BIO1802_Ex 2 DLN_Spring2024

BioCORE Trainee Ex 2 Digital Lab Notebook BioCORE™ Lab Notebook Ex 2: Aquatic Biology and Microbial Biodiversity Name: —- Course: Bio 1802 Section: — Course Mentor: — Date: 2/6/24

BioCORE Trainee Ex 2 Digital Lab Notebook Table of Contents This lab notebook is to be used for the following pre-lab activities through AsULearn, virtual labs in Connect, and in-lab activities: Topic: Page # or Range: 1. Background research: 3-12 IncludedActivitiess: Prep Resources Pre-lab Reading: The Life that Springs from Dead Leaves In Springs Activity 1 Proper Microscope Technique, Cell Theory, and Prokaryotes Virtual Lab Primer: Proper Microscope Technique Activity 2 Domain Bacteria Activity 3 Domain Archaea Activity 4 Eukaryotes and Kingdom Protista Virtual Lab Primer: Pond Water Wet-mount 2. Field Work: 12- 17 IncludedActivitiess: Ex 2 In-Lab Activities Activity 1 Proper Microscope Technique, Cell Theory, and Prokaryotes Activity 2 Domain Bacteria Activity 3 Domain Archaea Activity 4 Eukaryotes and Kingdom Protista 3. Analysis of Observations and Research: 17-19 Comprehension Assessment 4. Reflections and Discussions 5. (Optional) Study Notes: 19-20

BioCORE Trainee Ex 2 Digital Lab Notebook 5. How might the food web shift, specifically between bacterial and fungal decomposition of leaves and the availability of decaying leaf matter to macroinvertebrate shredders and gathers, as global climate change warms areas of the planet? What global impact(s) might this have? Warmth increases the rate of microbial activity so bacteria and fungi may become more prominent consumers of leaves in warmer waters. This may leave less food for insects and the predators of insects. Pre-lab Activity: Tree of Life Flow Chart As you complete your background research, you will be prompted to fill out the following flow chart. To do so, go to “Insert” and then “Drawing” and then “+ New”.On the drawing page, you can choose to insert text. Choose this option and type the word you want to insert into the flowchart. Save and close. An option box will appear, choose the option for “In front of text” and then move your text box to the correct location on the flowchart. Prokaryotic Bacteria Archaea Eukarya Fungi Plantae Animalia Protista Activity 1 Background Information: Proper Microscope Technique, Cell Theory, and Prokaryotes Pre-lab Mini-Lecture Pre-lab Video: Proper Biological Drawing Technique of Microscopic Specimen 6. What shape is the field of view and why? What impact does this have on your observation and documentation of microscopic specimens? The field of view is circular because it’s easier to focus light and for magnification

BioCORE Trainee Ex 2 Digital Lab Notebook 7. When recording your observations of a microscopic view using proper drawing techniques, what important information about the field of view should you include in your notes and/or title? Figure name, Common and scientific name, the view, magnification,n, and small description Pre-lab Video: Movement of Prokaryotic Flagella Pre-lab Video: Use of Flagella for Various Movements Prokaryotes are a grouping of organisms that have a prokaryotic cell type. These organisms are very small (microscopic!), lack membrane-bound organelles, and are most similar to the first life forms that originated on Earth . Prokaryotes cannot be seen with the naked eye; to view a prokaryote, we must use a compound microscope (or a fancier microscope with higher magnification), and typically, we must use the highest objective lens for the greatest amount of magnification. Prokaryotes include two of the Domains of life: Domain Bacteria and Domain Archaea. Prokaryotes often have hair-like filaments that project out of the surface of their cells, called flagella. At the base of the flagella is a “motor”, which spins turning a long rod of protein (the filament of the flagella) allowing it to rotate much as a plane propeller would. Watch the provided pre-lab videos and note the general movement of a prokaryotic flagellum. Depending upon the number of flagella, the direction of the rotation (clockwise or counterclockwise), and the angle of the flagella, prokaryotes can accomplish a surprising variety of movements: twitching, tumbling, forward and backward swimming! Remember, this flagella type is used by prokaryotes, which includes both bacteria and archaea. 8. How do prokaryotic flagella use various movements and angles to produce the movements indicated below? Forward movement: counter-clockwise rotation Backward movement: clockwise rotation Change in direction: Flick→ changes cell orientation about 90 degrees Getting unstuck: Reverse corkscrew → flagellum wraps around cell so that cell can unscrew itself out if it’s stuck 9. Reference Figure 5 from the Ex 2 Handout. Describe the “mechanics” of a prokaryotic flagella. What are the basic structural components (see labels in figure)? Flagella helps cells move through an aquatic environment. Filament, Hook, motor, and rod 10. How are prokaryotic flagella attached to the cell? They’re anchored to the cell by a basal body. 11. Are prokaryotic flagella membrane-bound (is the filament covered by the cell membrane)?

