycle, during which forms several special and characteristic stages. A. Prepared slides of Nostoc. This slide contains filaments only, not globular colonial organisms. Draw and describe the structure, cell types (vegetative, heterocyst and akinete) and their functions of Nostoc filaments. Figure 7: Nostoc filaments with heterocysts (arrow), general cell differences and Nostoc call cultures. B. Live Nostoc filaments. Make a wet mount and compare the structure and composition of these filaments to Anabaena. What are similarities? What are differences? What do these tell you about the age, state and function of these colonies? C. Live Nostoc ball cultures. If the balls are large enough, observe under a dissecting microscope first. Can you discern the filaments inside? Allow one of the balls to sit outside water for one hour. How has the ball changed? What is its appearance? 6

7 Make a wet mount and observe using the compound microscope at increasing magnifications. What is the cell composition of the filaments? What does that tell you about the functionality? II. Straight trichomes 1. Arthrospira - Unbranched, solitary and free-living (floating) cyanobacterial spiral trichomes, also in mats which are microscopic or macroscopic, covering the substrate, more or less regularly screw-like and freely coiled along the whole length, with unchanged or continually changing (decreasing or increasing) width of screws. Sheaths usually missing and trichomes composed of cylindrical cells, constricted or not at the visible crosswalls, not attenuated or slightly attenuated towards the ends, motile (rotation). Spirulina is a type of blue-green algae that is rich in protein (45-65% dry weight), vitamins, minerals, and carotenoids, antioxidants that can help protect cells from damage. It contains nutrients, including B complex vitamins, beta-carotene, vitamin E, manganese, zinc, copper, iron, selenium, and gamma linolenic acid (an essential fatty acid) and therefore provides a food source or nutritional supplement. A. Prepared Spirulina (Arthrospira) slide. What is the cell organization of Spirulina compared to Oscillatoria? Are the filaments constructed in a similar manner? Are the cells of Spirulina visually smaller or larger than those of Oscillatoria (make sure that you observe them at the same magnification or else you cannot compare approximate sizes)? What does cell size have to do with filament shape? B. Prepare a wet mount of Spirulina from a seawater culture and prepare a sketch. Can you view the cellular cross walls at all magnifications? If not, what magnification is required to view them? What are the structural similarities to Oscillatoria? Name all that may apply.what are visual differences between Spirulina and Oscillatoria? Observe the live specimen for 5 minutes. Is there another biological phenomenon that closely associates Spriulina with Oscillatoria? Figure 8: Arthrospira and Oscillatoria. 2. Oscillatoria Genus of blue-green algae most common in freshwater environments with some marine species (generally genus Trichodesmium). This unbranched filamentous alga, occurring singly or in tangled mats particularly found in polluted environments, derives its name from its motion, which is thought to result from a secretion of mucilage that pushes the filament away from the direction of excretion. Reproduction is by fragmentation in which dead concave cells (separation disks) separate sections of the filament (hormogonia). When present, the mucilage 7

8 sheath is very thin. A. Prepared Oscillatoria slide. Observe at increasing magnifications (up to 40X occular magnification). Draw the outline of a filament. How are the cells in a filament arranged? Are all cells equally colored? Oscillatoria reproduces asexually through a process called hormogonia (trichome becomes detached, glides away to form a new filament), is the coloration of the cells representative of this process, if so how? What about the cell arrangements within a filament? What are the shapes of the cells? What do you think their cross section will look like? Oscillatoria may also show asexual reproduction through fission. Observe cells at the tip of a trichome? Do they differ from the rest of the cells? If so, how? B. Observe the form, true color and movement (very slow) of Oscillatoria in a water culture. Prepare a wet mount (see procedure above for Gloeocapsa). Make your observations at 10x ocular magnification. Use a ruler to determine the diameter of the field of view at 10x ocular magnification. Examine and identify the millimeter markings along one edge of the ruler. Place the ruler on the stage so that it covers half of the stage opening. While on Low power (10x), look through the eyepiece. Focus on the edge of the ruler using the coarse adjustment. Adjust the position of the ruler so that the view starts with a ruler line. Count the squares across one field of view. One millimeter is the distance from the middle of one line to the middle of the next mark. Remember, 1mm = 1000 micrometers. Sketch the outlines of the trichomes (unbranched filaments) at time 0 and 5 minutes in the field of view. Have the filaments moved? If so what is their rate of movement? 3. Aphanizomenon - Filaments free-floating, solitary, or joint laterally, microscopic or macroscopic (up to 2 cm long) colonies with trichomes oriented in parallel; trichomes straight, slightly curved or slightly coiled, cylindrical or sharply or continually tapering to the ends. Cells at the ends, with the apical cells long, cylindrical and hyaline (at the end rounded or obtuse), or intensely elongated, narrowed, vacuolized up to hyaline and sharply or bluntly pointed, or conical. Trichomes with intercalary developed heterocytes, which are localized shifted to one end of a trichome. Vegetative cells cylindrical or barrel-shaped, pale blue-green or blue-green, usually with aerotopes,. Heterocytes barrel-shaped or cylindrical with rounded or obtuse ends, which can completely disappear under special conditions (under high N-content). Akinetes widely oval, usually oval to long, cylindrical with rounded ends, solitary or in short rows (in 2 or 3), close to heterocytes or slightly distant from them, usually in asymmetrical position in a trichome. Almost all species are planktonic, sometimes forming characteristic water blooms. Figure 9: Aphanizomenon colonies schematic drawing and picture. Do you notice and can you distinguish akinetes and heterocysts? 8

