Notes for Final Lab Exam

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University of Alberta
Biology (Biological Sciences)
Heather Proctor

LAB 1: Biology Tools and Techniques  Set up fungus culture: inoculate aspen wood shavings, wheat bran, gypsum, and water mixture with fungal mycelium (network of fungal hyphae, tubular filaments=basic growth form during vegetative phase) *Pleurotus sp. (Phylum- Basidomycota)  Set up fern spore culture: Using a serial dilution, *Ceratopteris sp. (Phylum- Pteridophyta)(C-fern) will later germinate and grown into reproductive structures gametophytes.  Set up Arabidopsis thaliana (Phylum-Angiosperm) seedlings in low phosphate or control phosphate levels. Parafilm around plate to maintain sterile internal environment and keep in moisture. * Genus species or G. species or Genus sp. or multiple Genus spp. 1. Aseptic technique: used to ensure that organisms in the environment do not contaminate cultures being studied, but also that the organisms studied are not released into the environment (contaminating the lab).  Use a Bunsen burner to sterilize the air  Open bottles in a tilted position  Hold the lid of petri plates at a 45° angle  Don’t breathe directly onto cultures, solutions, or medium 2. Compound Light Microscope:  3 lens system: condenser lens (focuses light on the specimen), objective lens (magnifies the image), ocular lens (magnifies image and inverts it – contains ocular micrometer)  Body tube: contains prism that allows light to pass from objective lens to ocular lens  Stage: where slide is placed – moves so you can relocate specimen if you record position of the rulers  Revolving nosepiece: holds the objective lenses, attached to the bottom of the body tube  Iris diaphragm: adjust amount of light on specimen. Field iris diaphragm – focus and center the light, adjust by turning ring. Aperture iris diaphragm – below the condenser, adjust using lever.  Condenser: lens under the stage that focuses light onto the specimen, move up or down by turning knob.  Coarse focus knob: raise/lower the stage to focus optics. Fine focus knob: fine- tune the focus.  Parfocal: microscope should be closed to focus even when you change lenses. Just fine tune using fine focus. Magnification:  Ocular micrometer gives measurement in eyepiece units. 1 epu at 10X = 10 micrometers 1 epu at 40X = 2.5 micrometers  Magnification (M) = size of drawing/actual size of object (*convert micrometers to millimeters!)  We made wet mount of cyanobacteria then used dichotomous key (paired statements, each statement is called a lead) to identify it = Gleocapsa. Lab drawing:  Drawing on left side, details and labels to the right. Include caption and magnification. 3. Dissecting Microscope: two eyepieces and two objectives – view material from slightly different angles with each eye = 3D view. Uses lower magnification than compound microscope.  Ocular lenses, focus knob, body tube, arm, zoom adjustment knob, stage, and base. Questions: 1. Was your microscope parfocal? YES – only needed to use the fine focus when switched to a higher power objective. 2. Switching from low power to high power, change in brightness of the field of view? YES – it goes darker because higher magnifications have thicker lenses (more light gets reflected). Adjusting the iris diaphragm improves the contrast. 3. Advantages to using dissecting microscope: Gives a 3D view, you can manipulate organisms with your hand rather than the toggle, you can view live organisms, and you can view larger organisms. Vocab: 1. Aseptic (sterile) technique 10. Fine focus 2. Body tube 11. Iris diaphragm 3. Compound light microscope 12. Objective lens 4. Condenser 13. Ocular micrometer 5. Course focus 14. Parfocal 6. Dichotomous key 15. Serial dilution 7. Dissecting microscope 16. Stage 8. Eye piece units 17. Wet mount 9. Eyepiece (ocular lens) LAB 2: Origin of Species  Species: group of individuals that can interbreed, or have the potential to interbreed, in nature.  Speciation: a lineage splitting event that produces 2+ separate species. Caused by a reduction of genes leading to a reproductively isolated population. 1. Allopatric speciation: geographic isolation 2. Sympatric speciation: reduction of gene flow 4 mechanisms of evolution: mutation, genetic drift, gene flow, and natural selection.  Phylogenies are used to depict species relatedness (genealogy – attempt to map the relatedness/kinship among family members). Internal nodes represent speciation events. Clade: shares common ancestor.  