BioCORE Trainee Ex 2 Digital Lab Notebook 14. Listed below are the metabolic tests listed in the Classification of Bacteria Using a Dichotomous Key video. Pick one to briefly describe and explain how it helps researchers classify bacteria. Oxygen tolerance? Only bacteria that tolerate oxygen will grow in a medium exposed to air. This growth is visible by cloudiness or turbidity Activity Resource: Important Lineages of Bacteria Note that important diagnostic features of each bacteria group/lineage are indicated by red boxes. Use these noted characteristics to fill in Table 2.1. Cell type refers to prokaryotic or eukaryotic. Table 2.1 Diagnostic Features of Lineages of Domain Bacteria Lineage Cell type: Diagnostic features: Mycoplasma Prokaryotic They lack a cell wall, are extremely small (0.1-0.3 micrometers), antibiotics are not effective against these infections, can be parasitic or saprophytic, cell shape is undefined, many are pathogens (agents of disease) like pneumonia, etc. Firmicutes Prokaryotic Important to gut flora (probiotic effect in yogurts), resistance to drying out (can exist in certain environments for extended times), some product harmful or helpful toxins, mainly cocci or bacillus shaped (collect into chains), disease-causing strains: anthrax, botulism, tetanus, etc. Cyanobacteria Photosynthetic Prokaryotic Forms long cellular chains, most abundant photosynthetic organism on earth (gave tons of oxygen to earth), endosymbiosis theory!!, also called blue/green algae, important to the nitrogen cycle, stain gram negative Actinobacteria Prokaryotic Decomposers of organic matter (help produce soil), a few are agents of disease, others produce chemicals that are sources of antibiotics, Stain-gram positive Spirochaetes Prokaryotic Extremely motile, important fermenters, agents of disease Lyme, and syphilis), play a major role in digestion for animals like cows or as symbionts for clams/mussels/oysters Chlamydiae Prokaryotic A small group of spherical, parasitic bacteria, Intracellular (must live inside another organism cells), very small size, Chlamydia, thin/difficult to detect cell wall: present, contains peptidoglycan, and stained gram-negative Bacteroidetes Prokaryotic Large/well studied, rod-shaped, good at degrading polymers (polysaccharides and proteins), mutualistic, non-endospore forming, all stain gram negative Proteobacteria Prokaryotic Large group and lots of diversity, Pathogens: E. coli, salmonella, etc. Others are free living and nonparasitic, a Large variety of lifestyles, grouped on a similarity of the

