Bio Lab Review What's covered in the final exam:

Bio Lab Review What's covered in the final exam:
The exam will contain 50+ questions (multiple choice, short answers, basic calculations): Microscopes: name and function of parts, setting and use, taking digital pictures.... Labs: all lab activities plus material covered during the labs or printed in the lab manual as well as content of lab assignments Lab manual: content of labs chapter and appendices (not the intro): how to graph data, trace a table, nomenclature rules.... You should be able to identify the organism, cell type or biological process shown on a picture (there will be a lot of pictures). Measurements, units and conversions: you should be able to convert SI units and to know how to measure samples on a microscope using all techniques covered in the labs. No calculator - no lab manual - no textbook allowed during the exam.

Introduction to Microscopy and Observation of Prokaryotic + Eukaryotic cells
Microscope should always be placed between the computer and the end of the counter During transportation it must always be kept upright to prevent the ocular lens from falling out

Olympus CX41-Compound microscope Parts:
1.Revolving nose piece 2.Stage 3.Coarse focus knob 4.Fine focus knob 5.Ocular/Eyepiece 6.Objectives 7.Condenser 8. Condenser height adjustment knob 9. Aperture iris diaphragm

Compound microscope

Revolving Nose piece -supports the various objective lenses and allows for simple changes of magnification Stage -supports the specimen being observed. A system of knows on the side of the stage allows use to move the specimen under the objective on the X, Y axis Coarse focus knob allows rapid change of distance between the specimen and objective, thus allowing for rough focusing (don’t use when focusing with the 40X obj!) Fine focus knob -allows small changes in distance between the specimen and the objective, thus allows for final focusing of image Ocular/Eyepiece - A magnifying element, usually 10X. It is through the ocular/eyepiece that we look at the specimen. Since our microscopes are parfocal the focus is not completely lost when we change to the next objective

Condenser System of lenses that concentrates the light from the illuminator but has no role in magnifying the object Condenser Height adjustment knob Allows us to focus the concentrated light onto the specimen Aperture Iris diaphragm Reduces glare from unwanted light by adjusting the angle of the cone of light that comes from the condenser Occular inscriptions: 40X/0.65 (magnification/numerical aperture, which determines the resolving power of the objective) OO/0.16 (………/maximum coverslip thickness to be used with this obj)

Most important part of the microscope is the objective
Most important part of the microscope is the objective. Best image is not the largest, but the clearest! Resolving power is the ability to see 2 objects that are close together and still be able to recognize that they are 2 separate entities. ( for human eye 100 μm) With compound microscope: Image is inverted Working distance/distance between the objective and specimen decreases when magnification increases Lens has a depth of focus: number of planes in which object appears to be in focus Increasing magnification decreases depth of focus Total magnification for any combination of objective and ocular is the product of the magnification of each lens i.e obj magnification – 4X, ocular magnification = 10X therefore total magnification and 40 X Magnification factor of a picture =

real size of obj A / real size of obj B = on- screen size of obj A / on-screen size of obj B
Therefore: real size of object A = (on-screen size of obj A x real size of obj B)/on-screen size of obj B *all you need is a reference object of known size Measuring an object using a scale bar file: Real size of obj = (on-screen length of obj/on-screen length of scale bar) x real size of scale bar* *real size of scale bar e.g. bar represents 0.2 mm at 10 X Measuring an object using field of view (FOV) Real size of obj = (real size of FOV/ on-screen size of FOV) x on-screen size of FOV *real size of FOV must be a given, for each magnification

Stereoscopic/dissecting microscope
used to view objects that are too large or too thick to observe under compound microscope They are equiped with 2 oculars which produce a stereoscopic/3-D image Not inverted! Can be used with either reflected or transmitted light Reflected light is directed unto opaque specimens from above and is reflected to the viewer Transmitted light is used with translucent specimens and passes through the specimen from beneath the stage into the viewer’s eyes

