Portions of this exercise have been adapted from Introductory Experiments in Cell Biology by K. Fleiszar and B. Wallace, Hunter Textbooks Inc., and Biology in the Laboratory by D. Helms, C. Helms, R. Kosinski and J. Cummings, Freeman and by LibreTexts licensed under CC BY-SA.
INTRODUCTION
The microbial world encompasses most of the phylogenetic diversity on Earth, as all Bacteria, all Archaea, and most lineages of the Eukarya are microorganisms. Microbes live in every kind of habitat (terrestrial, aquatic, atmospheric, or living host) and their presence invariably affects the environment in which they grow. Their diversity enables them to thrive in extremely cold or extremely hot environments. Their diversity also makes them tolerant of many other conditions, such as limited water availability, high salt content, and low oxygen levels.
Living things fall into three large groups: Archaea, Bacteria, and Eukarya. The first two have prokaryotic cells, and the third contains all eukaryotes. A relatively sparse fossil record is available to help discern what the first members of each of these lineages looked like, so it is possible that all the events that led to the last common ancestor of extant eukaryotes will remain unknown. However, comparative biology of extant organisms and the limited fossil record provide some insight into the history of Eukarya.
The earliest fossils found appear to be Bacteria, most likely cyanobacteria. They are about 3.5 billion years old and are recognizable because of their relatively complex structure and, for prokaryotes, relatively large cells. Most other prokaryotes have small cells, 1 or 2 µm in size, and would be difficult to pick out as fossils. Most living eukaryotes have cells measuring 10 µm or greater. Structures this size, which might be fossils, appear in the geological record about 2.1 billion years ago.
This lab will look at morphological differences between the domains Bacteria and Eukarya. Review the phylogenetic tree below and notice the evolutionary relationship between these domains.
Figure 1: In the evolution of life on Earth, the three domains of life—Archaea, Bacteria, and Eukarya—branch from a single point. (credit: modification of work by Eric Gaba)
The scientist has a variety of laboratory equipment which allows him to “see” what could not normally be observed with the naked eye. In this lab, your will be comparing the morphology of bacteria, protists and fungi. To view examples of this domain, we will use a microscope.
Part 1 of this lab will introduce you to the microscope.
In Part 2-4 of this lab, you will use the microscope to observe prokaryotic cells, protists and fungi and make some general observations about them.
PART 1 – The Compound Light Microscope
The compound light microscope (Fig. 2) must be used if you wish to observe smaller and thinner specimens. This type of microscope has much greater powers of magnification than the dissecting scope. The name of this microscope is derived from the fact that it utilizes two (i.e. compound) optical components and uses light as its source of illumination. Plug in the scope and turn on the light. The optical components are the ocular (eyepiece) and objective lenses. There are usually two – four objective lenses projecting from the revolving nosepiece. Each objective has a different power of magnification (indicated on the side of the objective; see Fig. 2). The LOW POWER OBJECTIVE is the shorter of the two and magnifies objects 4 to 10 times (4X or 10X). The other objective(s) is a higher power objective and will magnify 40, 43 or 45X. An oil- immersion objective may also be present (100 X). This objective is only used with oil. Since the ocular lens is located at the very top of the microscope and the objective lenses are located just above the stage, the object being observed is magnified first by the objective lens and then this
image is magnified ten times more by the ocular lens. Thus, if a 10 X low power objective is being used, the total magnification of an object is 10 x 10 or 100X. Magnification powers of 2,000 X are possible with more sophisticated compound light microscopes.
Figure 2: Compound light microscope.
Calculate the total magnification if you are using the highest power objective of your microscope:
In addition to the ocular lens and objective lenses, the optical system includes the light source (necessary to illuminate the specimen), condenser (which contains a system of lenses that focuses the light on the specimen), an iris diaphragm (which is used to adjust the amount of light striking the specimen), and the body tube (which can be rotated on some microscopes). Some microscopes do not have a condenser. Others have a movable or a fixed condenser. If your microscope is equipped with a movable condenser, locate the knob that raises and lowers the condenser and add it to the diagram (identify it as such). The mechanical system consists of the stage, a course adjustment knob (used for initial focusing of specimens under low power), a fine adjustment knob (used for precision focusing at higher power), stage clips (for holding a specimen in place), base and arm (to allow easy carrying). You will be expected to locate the optical and mechanical parts of the compound microscope and discuss the function of each part.