BioCORE Trainee Ex 2 Digital Lab Notebook nucleotide sequence in DNA, The Majority stain gram-negative *Fill in box B of the flowchart. Activity 3 Background Information: Domain Archaea Domain Archaea is a subset of prokaryotic organisms. They are prokaryotic cells with prokaryotic flagella, they have cell walls that do NOT include peptidoglycan, and the lipid bilayer of their cell membranes contains isoprenoid-based (not fatty acid) tails linked to the glycerol by ether linkages. Activity 3 Pre-lab Mini Lecture Pre-lab Video: Why Aren’t Archaea Pathogens? As a scientific community, we don’t know as much about Domain Archaea, but we have been able to determine that their phylogenetic relationships and cellular components are quite different from Domain Bacteria. Fill out the table below which compares these two seemingly similar, but vastly different, Domains. Cell type refers to prokaryotic or eukaryotic. Table 2.2 Characteristics of Bacteria versus Archaea Characteristics Domain Bacteria: Domain Archaea: Cell type: Prokaryotic Prokaryotic Phylogenetic relationships: Eukarya Cell membrane characteristics: DNA centralized in nucleoid region (not in an enclosed membrane)... exhibit cell membranes covered by cell walls covered by a layer of polysaccharides… lack organelles… DNA in nucleoid region (not enclosed in a membrane)... cell membranes by cell walls Cell wall characteristics: Have cell walls that contain peptidoglycan Have cell walls without peptidoglycan Cell size and morphologies: Single-celled but form biofilm colonies Single-celled but can live in biofilms Life strategies: short generation times allow for genetic recombination… ancient lineage Extremophiles (live in either low or high pHs, extreme pressures, aka extreme environments) Other notes: Tails of lipid bilayers are fatty acids… Pilus (hair-like) can facilitate the exchange of DNA Tails of lipid bilayers aren’t fatty acids… have isoprenoid-based

BioCORE Trainee Ex 2 Digital Lab Notebook 17. List the lineages of protists included in each subgroup: Unikonta: single-celled opisthokonta: choanoflagellates/fungi/ animals Bikonta: Excavata: Parabasalids/ Diplomonads/ Euglenoids/ Kinetoplastids Stramenopila is its lineage. 18. What is the difference between the subgroups of protists:uniokonta, bikonta, and group Stramenopila? Bikonta: parasitic, attached flagella runs the length of the body, photosynthetic Unikonta: Single flagellum at the base, possibly closest living relatives to animals, freshwater organisms, “animal-like” Stramenopila: contains flagella with hollow projections, both sexual and asexual reproduction, Mostly marine. 19. Convergent evolution is the process whereby distantly related organisms independently evolve similar traits to adapt to similar needs. Refer back to the How Flagella Impact Bacteria video and compare and contrast prokaryotic and eukaryotic flagella. Compare (find similarities between): Both eukaryotic and prokaryotic cells have flagella Contrast (find differences between): Mainly differ in structure and movement. Eukaryotic flagella are microtubule-based structures that are attached to the cell at the cell membrane through basal bodies while prokaryotic flagella are located outside of the plasma membrane. Eukaryotic are also arranged in a 9+1 arrangement whereas prokaryotic flagella consist of a filament having thousands of copies of flagellin 20. Note that important diagnostic features of each protist group/lineage are indicated by red boxes. Use these noted characteristics to fill in Table 2.3. Cell type refers to prokaryotic or eukaryotic. Table 2.3 Diagnostic Features of Lineages of Kingdom Protista Lineage Cell type: Diagnostic features Amoebozoa Prokaryotic Lack of cell walls, flexible/dynamic cell membranes, amoeboid movement with cytoplasmic streaming… common freshwater organisms… pseudopods (false feet)... considered animal-likee due to mobility… exhibit large, lobed pseudopods

BioCORE Trainee Ex 2 Digital Lab Notebook Opisthokonta Prokaryotic Representatives from animals, fungi: reproductive cells with a single flagellum at the base. A representative from protists: single-celled opisthokonts: choanoflagellates→ group of freeing living, unicellular, but colonially-living protists→ possibly closest living relatives to animals cells are ovoid with a collar a single flagellum at the bases→ relatives to these are important components of sponges Excavate Prokaryotic Excavated feeding groove on the side of the cell… most swim utilizing flagella… diplomonads have 2 nuclei… many euglenoids are photosynthetic… many lack classic mitochondria… most ancient basal group…pellicle= collection of protein strips under plasma membrane for structure Archaeplastida Prokaryotic Can be unicellular, colonial,l or multicellular… contain chloroplast… cell walls composed of cellulose… algae produced flagellated cells… all contain chloroplast descended from a common ancestor that engulfed cyanobacterium via endosymbiosis event… Rare, freshwater, microscopic algae… likely ancient lineage… Green algae= chlorophytes and charophytes… closely related to land plants… Rhizaria Prokaryotic Lack of cell walls but many have shell-like coverings… move by amoeboid motion via slender pseudopodia… shells of dead foraminiferans form extensive sediments on the bottom and can form limestone… shells normally made of calcium carbonate… mostly marine Alveolata Prokaryotic Ciliates, dinoflagellates, and apicomplexans… contain vesicles called alveoli that support the plasma membrane…

