Lab 2 - Microscopy, Kingdoms Prokaryotae and Protoctista
Be sure you know how to operate and care for your microscope. Be sure to use only the lens cleaning paper provided to clean the lenses. Use “kimwipes” for everything else. Your instructor will review the parts and operation of the scope with you, and demonstrate the proper technique for making slides of living material (“wet mount” ).
Know the following parts of the scope: stage, stage clips, coarse focus, fine focus, ocular lens, objective lens, diaphragm, dissecting microscope, compound light microscope.
Know how to calculate the power using any combination of lenses.
Look at a slide of the letter “e”. Focus your scope until it is crisp and clear. Now take the slide off the stage and hold it up to the light. What do you see? The image is upside down and backwards (inversion).
Look at a slide of colored threads. Focus up and down through the threads at lower, then at higher powers. Notice how you pass up and down through the stacked threads. Can you determine the order the threads are stacked in? (It varies from slide to slide.) This phenomenon is called depth of field, and it changes at different powers.
Make a wet mount slide of diatomaceous earth. Use a tiny bit of material. Have your instructor check your work.
Bacteria are the oldest group of organisms on Earth. They are very simple, in their physical structure. Although they are generally similar to higher organisms in their basic organization, they differ radically from higher organisms in their metabolic chemistry. Different types of bacteria also differ radically from one another in their metabolic pathways. Bacteria may represent a range of early evolutionary experiments in cellular chemistry.
Bacteria are extremely small, about 1/1000 of a millimeter, and are the most abundant organisms on the planet. All bacteria are haploid. Bacteria reproduce by asexual reproduction. They simply divide into replicas of themselves, a process called binary fission. Some can undergo a rudimentary form of sexual reproduction known as conjugation, an exchange of genetic material. Bacteria are solitary organisms in the sense that they do not form true social groupings or colonies. They often stick together after fission, to form long chains or clumps. This is not true colonial organization, because the cells do not communicate or interact in any complex way. Some forms are motile, they swim by means of a rudimentary flagella. There are three basic types of bacteria that we can easily recognize: Bacillus (-i) = rod shaped; Coccus (-i) = spherical; Spirillum (-i) = spiral shaped
All bacteria are prokaryotes (pro=first, karyote=kernel, i.e. nucleus). All higher organisms are eukaryotes (eu = true). Prokaryotes are unicellular, lack a nuclear membrane around the nucleus, and lack cellular organelles that are bound by membranes (no chloroplasts, no mitochondria). Eukaryotes can be unicellular, but are usually multicellular, have a nucleus bounded by a nuclear membrane, and have cellular organelles bound by membranes (chloroplasts and mitochondria).
Both chloroplasts, which contain the photosynthetic machinery, and mitochondria, which produce energy for the cells, function as little autonomous and self-replicating units inside eukaryotic cells. The theory of endosymbiosis (endo = within, sym = same or shared, biosis = life) suggests that these organelles were actually free-living bacteria in the distant past, which were captured and ingested by larger bacterial cells. Instead of being digested, they somehow took up residence, providing cells with new energetic pathways, and providing the organelles with nourishment and a relatively safe shelter. So in a fundamental sense, every cell in the body of a higher eukaryotic organism like ourselves is itself a colonial organism, the heritage of an ancient confederation between different types of bacteria.
Bacteria have a rigid cell wall made of polysaccharides and amino acids, which protects them against mechanical and osmotic damage. Some bacteria have a second cell wall, consisting of polysaccharides and lipids. This second cell wall makes these species of bacteria especially resistant to antibiotics, so this group of bacteria contain some of the most dangerous disease causing organisms.
Bacteria get their energy in a variety of ways. Some bacteria are autotrophs, or “self feeders”. They produce their own energy from sunlight (photosynthetic), or from inorganic compounds (like Hydrogen Sulfide, H2S). Other bacteria are heterotrophs, (= fed by others), they use energy produced by other organisms. Autotrophic bacteria can be photosynthetic (use H2O) or chemosynthetic (use H2S instead of water as an electron source). Photosynthetic bacteria, especially the cyanobacteria, played a major role in creating our oxygen atmosphere. Over 90% of all carbon dioxide produced in nature comes from the metabolism of bacteria and fungi.