IT IS IMPERATIVE THAT YOU LEARN AND ALWAYS PRACTICE THE FOLLOWING PROCEDURE FOR USING THE COMPOUND LIGHT MICROSCOPE:
Use cotton swabs and lens cleaner to clean the ocular and objective lenses before and after use (if necessary). Do not use paper towels, Kleenex tissue, cloth, etc. Using something other than cotton swabs could smudge or damage the lenses.
Always carry the microscope in an upright position. Use one hand to grasp the arm of the microscope; use the other to support the base. The eyepiece (ocular lens) slides into the body tube in many brands and could fall out if the microscope is tilted.
When you are finished with an observation, turn off the illuminator and rotate the low power objective into viewing position.
To observe a specimen:
Turn the illumination source on.
Move the stage downward to its lowest position with the coarse adjustment knob.
Place the microscope slide with specimen on the stage. Make sure the lowest power objective is in place (rotate the nosepiece until the low power objective “clicks” into place).
Adjust the light so that you have enough illumination but not too much. Excessive light can cause eye strain. As you increase magnification you will need to increase the intensity of the light.
If you wear glasses, remove them! Raise the stage to its highest position and, while looking through the ocular, scan slide until you see a region of color. If you have difficulty locating the specimen, use a systematic pattern to search the slide. Then, while looking through the ocular, slowly lower the stage until the object is in focus. Try to keep both eyes open. This will be less tiring for your eyes.
Use the fine adjustment knob to bring the specimen into sharp focus.
If necessary, readjust the amount of light with the light intensity control or iris diaphragm.
Then, and only then, can you observe the specimen with a higher objective.
Make sure to start on the lowest power objective lens and to focus the image at each magnification before advancing to the next one.
Part 2 - EXAMINING PROKARYOTIC CELLS
One of the fundamental features of life is that organisms are composed of many cells. The distinguishing feature prokaryotic and eukaryotic cells is an intracellular structure called the nucleus. The nucleus is a membrane- bound structure that encloses a cell’s genetic material (DNA). Prokaryotic cells lack a nucleus, and their DNA is only loosely confined to an area within the cell. Eukaryotic cells possess a nucleus.
Bacteria are single-cellular prokaryotic organisms. Present-day bacteria are extremely small (approximately 1 – 2 µm in diameter), and many are devoid of natural color. Morphologically, they are either round (cocci), rod-shaped (bacilli), or spiral-shaped (spirilla). They are often found in clusters or in chains. To view bacteria with the compound light microscope, the cells must be stained, and one must use an oil immersion lens (100X). Even then, not much more than their basic shapes will be visible.
Figure 3: Many prokaryotes fall into three basic categories based on their shape: (a) cocci, or spherical; (b) bacilli, or rod-shaped; and (c) spirilla, or spiral-shaped. (credit a: modification of work by Janice Haney Carr, Dr. Richard Facklam, CDC; credit c: modification of work by Dr. David Cox, CDC; scale-bar data from Matt Russell)
Observe a prepared slide of Escherich coli. Describe and draw the appearance (color, cell size, cell shape, etc.) of this organism using the 100X objective. NOTE: Instructor will set the 100X objective
Observe a prepared slide of Staphylococcus aureus. Describe and draw the appearance (color, cell size, cell shape, etc.) of this organism using the 100X objective. NOTE: Instructor will set the 100X objective
Observe a prepared slide of Spriilum volutans. Describe and draw the appearance (color, cell size, cell shape, etc.) of this organism using the 100X objective. NOTE: Instructor will set the 100X objective
Part 3 - EXAMINING PROTIST CELLS
Protists belong to the domain Eukarya and the kingdom Prostisa. There are over 100,000 described living species of protists, and it is unclear how many undescribed species may exist. Since many protists live as commensals or parasites in other organisms and these relationships are often species-specific, there is a huge potential for protist diversity that matches the diversity of hosts. As the catchall term for eukaryotic organisms that are not animal, plant, or fungi, it is not surprising that very few characteristics are common to all protists.