BioCORE Trainee Ex 2 Digital Lab Notebook amphibians to wading birds 2. Mountain bog: Big?- wet, spongy, poorly drained ground in which dead and or decaying plant material and vegetation has accumulated causing low oxygen, nutrient-poor acidic environments… mainly filled with precipitation… formed by naturally occurring depression in the ground that retains water… With saturated soil and dead/decaying matter of a bog… Bacteria and archaea, many of which are methanogens. The most abundant class of bacteria is peat bogs: alpha-proteobacteria… Few vertebrae permanently reside in snails due to lack of fish, snails, and mussels… Insect rich… brown/amber/reddish tint to water Activity 1: Proper Microscope Technique, Cell Theory, and Prokaryotes 23. How can we determine the diameter of the field of view? In what units of measure is it measured? By taking a ruler, placing it under the microscope, and counting the number of ticks seen. The units of measure are normally millimeters. 24. Following the instructions provided by your lab instructor, measure the diameter of the field of view for the microscopes you are working on within the lab. Diameter of the field of view under the 4x objective lens (in mm): 4 ½ Diameter of the field of view under the 10x objective lens (in mm): 2 Diameter of the field of view under the 4x objective lens (in mm): 0.5 25. Why do we always start by using the lowest power objective and working up to the highest power objective (if needed)? Hint: consider what you can see in the field of view under low power versus high. Depth of focus is greatest on the lowest power objective and each time you switch to a higher power depth of focus is reduced. The field of view is also higher on the lowest power objective 26. When is it okay to use the coarse adjustment focus knob? When should you avoid it? Should only be used for the lowest-power lenses Activity 2: Domain Bacteria

BioCORE Trainee Ex 2 Digital Lab Notebook Gram-staining takes advantage of the relative thickness and porosity of the various components of the cell wall to distinguish between two major types of bacteria: Gram-positive and Gram-negative bacteria. Examine Gram-stained slides from each culture of pond water and bog water bacteria. Using the 40x objective lens to get a clear view of individual cells, take a micrograph (a picture of a specimen under a microscope, of the field of view) from each environment and insert them in the spaces provided below. As micrographs are figures, be sure to add a figure title, which should include the cell wall status of these bacteria (Gram-positive or Gram-negative) and where they were isolated from. Figure titles go below the figure. 27. Insert your micrograph of the Gram-stained bacteria cultured from the pond environment. 28. Insert your micrograph (microscope picture) of the Gram-stained bacteria cultured from the bog environment.

BioCORE Trainee Ex 2 Digital Lab Notebook Activity 3: Domain Archaea Being that Archaea are extremely difficult to culture in a laboratory setting, we will not have the opportunity to examine live specimens. However, your lab instructor will share a prepared slide of mixed archaea cells. 33. Insert a micrograph (microscope picture) of the mixed archaea. Activity 4: Eukaryotic Cells and Kingdom Protista Examine water samples from two different aquatic ecosystems: a mountain bog and a freshwater pond. You are asked to make a wet-mount slide of each, working up to examining your samples using the 40x objective,e and take a micrograph from each water sample. Using an editing app (PPT works fine), label at least one prokaryote (we can assume it will be a bacteria and not an archaea) and one eukaryote (protist). Take a screenshot of your labeled micrographs and insert them in the spaces provided below. Be sure to give your figures informative titles (which should go below the Figure itself). 34. Insert your micrograph from the pond water sample: *** Include title and labels