Prokaryotic and Eukaryotic cells
Cells can be divided into 2 types prokaryotic and eukaryotic prokaryotic: absence of nuclear membrane + membrane bound organelles. Most genetic material consists of a single circular DNA molecule, and about 5% is found in small circular DNA plasmids Eukaryotic cells: true nucleus bound by a nuclear membrane + membrane bound organelles Plasma membrane  Semipermeable lipid bilayer that surrounds the cytoplasms of the cell (in animal cells) and physically separates the intracellular components from the extracellular environment. Cell wall  Rigid layer located outside plasma membrane that provides structural support and protection. In plants cellulose is the main component. Protoplast  plant, bacterial or fungal cell that had its cell wall partially or completely removed

Cytoplasm part of the cell enclosed within the plasma membrane. In eukaryotic cells it consists of the organelles. It is the site where most cellular activities occur. E.g metabolic pathways, cell division Vacuoles Enclosed compartments filled with water containing of organic + inorganic molecules, enzymes, or solids that may have been engulfed. It isolates materials that might be harmful the the cell, maintains turgor within cell, contains small molecules etc. Nucleus Double membrane enclosed organelle found in eukaryotic cells, that contains of most of the cell’s genetic material (chromosomes + histones). It controls activities of the cell by regulating gene expression. Nucleolus region in the nucleus responsible for transcription of ribosomal RNA Choroplasts  Organelle found in plant cells + other eukariotic organisms that conduct photosynthesis. They capture light energy, due to the presence of chlorophyll and generate carbohydrates.

Lyngbiya – cyanobacterium
Plasmolysis is the process in plant cells where the plasma membrane pulls away from the cell wall due to the loss of water through osmosis.  plant cell loses water and hence turgor pressure, making the plant cell flaccid (central vacuole shrinks too) Lyngbiya – cyanobacterium Choroplasts have a spherical shape They move due to cytoplasmic streaming/cyclosis Function is to aid the delivery of nutrients, metabolites and genetic info to all parts of the cell - When placing a cell in a strong salt solution, the hypertonic environment created, causes water to diffuse out by osmosis, thus decreasing cell volume  the space between the cell wall and the protoplast *cell minus cell wall* is visible Elodea leaf

- Review metric system + conversions

Lab #2 – Permeability of RBC’s
erythrocytes/ rbc’s contain large quantities of pigment hemoglobin When volume of cell exceeds a critical volume, cell ruptures and pigment is release to external environment  hemolysis Tonicity – relative concentrations of solutes in the fluid inside and outside the cell When cells are placed in a hypotonic environment, water enters the cells and their volume increases  reach a critical volume and hemolyze Hemolysis may also be induced by placing the cell into an isoosmotic solution of a penetrating substance (same osmotic pressure as the fluid inside the cells) As the penetrating substance enters the RBC, tonicity and osmotic pressure change The movement of water via osmosis is so that an equilibrium is mantained The time to hemolysis of a RBC in soln’s of penetrating substances is a reflection of the permeability of the membrane to those substances.

We determined the relative permeability of erythrocyte to:
Ethylene glycol (2 -OH) - Glycerol (3 -OH) – extremely polar, can’t penetrate membrane easily Thiourea – lower solubility due to the presence of the less electronegative sulfur which is unable to form H bonds with water, and its ability to cross the membrane is low Urea – oxygen atom can easily form H bonds with water Dextrose – smaller carb, fewer –OH groups attached to it, can more readily diffuse Sucrose – larger carb, high # of –OH groups attached, thus high capacity for H bonding in the extracellular space, and slower diffusion rate than dextrose Triton 100-X – great size, but increase the permeability of the membrane, enabling an accelerated entry Ethanol – (1 -OH) large non-polar portion, can easily dissolve in the non-polar fatty acid chains of the membrane Water – very small molecules can easily diffuse through the pores of membrane Factors affecting the rate of penetration of substance across cell membrane: Molecular size Lipid soubility Polarity Ability to form H bonds with water

RBC’s under 0.145 M conditions ( isotonic)