Bacteria are also of critical ecological importance, because they are at the base of many food chains. Both autotrophic and heterotrophic forms include species capable of nitrogen fixation. These nitrogen fixers can change atmospheric Nitrogen, N2, into a form that can be used by plants (NH3, Ammonia). Rhizobium is a common genus that forms nodules on the roots of legumes, like the common clover, alfalfa, and soybeans. Nitrogen fixation is essential for agricultural crops. and without special bacteria doing it for them, cows could not digest grass nor could termites digest wood. Bacteria can break down the cellulose found in the cell walls of plants. So bacteria do some very good things for the planetary ecosystem. Many of our common food products would not exist were it not for bacteria, foods such as yogurt, pickles and most types of cheeses.
Bacteria are also among the most dangerous organisms on planet Earth. Cholera, diphtheria, syphilis, botulism, strep throat, tetanus, scarlet fever, meningitis, toxic shock syndrome, dysentery, and bubonic plague, the Black Death-are only a few of the more memorable diseases caused by bacteria. And ironically, we also owe many of our most effective antibiotics to bacteria: streptomycin, aureomycin, and neomycin, to name a few.
Subkingdom Archaebacteria - methanogens, halophilic bacteria, thermophilic bacteria
Subkingdom Eubacteria - cyanobacteria, Nostoc, Anabena, Oscillatoria
Domain Archaea - Archaebacteria
Domain Bacteria - Eubacteria
Domain Eukarya - Everything else
Bacteria can be divided into two subkingdoms, now given by some bacteriologists the status of separate kingdoms, the Archaebacteria and the Eubacteria. The Archaebacteria are the most primitive forms. We now realize that Archaebacteria may be as distantly related to other bacteria as bacteria are to the modern eukaryotes. Some authorities have therefore proposed a new rank called Domain, a rank higher than Kingdom. In this system, there are three domains, Archaea, Bacteria, and Eukaryota
All bacteria that are not archaebacteria are Eubacteria. The Eubacteria contain an amazing diversity of species, including several multicellular forms. The cyanobacteria are an especially important and interesting group, and most of your lab work on bacteria will focus on these photosynthetic forms. There are several thousand living species. For about 1900 million years (2500 mya to 600 mya) cyanobacteria dominated the earth’s ecosystems. (Nostoc, Anabaena, Oscillatoria). This group was formerly classified as a primitive type of algae, the “blue-green algae”, after their distinctive coloration. We now recognize them as a type of photosynthetic bacteria. Filamentous forms may have an enlarged structure called a heterocyst, in which nitrogen fixation takes place.
Only about half of the cyanobacteria actually show the strong blue-green color we associate with this group. They can actually come in many colors (red, yellow, purple, and brown). The red color of the Red Sea is due to the red pigment in a species of cyanobacteria. Some of the earliest fossils we have are of large stacks of roughly circular plates called stromatolites. These are composed of enormous colonies of bacteria going back about 2.7 billion years ago in the fossil record. Paleontologists believe that these large formations of cyanobacteria were very important early habitats for a variety of ancient organisms.
Examine bacterial slides of:
The three common shapes/types: coccus, bacillus, spirillum.
The symbiotic bacteria Rhizobium which reside in nodules on the roots of legumes like peas, peanuts and clover and fix nitrogen for the plant (preserved or live material may also be on display)
Make wet mount slides of the cyanobacteria Oscillatoria and Anabaena. Compare the living organism with the prepared slides.
Know the difference between prokaryotic and eukaryotic cells.
Know the difference between autotrophs and heterotrophs.
Understand the difference between binary fission and conjugation in bacteria and protists.
Remember what nitrogen fixation is and why it is important.
Many bacteria are pathogenic, like those that cause syphilis, botulism, strep throat, tetanus, scarlet fever, meningitis, toxic shock, dysentery, and bubonic plague.
Ironically, we also owe many of our most effective antibiotics to bacteria: streptomycin, aureomycin, and neomycin, to name a few.
Bacteria are the basis for most food chains. Most of the animals you will see in the next several weeks include bacteria in their diet. We use them to make cheese and yogurt.
Bacteria and fungi are the primary decomposers of dead organic matter, recycling materials on a planetary scale for other organisms to use.
Many bacteria, like Rhizobium, can perform nitrogen fixation, creating fertile soil for plants.