9 Prepared slides of Aphanizomenon.Observe the different cell types and compare and contrast cyanobacterium to other cyanobacteria examined. 4. Gloeotrichia - Filamentous - colonial; trichomes heteropolar with basal heterocytes and apical hair-like ends with own sheaths, united radially into gelatinous, hemispherical or spherical colonies, which are microscopic up to several cm in diameter, olive green, yellow-green, brown or dark blue-blackish. The whole colony enveloped by a fine or firm slime; trichomes always oriented with heterocytes into the centre of the colony. Trichomes rarely false branched (during trichome division), the branches separate soon from the mother trichome, but remain parallel and radially located within the colonial slime and form their own gelatinous sheaths. Colonies are joined to the substrate or free floating. Trichomes uniserial, rarely with intercalar heterocytes (developing before or during trichome disintegration), constricted or unconstricted at the cross walls, more or less straight or coiled. Sheaths are always present, but sometimes gelatinize within the mucilage of colonies, especially near apical parts of trichomes (near the margin of a colony). A. Prepared slides of Gloeotrichia. Observe the filamentous colony on the slide. Find the basal heterocytes. What are their functions? Observe the apical hair-like ends. Are they enclosed in their own sheath or lacking mucilage? Do you observe any branching? B. Make a wet mount of a Gloeotrichia culture. After observing the true color, stain the colony with Azure B by removing the water from the slide (place Kimwipe to the edge of the coverslip to draw water out) and replacing it with Azure B (add a drop close to one side of a coverslip and hold a Kimwipe to the opposite end to pull the liquid across the slide without removing the cover) What is the true color of the colony? Are the filaments oriented parallel to one another or is there a different type of orientation? Is the colony mucilage covered or naked? How can you tell? Figure 10: Gloeotrichia trichomes and trichome arrangement. 5. Anabaena a differentiated, filamentous genus of nitrogen-fixing blue-green algae with beadlike or barrel-like cells and interspersed enlarged spores (heterocysts), found as plankton in 9

10 shallow water and on moist soil. There are both solitary and colonial forms, the latter resembling a closely related genus, Nostoc. In temperate latitudes during the summer months, Anabaena may form water blooms and can be used as an indicator of pollution state of an aquatic habitat. Anabaena trichomes resemble a pearl necklace with the vegetative cells of similar size. In addition, you may find larger, empty looking cells, heterocysts, may be visible. If the colony you are looking at is aging you may also find another cell type, the akinetes, which are larger than the vegetative cells and yellow to reddish in color. Prepare a wet mount of Anabaena. What are similarities between Anabaena, Oscillatoria and Spirulina. What are differences? Find a heterocyst on the colony and compare this cell type to the vegetative cells. What is the difference between these two cell types? Does their function and therefore their cellular requirements explain that difference? How? In your specimen, which cells are more frequent? Heterocysts or akinetes? What does this tell you about the age and functionality of the culture? View representative pictures of Anabaena akinetes. How can you explain the color of the cells? What is the function of akinetes? What does that tell you about the environmental conditions for the organism in the picture? 6. Anabaenopsis Filamentous cyanobacterium with solitary filaments or in free clusters with several tangled trichomes, free floating, without (rarely) or with constrictions at the cross walls, straight, arcuated or irregularly screw-like or spirally coiled, without sheaths, but sometimes with very diffluent, colourless, homogeneous slimy envelopes. The trichomes are originally metameric (the heterocytes develop intercalary in pairs in certain distances one from another), but trichomes often disintegrate soon after heterocyte formation between heterocytes, the position of which is finally terminal in short disintegrated fragments of an original trichome (usual stage of population). Cells cylindrical or barrel-shaped, shorter up to several times longer than wide, pale blue-green, usually containing aerotopes facultatively missing). Heterocytes develop after the asymmetrical, mirror-like division of two neighbouring proheterocytic cells, spherical or widely oval, rarely ovoid or elongated, rounded conical, usually slightly greater than vegetative cells. Akinetes spherical or oval, solitary or several in a short row, intercalar (rarely occur terminal after disintegration of trichomes), arise always paraheterocytic, but usually a slightly distant from heterocytes. Cells divide cross-wise, sometimes asymmetrically, and grow to the original size before the next division; all cells capable to divide; without meristematic zones. Reproduction by trichome fragmentation and by akinetes. Prepared slides What are similarities between Anabaenopsis and Anabaena. What are differences? Find a heterocyst on the colony and compare this cell type to the vegetative cells. What is the difference between these two cell types? Does their function and therefore their cellular requirements explain that difference? How? In your specimen, which cells are more frequent? Heterocysts or akinetes? What does this tell you about the age and functionality of the culture? Did segmentation of the specimen occur? If so, what is the reason for this fragmentation? 7. Scytonema filamentous, thallous cyanobacterium forming solitary branched filaments or 10

