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BOTANY LABORATORY LBBBION | Term 1 | 2024 - 2025 COMPOUND MICROSCOPE MICROSCOPE Basic tool in science. Indispensable tool in Botany or all Biological Sciences. Enables people to see plants structures too small to be seen by the unaided or...

BOTANY LABORATORY LBBBION | Term 1 | 2024 - 2025 COMPOUND MICROSCOPE MICROSCOPE Basic tool in science. Indispensable tool in Botany or all Biological Sciences. Enables people to see plants structures too small to be seen by the unaided or naked eye. Since the invention of the light microscope in the 1590s, there have been numerous improvements and modifications on the instrument's performance. However, part of this improvement depends on the development of new techniques for specimen preparation. CYTOLOGY DIFFERENCES BETWEEN MICROSCOPES Branch of science dealing with microscope design, slide preparation, and examination. LIGHT MICROSCOPE Uses glass lenses to focus on the THREE ELEMENTS NEEDED TO FORM AN IMAGE specimen. 1. Source of illumination 2. Specimen to be observed TRANSMISSION ELECTRON MICROSCOPE 3. System of lenses to focus the illumination (TEM) on the specimen and to form the image Uses electromagnetic lenses to focus electrons that passes through the TWO TYPES OF MICROSCOPE specimen to produce an image in the fluorescent screen. LIGHT MICROSCOPE ELECTRON MICROSCOPE SCANNING ELECTRON MICROSCOPE (SEM) Uses a narrow beam of electrons to scan Light is the Beam of electrons is the over a specimen that is coated by a thin illuminating source electron source layer of heavy metals. Specimen preparation Specimen preparation usually takes a few usually takes a few days ELECTRONS minutes to hours Subatomic particles that have a negative electric charge. Live or dead Only dead or dried Found surrounding the nucleus of an atom. specimens may be specimen are seen When a metal becomes hot, some of the seen electrons gain so much energy that they escape from their orbits. Condenser, objective All lenses are Free Electrons and eyepiece lenses electromagnetic ○ The energy is associated with the are made up of proportion of waves. glasses ○ Higher the energy, shorter the wavelength. Specimen is stained Specimen is coated with A very suitable form of radiation for two by colored dyes heavy metals to reflect major reasons. electrons 1. Wavelength is extremely short. 2. Because they are negatively charged, It has low resolving It has high resolving they can be focused easily using power (0.25μm to power (0.001μm) electromagnets. 0.3μm) Electromagnets ○ Can be made to alter the path of the It has a magnification It has a magnification of beam, similar to how a glass lens of 500x to 1500x more than 100000x made to bend the light. Vacuum is not Vacuum is required required TYPES OF ELECTRON MICROSCOPE TRANSMISSION ELECTRON MICROSCOPE Image is seen by eyes image is produced on The beam of electrons is based through the through ocular lens fluorescent screen or specimen before being viewed. photographic plate Only those electrons that are transmitted are seen. Enables to see di erent components inside the cell. Sir John Paul Domingo BOTANY LABORATORY LBBBION | Term 1 | 2024 - 2025 SCANNING ELECTRON MICROSCOPE Electron beam is used to scan the surfaces of structures. Only the reflected beam is observed. Advantage ○ Surface structure like the chitinous outer body of insects can be observed in great detail. PARTS OF THE MICROSCOPE MECHANICAL PARTS Base Pillar Handle/Arm Inclination Screw Body Tube Ocular Tube/Draw Tube Revolving Nosepiece Dust Shield Adjustment Screws ○ Coarse Adjustment Screw ○ Fine Adjustment Screw Stage Mirror Rack MAGNIFYING PARTS Ocular/Eyepiece Objectives ○ Low Power Objectives (LPO) ○ High Power Objectives (HPO) ○ Oil Immersion Objectives ILLUMINATING PARTS Mirror Diaphragm Condenser MECHANICAL PARTS Those parts concerned with the support and adjustment of the optical parts. MAGNIFYING PARTS Those parts concerned with image enlargement of the specimen. FIELD OF VIEW Diameter of the circle of light seen through ILLUMINATING PARTS a microscope. Those parts concerned with light provision Usually measured in micrometers (μm). and regulation to the specimen. Area of the slide which can be seen when looking through the microscope. TYPES OF MICROMETER Low power objective lens is always used OCULAR MICROMETER first since it allows a larger area of the slide Measures the size of the cells. to be seen. Ruler in ocular lens. ○ It then allows you to choose which 100 equally spaced division marks. part of the specimen on the slide you want to view in further detail at STAGE PARTS higher magnifications. Ruler image on slide. 