The Kingdom Protoctista includes an incredible diversity of different types of organisms, including algae, protozoans, and slime molds. No one even knows how many species there are, though estimates range between 65,000 to 200,000. (fr. Greek protos = first, ktistos = first established). All protists are eukaryotes, complex cells with nuclear membranes and organelles like mitochondria and chloroplasts. They can be either unicellular or multicellular, and in this group we find the first inkling of what is to come in evolutionary history, the union of eukaryotic cells into a colonial organism, where various cell types perform certain tasks, communicate with one another, and together function like a multicellular organism.
Some protists are autotrophs, a photosynthetic group of phyla referred to as the algae. Autotrophs manufacture their own energy by photosynthesis or chemosynthesis. Algae use various combinations of the major chlorophyll pigments, chlorophyll a, b, and c, mixed with a wide array of other pigments that give some of them very distinctive colors. Some protists are heterotrophs, a group of phyla called the protozoa. Heterotrophs get their energy by consuming other organisms. Protists reproduce asexually by binary fission, and a few species are capable of sexual reproduction. Many have very complex life cycles.
Protists are so small that they do not need any special organs to exchange gases or excrete wastes. They rely on simple diffusion, the passive movement of materials from an area of high concentration to an area of low concentration, to move gases and waste materials in and out of the cell. Diffusion results from the random motion of molecules (black and white marble analogy). This is a two-edged sword. They don’t need to invest energy in complex respiratory or excretory tissue. On the other hand, diffusion only works if you’re really small, so most protists are limited to being small single cells. Their small size is also due to the inability of cilia or flagella to provide enough energy to move a large cell through the water.
Protists eat by phagocytosis - they engulf their food in their cell membrane, and pinch off a section of membrane to form a hollow space inside the cell. This hollow space, now enclosed by membranes, is called a vacuole. Vacuoles are handy little structures. Protists also use them to store water, enzymes, and waste products. Paramecium and many other protists have a complex type called a contractile vacuole, which drains the cell of waste products and squirts them outside the cell.
All protists are aquatic. Many protists can move through the water by means of flagella, or cilia, or pseudopodia (= false feet). Cilia and flagella are tiny movable hairs. Motile cells usually have one or two long flagella, or numerous shorter cilia. The internal structure of cilia and flagella is basically the same. All of the characteristics that this group shares are primitive traits, a perilous thing to base any classification on, because convergent evolution may be responsible for these superficial similarities. So the concept of the Kingdom Protoctista has been justly criticized as a “taxonomic grab bag” for a whole bunch of primitive organisms only distantly related to one another.
Protists are mainly defined by what they are not - they are not bacteria or fungi, they are not plants or animals. Protists gave rise to all other plants and animals. But where did protists themselves come from? The earliest protists we can recognize in the fossil record date back to about 1 billion, 200 million years ago. We do not know how the various groups of protists are related to one another. We assume they arose from certain groups of bacteria, but which groups and when are still investigating. Different phyla of protists are so unlike one another, many probably evolved independently from completely different groups of bacteria. Lynn Margulis recognizes nearly 50 different phyla of protists, or Protista, as this kingdom was formerly called. We will take a more conservative approach, and focus on nine important phyla of protists.
Kingdom Protoctista (Protista)
Protozoa = heterotrophic protists:
Phylum Ciliophora - (Paramecium, Blepharisma)
Phylum Sarcodina - (Amoeba, foraminiferans)
Phylum Sporozoa - (Plasmodium - malaria)
Phylum Phaeophyta - brown algae (Fucus)
Phylum Rhodophyta - red algae (Polysiphonia)
Phylum Chrysophyta - diatoms
Phylum Euglenophyta - (Euglena)
Phylum Pyrrophyta - dinoflagellates (Ceratium)
Phylum Chlorophyta - green algae (Spirogyra, Volvox, Chlamydomonas)
Phylum Ciliophora (8,000 sp., fr. Latin cilium = eyelash, Greek phorein = to bear) - Blepharisma, Paramecium
These ciliates move by means of numerous small cilia. They are complex little critters, with lots of organelles and specialized structures. Many of them, like Paramecium, even have little toxic threads or darts that they can discharge to defend themselves. Typical ciliates you may see in lab include Paramecium and Blepharisma.