11 mats on the substrate. Filaments free or in fascicles, sometimes densely coiled, creeping on the substrate or with erected branches, commonly falsely branched, with one or (obligatory) two lateral branches. Branching initiates after trichome disintegration by help of necridic cells between two heterocytes, usually not at heterocytes; both branches grow parallel, aside or in crossing position; the filaments make sometimes typical loop-like lateral formations before branching, in which tops of the trichomes later divide. Trichomes isopolar, cylindrical, not diversified in basal and apical parts, uniseriate, usually with solitary heterocytes, constricted (rarely not constricted) at cross walls; terminal parts of branches cylindrical or slightly widened, with rounded apical cell; middle parts of trichomes sometimes with elongated, cylindrical cells. Sheaths firm, limited, parallel or diverging, lamellate, usually yellow-brown (colored by scytonemin) in some parts. Cells pale or olive-green, usually with solitary, irregularly disposed granules or with granular content, rarely yellowish or pinkish colored; apical cells sometimes with large vacuoles. Heterocytes intercalar, solitary, rarely in pairs, cylindrical or barrel-shaped. Akinetes were several times mentioned, but not proved and well described. Prepared slide of Scytonema. How are the cells compared to Anabaena, Spirulina and Oscillatoria? Are the filaments straight, helical or in part falsely branched? Do you find heterocysts? If so, how does the structure of the heterocysts compare to those of Anabaena? What does that tell you about the overall shape of the heterocysts in different species? (View also pictures provided in class to answer this question) Figure 11: Scytonema and Stigonema. 8. Stigonema - filamentous thallous cyanobacterium; thallus wooly or crusty, composed from free, coiled, true branched filaments, usually attached to the substrate, not diversified distinctly in basal filaments and branches. Trichomes two or multiseriate (only in young trichomes and at ends of branches uniseriate), sometimes very thick, irregularly laterally true branched (T- and V- type of branching), irregularly coiled, sometimes narrowed (with less number of cell rows) towards the ends, apical cell is sometimes smaller than the other ones. Sheaths thin or thick, limited, later wide, lamellate and usually yellowish-brown; around cells in old parts of filaments sometimes special envelopes (trichomes disintegrating in separated cells within filaments). Cells barrel-shaped or roundly irregular, connected usually by one pore ("pit connections") one with another, which disappear in some segments of trichomes; cell content blue-green or olive green, usually with prominent solitary granules. Heterocytes intercalar, rarely lateral, in a similar form as neighboring vegetative cells. Akinetes not known. Sometimes chroococcoid cell clusters arise. 11

12 Prepared slide of Stigonema. Are the cells similar or different compared to the other thallous form (Scytonema)? If so, how? How do the filaments differ compared to Scytonema. Are heterocysts present or absent? What does that tell you about the abilities of the organisms. 9. Rivularia - heteropolar filaments, differentiated into basal and apical parts, simple, joined parallel into firm strata. At the beginning of the vegetation cycle hemispherical or spherical, later sometimes vast, flat, macroscopic, irregular strata, up to several cm or dm in diameter and several mm thick strata are layered (sometimes with several layers), with densely agglomerated trichomes, oriented by their bases with heterocytes to the substrate and by the apical hair-like parts to the surface of the colony. Strata are gelatinous up to leathery or intensely incrusted by calcium carbonate, always covering the substrate. Trichomes +/- cylindrical, constricted or unconstricted at the cross walls, dividing at intercalar heterocyst: the divided trichomes separate one from another, but remain parallel located within the mother sheaths in the colony. Hairs are composed from the narrow, long, hyaline cells. Sheaths firm, sometimes lamellate, colorless or yellow-brown, enveloping all the "daughter" trichomes. View prepared slides. View the slides and compare the cell structure, shape and composition (i.e. what cell types) to the structure of Anabaena, Spirulina and Oscillatoria. What distinguishes this colony from the shape and form of the other colonies? Figure 12: Rivularia colonies, trichome arrangement and trichome structure. 12