Constructed a phylogenetic tree for the Great Apes. 1. Listed binary traits (only two possibilities) – eyebrow ridge, relative size of fist incisors, diff. shape between 1 and 2 incisures, relative size of tarsal bones, hindlimb length. 2. Polarize characteristic relative to the outgroup (close relative that we know doesn’t belong to group being studied) 3. Mark character states for each one. 0=outgroup, 1=derived 4. Eliminate uninformative characters (autapomorphies – only 1 lineage is derived, synapomorphies – shared within all members) 5. Construct simplest tree = maximum parsimony  Used necklace with 4 diff. beads to represent 4 diff. amino acids. You can tell by the end results which groups had common ancestors. Two important elements: 1. There is a source of change – mutation, single-base substitution - Continuous accumulation of mutations can be used to estimate how long ago the ancestors of currently living species split into different lineages = molecular clock hypothesis. - Mutations must be neutral – can’t have a +/- effect on survival or reproductive output - Genetic marker used is cytochrome b – encoded on mitochondrial DNA (mtDNA) and acquires neutral mutations quickly. 2. Populations split/branch once in a while  We can also compare genotypes to make phylogenies based on DNA bases. We took a multiple sequence alignment for the cytochrome b protein in the 8 great apes (downloaded from NCBI). Orangutans split off first, then gorillas, then humans and chimpanzees split. Questions: 1. Why must the mutations used to determine an evolutionary rate be neutral? Because they cannot have a +/- effect on survival or reproduction that would change how quickly the species changed. 2. Why is mtDNA used in phylogenetic studies? It only comes from the mother, therefore only changes due to mutations. Nuclear DNA gets recombined every generation. Vocab: 1. Branch 11. Natural selection 2. Character states 12. Neutral mutation 3. Clade 13. Node 4. Consensus tree 14. Outgroup 5. Gene flow (due to migration) 15. Phenotype 6. Genetic drift (due to random 16. Phylogenetic tree sampling) 17. Phylogeny 7. Genotype 18. Single-base substitution 8. Molecular clock hypothesis 19. Speciation 9. Most recent common ancestor 20. Species 10. Multiple sequence alignment 21. Taxa LAB 3: Plants Pt. 1 – Plant Form and Function  Plants are dynamic organisms – influenced by the environment in which they reside. o Acquire resources by the root and shoot systems connected by vascular tissues (continuous network of veins). o Shoot system: apical bud axillary bud node (point of attachment) leaf (petiole, attaches blade to stem, and bud) – capture light energy via photosynthesis. Arranged in alternate, opposite, or whorled (3 or more) pattern. Shape can be simple or compound (divided into leaflets). stem – maintain plant structure and transport o Root system: absorption of water and nutrients. Taproot vs. fibrous system. Taproot (largest and most important) lateral roots o 3 types of tissue: 1. Dermal tissue – outer layer of cells, covers entire plant, composed of epidermis and the cuticle, occasionally trichomes/hairs present 2. Vascular tissue – run throughout whole plant, composed of xylem (carries water up) and phloem (carries photosynthesis products down) 3. Ground tissue – background tissue, fills space between epidermis and vascular tissue, may contain specialized cells  Internal Tissues (examined Helianthus sp. Phylum-Angiosperm) aka sunflower. o Epidermis – outermost layer. Include trichomes/hairs that project. Cuticle is the waxy outside covering. o Cortex – tissue just interior to epidermis. Contains specialized cells; starch storage, add support, contain chloroplasts for photosynthesis. o Pith – central part of stem composed of storage cells o Vascular bundle (xylem and phloem) – conductive tissue, also includes fibers that serve as support and protection.  Internal structure of a leaf (examined Syringa sp. Phylum Angiosperm) aka lilac. o Upper epidermis – outer layer on upper surface of leaf, covered by non- cellular cuticle o Palisade mesophyll – dense layer composed of elongated cells. PRIMARY SITE FOR PHOTOSYNTHESIS. o Spongy mesophyll – next photosynthetic layer, fewer chloroplasts and large air spaces o Lower epidermis – outer layer of cells on underside of leaf, contains stomatal apparatus o Stomata – two guard cells and the aperture/pore = gas exchange o Veins/vascular bundle – conducting and supporting tissue of the leaf, xylem and phloem, and supportive cells.  