Phylum Sarcodina (over 300 sp.) - Amoeba, radiolaria, foraminifera
These ciliates have a most unusual way of getting about. They extend part their body in a certain direction, forming a pseudopod or false foot, and then flow into that extension (cytoplasmic streaming). Many forms have a tiny shell made from organic or inorganic material. They eat other protozoans, algae, and even tiny critters like rotifers. Amoeba is a typical member of this phylum. Many sarcodines are parasites, such as the species Entamoeba histolytica, which causes amoebic dysentery. 10 million Americans are infected at any one time with some form of parasitic amoeba, and up to half of the population in tropical countries. Somewhat more unusual sarcodines are the Foraminiferans. These “forams” can have fantastically sculptured shells, with prominent spines. They extend cytoplasmic “podia” out along these spines, which function in feeding and in swimming. Forams are so abundant in the fossil record, and have such distinctive shapes, that they are widely used by geologists as markers to identify different layers of rock. The Pyramids are constructed of limestone formed from the shells of billions of foraminiferans.
Phylum Sporozoa (3,900 sp.) - Plasmodium
This last group of protozoans is non-motile, and parasitic. They have very complex life cycles, involving intermediate hosts such as the mosquito. They form small resistant spores, small infective bodies that are passed from one host to the next. Plasmodium, the parasite that causes malaria, is typical of this group. In more general terms, spores are haploid reproductive cells that can develop directly into adults.
Phylum Phaeophyta (1,500 species, fr. Greek phaios = brown) - Fucus
This phylum contains the brown algae, such as Fucus (rockweed), Sargassum, and the various species of kelp. Brown algae are the largest protists, and are nearly all marine. Kelp blades can stretch up to 100 meters long. Brown algae have thin blades with a central midrib or stipe. Like all algae, their blades are thin because they lack the complex conductive tissues of green plants (phloem), and must rely on simple diffusion, though some kelp have phloem-like conducting cells in the midrib. Kelp form the basis of entire ecosystems off the coast of California and in other cool waters. In the “Sargasso Sea”, the Atlantic Ocean northeast of the Caribbean Islands, the brown algae Sargassum forms huge floating mats, said in older days to trap entire ships, holding them tight until the crew met a watery grave.
Phylum Rhodophyta (fr. Greek rhodos = red, 4,000 sp.) - Polysiphonia
Like brown algae, the red algae also contain complex forms, mostly marine, with elaborate life cycles. Chloroplasts in this group show pigments very similar to those found in cyanobacteria, and ancient red algae may have engulfed these cyanobacteria as endosymbionts. Red algae have many important commercial applications, such as the agar used for culture plates. Its cell walls contain carrageenan, a polysaccharide used in the manufacture of ice cream, paint, and cosmetics.
Phylum Bacillariophyta (11,500 sp., many more fossil sp., fr. Latin bacillus = little stick) - diatoms
Diatoms have a golden-brown pigment. Some books still place them with the Chrysophyta, the golden-brown algae, but they are now recognized as an entirely separate group. Diatoms have odd little shells made of organic compounds impregnated with silica. The shells fit over the top of one another like a little box. Diatoms usually reform the lower shell after they divide This means they become smaller and smaller, and when they become too small they leave their shells and fuse through sexual reproduction into a larger size and start over again. They are one of the most important organisms in both freshwater and marine food chains. Diatoms are so abundant that the photosynthesis of diatoms accounts for a large percentage of the oxygen added to the atmosphere each year from natural sources. Their dead shells form huge deposits, that are mined for commercial uses. Diatom shells are sold as diatomacious earth, and used in abrasives, talcs, and chalk. Diatoms are so numerous that their shells form thick deposits all over the world. A single quarry in Lompoc, California, yields over 270,000 metric tons per year. One bed in the Santa Monica Ca. oil fields is over 900 meters thick! Various species of diatoms are also widely used as indicator species of clean or polluted water.
Phylum Euglenophyta (800 sp.) - Euglena
Is it a plant, or is it an animal? It moves around like an animal, and sometimes eats particles of food, but a third of them are also photosynthetic, a nice bright green pigment like a green algae (which it used to be called). This organism may actually have resulted from endosymbiosis, in which an ancestral form engulfed a green algal cell.