BioCORE Trainee Ex 2 Digital Lab Notebook 35. Describe the types of organisms (and maybe the biodiversity) of the pond water sample: Looks like there are some algae and maybe some bacteria and possibly some air bubbles. 36. Insert your micrograph from the bog water sample: Include title and labels **** 37. Describe the types of organisms (and maybe the biodiversity) of the bog water sample: It looks like some kind of funky plant, some bacteria/prokaryotic cells Analysis of Observations and Research: analysis and application of the observations and results from your research; post-lab assignments have been provided to help you in your analysis. 38. When comparing prokaryotic and eukaryotic cells under a compound microscope, what will be the main difference between them (regardless of whether you are examining them under the 4x, 10x, or 40x objective lens)? Eukaryotic cells should be larger. They should have a nucleus around their membrane and organelles within a cell membrane… Prokaryotic cells should be a lot smaller and wouldn't have a nucleus keeping their DNA in one place. 39. How does this difference impact your use of the microscope when looking at a prokaryotic organism? A eukaryotic one? The bigger the cell, the less magnification you may need versus a smaller (prokaryotic) cell which would need more magnification

BioCORE Trainee Ex 2 Digital Lab Notebook Reflections and Discussion: Once you have completed the in-lab lab exercises, 1) refer back to the Course Learning Objectives, 2) consider the background knowledge you gained from the pre-lab assignments and content, and 3) consider the concepts reviewed and results gained from the in-lab experiments. Reflect on what (knowledge, skill, or data) you have gained from completing Activities 1-4. Activity 1 Proper Microscope Technique, Cell Theory, and Prokaryotes This is important to not break the equipment. One thing to pay attention to is the coarse adjustment knob on the 4x power magnification setting. The fine coarse adjustment knob should be used for the 10x and 40x power magnification lenses. Prokaryotic cells are very small. Some components: DNA free floating in the cytoplasm without a nucleus, no membrane-bound organelles, flagella that have a single hook and motor filament that rotates for movement Activity 2 Domain Bacteria Bacteria are prokaryotic cells. They have cell membranes that are composed of fatty acid tails. Their cell walls contain peptidoglycan. They can also create biofilms which are small communities of bacteria. Some shapes: cocci, bacillus, and spirillum. Activity 3 Domain Archaea Archaea are prokaryotic cells, They have cell membranes that are made of isoprenoid tails. Their cell walls don’t contain peptidoglycan. They have different shapes, sizes, and lifestyles. They can also live in extreme environments. Activity 4 Eukaryotes and Kingdom Protista Eukaryotic cells have membrane-bound organelles. Eukaryotic cells also have flagella that are made of several microtubules. The kingdom Protista includes Amoebozoa, Opisthokonta, Excavate, Archaeplastida, Rhizaria, Alveolata, and Stramenopila. (Optional) Study Notes: 2/6/24 - Pond → small bodies of fresh, shallow. Standing water where light penetrates to the bottom of the water body… formed by naturally occurring depressions in the ground that retain water - components= other than sediment, you'll find bacteria and other microorganisms, numerous plants and algae species, vertebrates, from dish and amphibians to wading birds - Submerged, partially submerged, - Green, brown tint to water… may exhibit turbidity from sediment and or algae but oftentimes more clear… mostly uniform with temperature… pretty oxygen-rich - Big?- wet, spongy, poorly drained ground in which dead and or decaying plant material and vegetation has accumulated causing low oxygen, nutrient-poor

BioCORE Trainee Ex 2 Digital Lab Notebook acidic environments… mainly filled my precipitation… formed by naturally occurring depression in the ground that retains water - With saturated soil and dead/decaying matter of a bog - Bacteria and archaea, many of which are methanogens. Most abundant class of bacteria is peat bogs: alpha-proteobacteria - Few vertebrae permanently reside in snails due to lack of fish, snails, and mussels - Insect rich… brown/amber/reddish tint to water - Often covered in iridescent or oiling looking films (normally bacteria or archaea)... contains dissolved organic material (tannins, natural acids) - -