Photosynthetic protist are called algae. They can photosynthesize like land plants and contain chlorophyll. A model organism for the green algae is Spirogyra. Spirogyra is a unicellular green algae that grows in long, filamentous colonies, making it appear to be a multicellular organism. Even though it is technically unicellular, its colonial nature allows us to classify its life cycle as haplontic. In the haploid vegetative cells of the colony, the chloroplasts are arranged in spirals, containing darkened regions called pyrenoids where carbon fixation happens. Each haploid cell in the filament is an individual, which makes sexual reproduction between colonies an interesting process.
When two colonies of Spirogyra meet that are of a complementary mating type (+/-), sexual reproduction occurs. The two colonies align, each cell across from a complementary cell on the other filament. A conjugation tube extends from each cell in one colony, inducing formation of a tube on the cells in the other colony. The conjugation tubes from each colony fuse together.
Figure 4: Spirogyra conjugation
1. Obtain a prepared of spirogyra. Describe and draw the appearance (color, cell size, cell shape, etc.) of this organism using the 40X objective.
Volvox is a green alga as well that is motile because of the cells that make up the colony have flagella. Each cell in the colony is connected by delicate cytoplasmic extensions. Volvox can reproduce both asexually and sexually. The cells in an adult colony will have a denser circular area known as daughter colonies which is a means of asexual reproduction. Daughter colonies can separate from the parental colony by means of enzymes to form a new volvox colony.
2. Obtain a prepared of Volvox. Describe and draw the appearance (color, cell size, cell shape, etc.) of this organism using the 40X objective.
Diatoms are another photosynthetic lineage that was derived from the secondary endosymbiosis of the red alga. Diatoms are an incredibly diverse group of unicellular organisms containing anywhere from 20,000 to 2 million species. These organisms are unicellular and surrounded by a frustule, a silica shell made from two distinct valves that enclose the plasma membrane. Frustules are amazingly intricate, covered with small pores in an arrangement specially adapted for capturing sunlight (Figure 5). They have golden chloroplasts due to the carotenoid pigment fucoxanthin (Figure 6).
Figure 5: A diatom filled with golden organelles (chloroplasts). Photo by Vicente Franch Meneu, CC-BY-NC.
Figure 6: An Isthmia nervosa frustule showing the intricate pattern of pores. There appear to be multiple layers with a different pattern of pores to each. Photos by Lama Mark Webber, CC-BY-NC.
We are still trying to figure out how to determine what a diatom "species" is and, so far, they have been classified based on the morphology of their frustules. Using this classification, historically there were two major groups of diatoms: centric (have radial symmetry) and pennate (have bilateral symmetry). In addition to morphology, diatoms can also be classified by where they occur. Free-floating diatoms are planktonic. Diatoms attached to other organisms (like giant kelp) are epiphytic. Benthic diatoms tend to dwell toward the bottom of a body of water.
Figure 7: These images show centric diatoms. You can draw several lines of symmetry through each of these organisms. Centric is still a morphological description used for diatom genera. First: Triceratium, photo by Ryan Watson, CC-BY. Second: Arachnoidiscus ehrenbergii found on Ulva, photo by Randall, CC0.
3. Obtain a prepared of diatom. Describe and draw the appearance (color, cell size, cell shape, etc.) of this organism using the 40X objective.
Protists and Human Disease: Most protist diseases in humans are caused by animal-like protists, or protozoa. Protozoa make us sick when they become human parasites. Three examples of parasitic protozoa are described below.
A. Trypanosoma Protozoa
Members of the genus Trypanosoma are flagellate protozoa that cause sleeping sickness, which is common in Africa. They also cause Chagas disease, which is common in South America. The parasites are spread by insect vectors. The vector for Chagas disease is shown in Figure below. Trypanosoma parasites enter a person’s blood when the vector bites. Then they spread to other tissues and organs. The diseases may be fatal without medical treatment. In Chagas disease, the Trypanosoma parasite is spread by an insect commonly called the “kissing bug.” A bite from this bug could be the kiss of death.