RBC’s under 0.35 M conditions (hypertonic) -shrinkage

RBC’s under 0.065 M conditions (hypotonic) - swelling

Cellular processes in Amoeba proteus
Amoeba proteus is a protozoan, single celled, uninucleate, common at the bottoms of freshwater ponds and lakes Famous for continuously changing shape, it may even obtain a length of 600 micro Small chambers made of parafilm melted onto glass slides were used to prevent crushing the amoebas They are very sensitive to light + heat (used low intensity light + yellow filter) We measured the diameter of a contractile vacuole during its cycle and plotted it against time Time zero = amoeba emptied the contents of its contractile vacuole i.e systole Pictures every 30 s Small vacuoles began to aggregate and fuse forming an increasingly larger vacuole Finally when it became large enough, it migrated to the uroid and emptied its contents

Bulk-phase (non-specific endocytosis)
Principal regions of the cell: Hyaline Ectoplasm Granular endoplasm Plasmagel Plasmasol Hyalin cap Uroid end = posterior end of the amoeba Bulk-phase (non-specific endocytosis) Pinocytosis – with the aid of bovine plasma albumin

Mitosis bacteria, protists and some fungi rely on cell division to produce 2 new individuals through asexual reproduction Cell division plays an important role in replacement + repair of worn-out cells Organized cell division ensures that the nuclear material which carries the genetic info needed for maintenance of cell + cytoplasm are correctly divided out Cell cycle Series of events that may last a few hours or even years Cell cycle = M phase (Mitosis + cytokinesis) + Interphase (G1 + S + G2) M phase – nuclear division ( mitosis) + cytoplasmic division (cytokinesis)

Interphase: phase separating 2 cell divisions, period of growth + preparation for M phase
G1 – period of growth + active sysnthesis of all groups of macromolecules e.g RNA, proteins. Cytoplasmic organelles are duplicated. S (synthesis) phase – replication of all DNA + synthesis of DNA associated proteins or microtubule-associated proteins at the end of S phase, chromosome consists of 2 chromatids joined at centromere region Kinetochore - button like structure linking the chromosome to mitotic spindle G2 – protein synthesis + production of structures needed for mitosis like spindle fibres *2 examples of cells that stop their cell cycle during interphase are central nervous system + muscle cells  they stop dividing once their growth and differentiation is completed

Mitosis: process of nuclear division where chromosomes are equally distributed between 2 daughter nuclei Both daughter cells are identical to each other and parental cell Cytokinesis: division of cytoplasm; begins in late anaphase and continues through telophase  seen as cleavage furrow that forms in the middle of animal cells or cell plate Cell cycle phases in plants Interphase Growth, synthesis of macromolecules, assembly of organlles, DNA replication Clear-cut nucleus, darkly stained nucleoulus, and heterochromatin Most cells appear to be in this stage

Prophase – chromosomes shorten and thicken Each consists of 2 chromatids joined at the centromere region (each chromatid contains identical info) Microtubules of cytoskeleton disassmble into tubulin subunits which reassemble intoo mitotic spindle Nucleoli disappear * Centrosome (including its 2 centrioles) duplicate just before S phase 2 centrosomes separate and migrate towards opposite poles of the cell

Prometaphase Breakdown of nuclear membrane Centrosomes are found at opposite poles of cell (spindle poles) On the centromere of each chromatid, a protein complex forms aka kinetochore (2 kinetochores, 1 per chromatid) Microtubules from each pole (polar microtubules) attach to corresponding kinetochore Chromosomes migrate towards metaphase (equatorial) plate

Metaphase Mitotic spindle microtubules are fully formed between poles Centromeeres of all chromosomes are located at metaphase plate Centromeres start to separate

Anaphase -centromeres separate and each chromatid (daugther chromatid) moves to opposite pole of cell due to shortening of kinetochore-pole microtubules Citokinesis begins

Telophase Chromosomes at the poles decondense, become longer and thinner Nuclear membrane reappears Nuclei reform Spindle disappears Cytokinesis occurs *Plant cells: phragmoplast guides formation of new cell wall across centre of cell *Animal cells: cleavage furrow pinches cell in 2

Mitosis in Onions

Plants have growth confined to growth centres primarily found in root tips and stem tips (apical meristems) Preparing a Feulgen squash Squashed beneath a coverslip  flattened intact cells layer Feulgen Stain Colors DNA containing structures magenta red (specifically colors nucleic acids and chromosomes) Qualitatively indicate presence or absence of DNA in cell Quantitatively measures the amount of DNA present in cell Consists of a colourless subtsance “leucobasic fuschin”  reacts with aldehyde groups to form a colored reaction product *intact DNA does not contain free aldehyde groups, but it does contain deoxyribose sugar molecules chemically linked through aldehye groups to purine and pyrimidine bases