Phylum Pyrrophyta (3,000 sp., fr. Greek dinos = whirling, Latin flagellum = whip) - dinoflagellates, Ceratium
Dinoflagellates are named after their two flagella, which lie along grooves, one like a belt and one like a tail. Many species have a heavy armor of cellulose plates, often encrusted with silica. This species is very important both ecologically and economically. Some species form zooxanthellae, dinoflagellates which have lost their flagella and armor, and live as symbionts in the tissues of mollusks, sea anemones, jellyfish, and corals. These dinoflagellates are responsible for the enormous productivity of coral reefs. They also limit coral reefs to surviving in shallow waters, where sunlight can reach the dinoflagellates. Some dinoflagellate species often form algal blooms in coastal waters, building up enormous populations visible from a great distance. The amazingly potent toxins, that about 20 species produce, poison shellfish, fish, and marine mammals, causing the deadly red tide. This is the organism that can make Louisiana oysters your last meal on Earth!! One outbreak in 1987 killed half of the entire bottlnose dolphin population in the Western Atlantic.
Phylum Chlorophyta (7,000 sp., fr. Greek chloros = yellow-green) - Volvox, Spirogyra, Chlamydomonas
Several multicellular organisms have arisen from this very diverse group of algae, including the unknown ancestor of all green plants. Like higher plants, they: use chlorophyll a and b for photosynthesis; have cell walls of cellulose and pectin; and store food as starch. There are several colonial forms, such as Volvox. Groups of cells unite to form a colonial organism, in which certain groups of cells perform certain tasks. It is one of the simplest organisms to show a true division of labor, true multicellularity. Volvox colonies can contain 500-60,000 vegetative cells. The colony has polarity, a head and tail end. It even has special reproductive cells concentrated at its tail end. The flagella that stick out from its surface cells moves the colony forward by causing it to spin clockwise. Volvox crosses a major evolutionary boundary. When Volvox reproduces, the new daughter colonies form inside the parent colony. The only way they can be released is for the parent colony to burst open and die. It is this final act of sacrifice that tells us an invisible line has been crossed. Single celled bacteria and protists are immortal. They can go on dividing in two forever, and so never truly die. But in the Kingdom Protoctista, we see the beginnings of specialization among groups of cells, specialization which entails the death of certain cells so that other cells can survive. As Volvox reminds us, the price of complex multicellularity is death.
Examine prepared slides of these protists: Amoeba, Foraminifera, Diatoms, Polysiphonia, Dinoflagellates
Make wet mount slides of these protists: Blepharisma, Euglena, Chlamydomonas, Spirogyra, Volvox
You should also look at the prepared slides of these live organisms (Paramecium is very similar to Blepharisma). You may get either one on your lab exams.
Hint: One drop of “detain” on top of your drop of water will slow down little critters like Euglena and Blepharisma to sub-light speed. You will need to use prepared slides and higher power lenses to view the fine structure of these organisms.
Protists are so small that they do not need any special organs to exchange gases or excrete wastes. They rely on simple diffusion, the passive movement of materials from an area of high concentration to an area of low concentration, to move gases and waste materials in and out of the cell.
Protists eat by phagocytosis - they engulf their food in their cell membrane, and pinch off a section of membrane to form a hollow space inside the cell. This hollow space, now enclosed by membranes, is called a vacuole.
Algae and protozoa are important prey in food chains. Even humans eat algae.
Many protozoans are important disease causing organisms (malaria,
Dinoflagellates cause billions of dollars in damage to the seafood industry, and are important symbionts in corals and other marine animals.
An extract of red algae is used to make paint, cosmetics, and ice cream.
Protozoans gave rise to all higher forms of animal life.
Green algae gave rise to all higher plant life.
Bacteria first mastered the fine art of photosynthesis. Cyanobacteria established the oxygen atmosphere we breathe today. But diatoms are mainly responsible for current oxygen input from photosynthesis).
How does size affect basic processes like respiration, ingestion, or excretion?
What role did endosymbiosis play in the early evolution of cells?
Why is Kingdom Protoctista usually considered an “artificial” classification?
Why is it never a good idea to classify organisms together on the basis of primitive traits?
What does the life cycle of Volvox tell us about division of labor and coloniality?
Kingdom Prokaryotae (Monera)
Cyanosite offers cyanobacteria researchers a small slice of heaven:
UCMP's introduction to cyanobacteria is available at:
Take a tour of the Microbe Zoo at the University of Michigan. Be sure to visit Dirtland:
Search the Microbial Underground for anything microbial and medical:
Kingdom Protoctista (Protista)
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