B. Plasmodium Protozoa
Plasmodium protozoa cause malaria. The parasites are spread by a mosquito vector. Parasites enter a host’s blood through the bite of an infected mosquito. The parasites infect the host’s red blood cells, causing symptoms such as fever, joint pain, anemia, and fatigue. Malaria is common in tropical and subtropical climates throughout the world (see Figure below). In fact, malaria is one of the most common infectious diseases on the planet. Malaria is also a very serious disease. It kills several million people each year, most of them children. A vaccine to malaria is a possibility.
Figure 8: Worldwide Distribution of Malaria. This map shows where malaria is found. The area is determined by the mosquito vector. The mosquito can live year-round only in the red-shaded areas.
4. Obtain 1 slide from examples A and B above. Describe and draw the appearance (color, cell size, cell shape, etc.) of this organism using the 40X objective.
Trypanosoma Protozoa
Plasmodium Protozoa
Part 4 - EXAMINING FUNGAL CELLS
Figure 9 : The poisonous Amanita muscaria is native to temperate and boreal regions of North America. (credit: Christine Majul)
Figure 10: Comparison of hyphae verses mycelium Credit: https://thealevelbiologist.co.uk/kingdoms-prokaryotae-protoctista-plantae-fungi-animalia/
Fungi are eukaryotes, and as such, have a complex cellular organization. As eukaryotes, fungal cells contain a membrane-bound nucleus. Unlike plant cells, fungal cells do not have chloroplasts or chlorophyll. Many fungi display bright colors arising from other cellular pigments, ranging from red to green to black. Most fungi are multicellular organisms. They display two distinct morphological stages: the vegetative and reproductive. The vegetative stage consists of a tangle of slender thread-like structures called hyphae (singular, hypha), whereas the reproductive stage can be more conspicuous. The mass of hyphae is a mycelium. It can grow on a surface, in soil or decaying material, in a liquid, or even on living tissue. Although individual hyphae must be observed under a microscope, the mycelium of a fungus can be very large, with some species truly being “the fungus humongous.” The giant Armillaria solidipes (honey mushroom) is considered the largest organism on Earth, spreading across more than 2,000 acres of underground soil in eastern Oregon; it is estimated to be at least 2,400 years old.
1. Obtain a prepared slide of Penicillium. Describe and draw the appearance (color, cell size, cell shape, etc.) of this organism using the 40X objective.
2. Obtain a prepared slide of Rhizopus. Describe and draw the appearance (color, cell size, cell shape, etc.) of this organism using the 40X objective.
3. Obtain a prepared slide of Aspergillus. Describe and draw the appearance (color, cell size, cell shape, etc.) of this organism using the 40X objective.
Yeasts in general are unicellular fungi and in form and size very similar to bacteria. Like all fungi, they have a cell wall composed of chitin and possess a nucleus and other organelles, in particular, mitochondria. In many ways they represent fungi that have evolved to become 'bacteria-like' in their form and ecology. Baker's yeast is typical of yeasts in generally — they typically are roughly spherical and around 5 um in diameter. Brewer's (aka baker's yeast or commercial yeast), is the organism that is used to make bread rise and produce wine from the fruits of grape. It also is extremely important as a 'model organism' in biology. It was the first eukaryote to have its entire genome sequenced and studies using S. cervisiae have been highly significant in developing our understanding of meiosis, mitosis and cancer. The translation of the scientific name is: saccharo = sugar, myces = fungus, cerevisiae = beer, reflecting its ability to make beer out of sugar water. It accomplishes this feat in a manner that several organisms can, by carrying out a processes termed fermentation, an anaerobic respiration process that releases carbon dioxide while converting six-carbon sugars (glucose and/or fructose) into ethanol.
2. Obtain a prepared slide labeled yeast. Describe and draw the appearance (color, cell size, cell shape, etc.) of this organism using the 40X objective.