DNA is first hydrolised with hot HCl to remove free purine bases and form free aldehyde groups on deoxyribose sugar Result? Apurinic acid (DNA lacking purines)  this material reacts with Feulgan stain *hydrolysis of short duration does not free all aldehyde groups *excessive hydrolysis causes destruction of apurinic acid DNA (in tissue) -----> (HCl + heat) ----- apurinic acid + purines Apurinic acid + Feulgen stain ---- colored apurinic acid

Root growth Root cap – mass of irregular dead cells with thicker walls which protect apical meristem of the root tip as it pushes into the soil. Also thought to be the site that detects gravity + controls direction of root growth Region of apical meristem: zone above root cap, where cell division takes place Quiescent centre – zone at the base of apical meristem, relative inactive region where cells are arrested in G1 phase Region of elongation – above region of cell division. Elongation of cells results Is most of the increase in length of root Region of maturation – most of the cells of the primary tissues mature; root hairs are produced in this region Xylem cells: bundles that transport water and salts from soil to rest of plant Phloem cells: transport carbs from photosynthetic portion of plant to roots *label the diagram they gave us in the lab manual * Roots do not have chloroplasts and therefore can’t make their own food

Onion root tip ( Allium cepa)

Onion (TA pic)

Whitefish blastula (Coregonus clupeaformis)

White fish ( Coregonus clupeaformis)TA picture

Broad bean (Vicia faba) root tip

In all 3 species (Allium cepa, Vicia Faba, Coregonus clupeaformis), most cells were seen in interphase. And the least amount of cells in anaphase + telophase Prophase – longest phase of mitosis *duration of each phase of the cells is proportional to the number of cells observed in any given phase Animal mitosis No cell wall around cell membrane, therefore no cell plate formed at telophase i At the end of nuclear division, membrane pinches in the middle Aster (only in animal cells): semicircle of fibirls around each end of spindle

Meiosis Process of nuclear reduction division, that only occurs is sexually reproducing organisms DNA duplication is followed by 2 divisions Net result is 4 instead of 2 daughter cells that are not genetically identical to each other Each daughter cell contains half the number of chromosomes in the mother cell Upon fertilization the 2n number of chromosomes restored Meiosis assures: Chromosome # remains stable from generation to generation Each offspring resulting from sexual reproduction receives 2 entire setss of genetic instructions Genetic diversity is promoted among the products

Plants: Life cycle is characterized by an alternation of generations between diploid and haploid stages Diploid generation (2n) – sporophyte – undergoes meiosis to form haploid spores Spores divide mitotically and become multicellular haploid individuals aka gametophytes Gametophyetes produce gametes which fuse to form diploid zygotes Zygotes divide mitotically, developing into a multicellular diploid organism (sporophyte)  This type of meiosis is called sporic meiosis

Animals: Alternation of ploidy level like in plants Diploid individuals produces haploid gametes by meiosis (gametogenesis) Male and genale gametes fuse to form a diploid zygote which divides mitotically and becomes a multicellular diploid organism  Gametic meiosis

Stages of Meiosis I Premeiotic Interphase Substages similar to mitotic interphase (G1, S, G2) Differences between premeiotic and premitotic interphase: Premeiotic S may be 20 X longer than premitotic interphase (in same species) Sex chromosomes are replicated at the beginning of S ( mitosis: end of S) Nucleus grows to a greater volume than in mitotic interphase 2 identical sister chromatids attached at centromere