Role of essential nutrients on plant growth – Phosphate’s role on root structure in Arabidopsis sp. (Angiosperm) o Macronutrients needed: Carbon, Oxygen, Hydrogen, Nitrogen, Phosphorus, Sulfur, Calcium, Potassium and Magnesium. o Low phosphate gave shorter roots, darker green leaves, and less branching.  All hypotheses are testable, falsifiable, applicable to multiple cases, and based on existing knowledge. Prediction is what you expect outcome to look like.  We use stats to demonstrate results are meaningful and not due to chance. o Mean – average. Variability – how much values differ from mean. o Use two-sample t-test – used because of continuous data. Null hypothesis – pattern is due to chance alone. P-value is the probability of Ho being true (we used p<0.05), if t-value is less, differences are not due to chance. Alpha- level is that cut-off (0.05). Degrees of freedom, relates the number of different categories being studied. o The larger the difference between means, the higher the value of the t- statistic. The larger the variance within samples, the smaller the t-statistic. o Df=(n1-1)+(n2-1)… Questions: 1. Is leaf shape important for photosynthesis? Yes, flat leaves would be better to capture sunlight. 2. What do the veins on the leaves represent? They represent vascular tissues. 3. How to germination of Arabidopsis compare? No appreciable difference. Vocab: 1. Compound leaf (divided into 19. Phloem leaflets) 20. Pith (central part of stem, storage 2. Continuous data cells) 3. Cortex (tissue just interior of 21. P-value epidermis – specialized cells incl. 22. Qualitative chloroplasts) 23. Quantitative 4. Count data 24. Root 5. Critical value 25. Simple leaf 6. Cross section 26. Spongy mesophyll (layer below 7. Cuticle (waxy, covers epidermis) palisade mesophyll, less 8. Degrees of freedom chloroplasts, lots of space) 9. Dermal tissues (outer layer: 27. Statistical null hypothesis epidermis, cuticle, trichomes) 28. Stem 10. Epidermis (outer layer) 29. Stomata/stomatal apparatus 11. Ground tissues (background tissue 30. Taproot (primary root is largest that fills up space, may contain and most important, as opposed to specialized cells) fibrous root) 12. Lateral roots 31. Trichome 13. Leaf 32. T-statistic 14. Leaf buds (apical vs. axillary) 33. T-test 15. Mean 34. Variance 16. Null hypothesis 35. Vascular bundle 17. Palisade mesophyll (layer below 36. Vascular tissues upper epidermis, primary site of 37. Veins photosynthesis) 38. Xylem 18. Petiole (attaches blade of leaf to 39. Alpha-level stem of plant) LAB 4: Plants Pt. 2 – Plant Development and C-Fern Life Cycle Four major groups of land plants derived from green algae: 1. Non-vascular plants (liverworts, mosses, hornwarts) 2. Seedless vascular plants (lycophytes, pterophytes) 3. Vascular seed plants (gymnosperms) 4. Vascular seed plants (angiosperms)  Alternation of generations: Underlying pattern between all variations of land plants. MEIOSIS -> haploid spores -> MITOSIS -> multicellular gametes (gametophyte (n)) -> FERTILIZATION -> formation of zygote = diploid (sporophyte (2n))  Sporophyte = visibly dominant. Gametophyte develops independently of sporophyte in ferns.  Homospory – one spore develops into sporangia and are bisexual (ex. mosses, ferns) Heterospory – two types of spores (male and female) (ex. seed plants)  In Ceratopteris sp. = homosporous, simple haploid gametophyte -> diploid sporophyte o Antheridia produce sperm that need water to swim and archegonia produce eggs (water causes neck to open and release chemical). o Two types of gametophytes: hermaphroditic (have archegonia and antheridia) and male (have only antheridia). Sexual differentiation is controlled by pheromone antheridiogen Ace. o Hermaphrodites – absence of ACe, meristematic region, indeterminate growth – releases ACe, so if on a plate with other spores, it inhibits growth of other hermaphrodites. o Males – presence of ACe, smaller, determinate, lack a meristem  To observe impact of population density on sexual expression, calculated density of gametophytes on the plate and then quantified amount of hermaphrodite vs. male. o Graphed average density of gametophytes (x) vs. percentage of sexual type (y) Questions: 1. Why do some spores not germinate? Some just die, local contamination, some die during spreader flaming. 