Prophase I Longest stage of meiosis (can last from weeks to years) Homologous chromosomes pair Recombination occurs Leptotene – initial condensation of chromosomes  appear as single threads. Telomeres *tips of chromosomes* are attached to nuclear membrane 2) Zygotene – maternally and paternally derived copies of each homologous pair of chromosomes line up along their lengths (process = synapsis) Chromatids of homologous chromosomes interwine and form a synaptonemal complex 3) Pachytene – adjacent chromatids break and join in a process called crossing over Fully pairred homologues are called tetrads (4 chromatids)/ bivalents 4) Diplotene – chiasmata (region of crossing over) becomes visible Synaptonemal complex disappears, and chrimosomes detach from nuclear membrane 5) Dikinesis - Condensation of chromosomes finishes, final modifications occur, chromosomes are ready for division; nucleoli disappear

Metaphase I Nuclear membrane breaks down Paired homologues line up at the equatorial plate Ordering of maternal and paternal homologues occurs at random 2 sister chromatids of one homologue attach their kinetochores via microtubules to the same spindle pole ( same happens with the other 2 sister chromatids) Anaphase I Centromeres do not split as in mitosis. Homologues are pulled away from each other towards opposite poles (*key difference between meiosis and mitosis) Late anaphase: cytokinesis begins

Telophase I -Chromosomes decondense -spindle breaks down Nuclear membrane reforms Cytokinesis continues Result? 2 daughter cells each with half the # of chromosomes, but with each chromosome containing 2 chromatids Interkinesis interphase-line stage between meiosis I and II No DNA replication occurs Centrioles do not duplicate

Meiosis II – virtually the same as mitosis
Prophase II nuclear envelope breaks down Chromosomes recondense Spindle forms  kinetochores become attached to spindle microtubules Chormosomes begin to move to equator Metaphase II - chromosomes each consisting of 2 chromatids line up at the equatorial plate Anaphase II Sister chromatids separate and move to opposite spindle poles, cytokinesis begins Telophase II - chromosomes decondense, nucleoli reform, nuclear membrane forms around each product, cytokinesis finishes  4 haploid cells, genetically different from each other, result

Meiosis in plants Male spores: microspores Female spores: megaspores Male gametophytes in flowering plants occur in anthers Microspores are produced by a reduction division within 4 microsporangia (pollen sacs) of the anther Sporogeneous cells become surrounded by layers of sterile cells  innermost layers develop into cells that provide nutrition to the developic microscospore (tapetum) + outermost layer forms wall of microsporangium -the sporogeneous cells develop into microsporocytes (microspore mother cells) These diploid cells divide by meiosis and produce 4 haploid single celled microspores Microspore divides mitotically to form a tube cell and a generative cell (microgametophyte)  these 2 cells - These 2 cells + the spore wall = pollen grain

Lilium

Lilium anther, early prophase, c.s.
Pollen sac Epidermis Tapetum (tapetal cells) Developing pollen grains Parenchymatous tissue Cell wall Cytoplasm Nuclear membrane Chromsomes Nucleolus (possibly)

Lilium anther, late prophase, c.s.
Pollen sac Epidermis Tapetum Developing pollen grains Parenchymatous tissue Cell wall Cytoplasm Nuclear membrane Chromosomes (homologous pairs, chiasmata may be visible)

Lilium anther, pollen tetrads, c.s. or Lilium
anthers, 2nd division, c.s. Pollen sac Epidermis Tapetum Developing pollen grains Parenchymatous tissue Cell wall Cytoplasm Transitory nuclear membrane Chromsomes Spindle Tetrad of four haploid cells I am really not sure which image would apply here.

Lilium anther http://images.iasprr.org/lily/male.shtml
- Excellent site

Meiosis in animals Spermatogenesis = formation of male gametes, 4 viable sperm cells are produced Oogenesis = formation of female gametes, 1 viable egg Cytoplasm + stored food must be retained in egg for the use of the developing embryo 2 products of first meiotic division: egg, and a small polar body 2nd meiotic division: one large egg + 2 small polar bodies Ascaris Parasitic roundworm with diploid number of chromosomes = 4 Spermatozoa complete meiosis in testis of male - Eggs are shed from ovary during prophase I and travel down the oviduct without going into metaphase I Male and female copulate  spermatozoa reach eggs in the oviduct and only one penetrates each egg and loses its membrane The sperm entrance stimulates egg to continue into metaphase I

For excellent images of Ascaris:

Ascaris megalocephala, sperm entrance, c.s.
(SLIDE) Oocyte plasma membrane (vitelline membrane) Primary oocytes(with vacuoles) Sperm (nucleus) Uterus (uterine wall and lumen of uterus, may not be visible)

Ascaris, c.s. Metaphase 1 (no polar body in
perivitelline space) (PPT) Tetrad (chromosomes at equator of spindle) Meiotic spindle Shell Male pronucleus Perivitelline space

Ascaris, c.s. Anaphase 1 (no polar body in
perivitelline space) (PPT) Chromosomes migrating to spindle poles Meiotic spindle Shell Male pronucleus

Ascaris, c.s. Metaphase 2 (one polar body in
perivitelline space) (PPT) Secondary oocyte Oocyte plasma membrane Chromosomes at equator of spindle Meiotic spindle Shell (inner ascaroside layer, chitinous layer, fertilization membrane) Male pronucleus Polar body

Ascaris, c.s. Anaphase 2 (one polar body in
perivitelline space) (PPT) Chromosomes migrating to spindle poles Secondary oocyte Oocyte plasma membrane Meiotic spindle Shell (inner ascaroside layer, chitinous layer, fertilization membrane) Male pronucleus Polar body

The two haploid pronuclei (one male and one female) are seen in the centre of the cell. The nuclear membranes of both pronuclei will now break down releasing their chromosomes. This forms the diploid zygote which proceeds immediately into the first cleavage division. Note the presence of two polar bodies, the second having been released following the completion of the second meiotic division. The dark structure along the bottom of the picture is the wall of the uterus. The fertilization shell layers are labelled as follows: 1. The fertilization membrane with outer protein layer 2. The chitinous layer 3. The ascaroside layer and perivitelline space Ascaris megalocephala, pronuclei, c.s. (Slide) Male and female pronuclei Shell (inner ascaroside layer, chitinous layer, fertilization membrane) Oocyte plasma membrane Perivitelline space Polar body 1 (+possibly 2)

Ascaris megalocephala, cleavage, c.s. (slide)
Mitotic metaphase Shell (inner ascaroside layer, chitinous layer, fertilization membrane) Zygote plasma membrane Perivitelline space Polar body 1 (+possibly 2) Chromosomes (at equator of spindle) Mitotic spindle Possibly two-celled or four-celled zygote

Spermatogenesis in mammals
Spermatogonia (2n) – large darkly stained outer cells with well defined nuclei - divide by mitosis to produce more spermatogonia - half of the spermatogonia undergo meiosis to become sperm cells, and the other half divide too replenish the spermatogonia population Primary spermatocyte (2n) – located just below spermatogonia, not as darkly stained. - large cells undergoing first meiotic division Secondary spermatocyte (n) – product of the first meiotic division and rapidly undergo meiosis II to produce spermatids Spermatids (n) – very small circular cells that differentiate into functional spermatozoa Spermatozoa (n) – possess very long thin flagellum, located near the lumen Nurse cells (sertoli cell)- large nurse cells found in the walls of the seminiferous tubules which feed and regulate the differentiation of spermatids into mature spermatozoa

This is a much better picture than what we managed to take in the lab itself – much better level of detail.

Rat testis – 10 X - Labels

Rat testis – 40 X - Labels

Oogenesis in mammals Primary follicles – numerous small, round structures at the periphery of the ovary. - each contains one oogonium (2n) surrounded by a layer of follicular cells - at onset of meiosis I, oogonium enlarges to become primary oocyte - Cytokinesis of primary oocyte at the end of Meiosis I produces a large secondary oocyte + a polar body 2) Growing follicles – larger follicles with a few layers of follicular cells - each contains a primary oocyte (2n) or a newly formed secondary oocyte (n) 3) Graafian follicles – very large follicles. Most space is occupied by a large fluid filled cavity. The mature oocyte (2ndary oocyte (n) ) would be found nestled at the centre of the follicle - At this point the secondary oocyte is called a mature oocyte - When mature follicle ruptures at ovulation, the mature oocyte is release

Rabbit ovary – 10 X http://www.ouhsc.edu/histology/
- To help you label it

Rabbit ovary – 40 X - To help you label it

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