2. Relationship between gametophyte density and sex expression? Higher density gives more males. If few gametophytes, it is advantageous to self-fertilize; if lots of gametophytes, hermaphrodites would want more males to breed with and it promotes genetic variability. 3. How does sex expression occur? Thanks to pheromone-like ACe. In presence – males develop. 4. Dilution experiment – control? Undiluted plate. Treatment? Diluted plates. 5. What happens when you add water to fern gametophytes? Mature antheridia will release sperm that swim and congregate at the archegonia. Some will swim down the neck of archegonium. 6. Where in the fern life cycle is organism most vulnerable? At fertilization. Sperm is motile – it needs continuous and still water. 7. Life cycle: Spore (n) -> MITOSIS -> male/female gametophyte (n) -> MITOSIS -> FERTILIZATION -> zygote (2n) -> MITOSIS -> sporophyte (2n) -> MEIOSIS -> spore (n) Vocab: 1. Alternation of generations 12. Sperm 2. Antheridium 13. Gametophyte 3. Archegonium 14. Haploid 4. Determinate growth 15. Hermaphroditic 5. Diploid 16. Heterospory 6. Egg 17. Homospory 7. Embryo 18. Indeterminate growth 8. Fertilization 19. Meiosis 9. Meristematic region (cells grow, 20. Meristem are indeterminate) 21. Spore 10. Mitosis 22. Sporophyte 11. Pheromone 23. Zygote LAB 5: Kingdom Fungi Fungi include multicellular and unicellular forms. More closely related to ANIMALS than plants. 5 major groups: 1. Chytrids 2. Zygomycota 3. Glomeromycota 4. Ascomycota 5. Basidiomycota  All fungal cells have a rigid wall external to plasma membrane, the ability to absorb compounds for metabolism, and the ability to reproduce by forming spores.  Composed of tubular filaments (hyphae, nucleus=haploid), which form a network (mycelium). Some mycelium differentiate into fruiting bodies = mushrooms/puffballs/etc that we see. Purpose= provide protection, a durable enclosure, and a dispersal device for haploid spores.  Sexual reproduction: 1. Plasmogamy: fusion of cytoplasm from two opposing haploid hyphae (haploid nuclei from each parent into 1 cell). Nuclei pair up => dikaryon (n+n) 2. Karyogamy: fusion of nuclei, forming a diploid zygote nucleus (2n) 3. Meiosis: produces four haploid (n) nuclei, which continue to germinate (mitosis) until plasmogamy or asexual reproduction.  Life cycle of Basidiomycota (Pleurotus sp. = Phylum Basidiomycota) o Basidiocarp = mushroom = fruiting body o Basidia line the gills, where spores are produced through meiosis.  Asexual reproduction in Ascomycota (Penicillium sp. = Phylum Ascomycota) o Mycelia can produce asexual spores = conidia that are on the tips of modified hyphae (conidiophores). o Fungus found in blue cheese – part of fungi imperfecti (multicellular, but only found in asexual state). Conidiophore looks like a broom, spores are found at the tip and grow in strings. o Yeast is a unicellular fungus that reproduces asexually by budding – outgrowth of parent cell, grows and forms a new cell.  Mycorrhizae – mutualistic symbiotic associations between fungi & roots of vascular plants, allow plants to survive in competitive communities by increasing the physiologically active area of the root system (increases ability to capture water and nutrients, increases tolerance to environmental extremes, and provides protection from disease and pests). Fungus benefits because they receive photosynthetic products and vitamins from the plants. o Arbuscular: penetrate roots cells and form structures inside the cells. o Ectomycorrhizal: do not penetrate, wraps around the root cells.  Lichen - mutualistic association between fungal partner and algae/cyanobacterial cells (fungal=Ascomycota, photosynthetic partner=unicellular Chlorphyta or Cyanobacteria) o It is the algal layer that does photosynthesis, allows lichen to live. Lichen gets organic C from algae, and N from cyanobacteria. o Growth is super slow and dominate in mountain and arctic regions. o 3 common growth forms: 1. Crustose – thallus forms a thin crust that grows right on top and is entirely stuck to the surface (cannot be removed). 2. Foliose – thallus is flat and has leaf-like loves, attached with rhizines or is circular and attached by a single cord (can be peeled off) 3. Fruticose – thallus is only attached at the base and grows outward, either shrub-like or strand-like. o Asexual reproduction =fragmentation of the thallus. o Sexual reproduction = formation of ascocarps (confined to fungal partner) o Used dichotomous key to identify different lichens.  Types of fungi:
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