1 division = 0.01mm (10μm) Before increasing magnification, the area you 100 equally spaced division marks have chosen should be moved to the center of the field of view. Sir John Paul Domingo BOTANY LABORATORY LBBBION | Term 1 | 2024 - 2025 STANDARD VALUES OF FIELD OF VIEW Used when there is no MICROMETER. MAGNIFICATION OF IMAGES Number of times an object is enlarged by the magnifying lens. Number of times a drawing or image is enlarged or reduced from the original size of the object. FINDING FIELD OF VIEW UNDER LOW POWER Total Magnification = Magnification of Eyepiece x 1. Place the transparent plastic ruler on the Magnification of Objective stage so that the ruler's edge is centered in your field of view under low power: 2. Position the ruler so one of the millimeter markings is just visible to the left side of your circle in your field of view. CHEMICAL COMPONENTS BIOLOGICAL ORGANIZATION How life is constructed. 3. Count the number of whole millimeters that ORGANIC + INORGANIC you see, and estimate the fractions of Compounds comprising life. millimeters that you see. In the above Example is plant cells. diagram, we see 4 whole millimeters, and about ½ of a millimeter. INORGANIC Water, oxygen, minerals, etc. So, in the above example the FOP is approximately 4.5mm. ORGANIC Biomolecules that have a C-backbone or Carbon Backbone that influences the shape of the compounds. Carbohydrates, proteins, lipids, nucleic acids. Diverse because of the attachment of various functional groups to the Carbon Skeleton. 4. It becomes very di cult to accurately PERCENT COMPOSITION OF ORGANIC AND estimate the diameter of the FOV when we INORGANIC COMPOUNDS IN PLANTS switch to higher power. Therefore we can perform a simple calculation to determine Fresh Weight = living weight the FOV under high power: Dry Weight = after oven drying Ash Weight = after incinerating Sir John Paul Domingo BOTANY LABORATORY LBBBION | Term 1 | 2024 - 2025 CARBOHYDRATES Universally used as an energy source of all organisms. Contains Carbon, Hydrogen, and Oxygen or CnH2nOn. Basic unit is Monosaccharide. ○ Simple sugars that consist of only a single sugar molecule. Cu2+ will react to Aldehyde. Aldehyde will give Starch: Plant storage food electrons to Cupric Ions leading to Carboxylic Cellulose: Structural (cell wall) Acid. Cupric Ions will become Cu2O that are Presence of carbohydrates can be detected insoluble in water. using either of the following tests: ○ Benedict’s REDUCING SUGAR ○ Barfoed's Any monosaccharide or disaccharides sugar ○ Iodine containing a hemiacetal. ○ Not complex carbohydrates such as BENEDICT’S TEST starch, glycogen, and cellulose. Test to detect the presence of reducing sugars that are mostly monosaccharides or disaccharides. DIFFERENCES BETWEEN Reducing Sugars HEMIACETALS AND ACETALS ○ Sugars with open aldehydes and/or ketones. HEMIACETAL ○ For ring reducing sugar, they will One OH group and one O-R group react to hemiacetals. attached to same carbon Composed of Copper Sulfate or CuSO4. In equilibrium with aldehyde/ketone. Blue in color as a solution. Red colored solution means a large amount ACETAL of reducing sugar due to the RedOx Two O-R groups attached to same carbon reaction. Not in equilibrium with aldehyde/ketone RedOx Reaction ("locked") ○ Involves reduction and oxidation reactions. REDUCING AGENT Sugars with a hemiacetal are in equilibrium with a ring-opened form containing an aldehyde and will react as a reducing agent toward certain (oxidizing) metal salts such as Cu2+ (Benedict's test, Fehling's solution) and Ag+ (Tollens test). Examples: ○ Other monosaccharides such as galactose, mannose, and ribose. ○ Certain disaccharides such as lactose and maltose. Sir John Paul Domingo BOTANY LABORATORY LBBBION | Term 1 | 2024 - 2025 PROTEINS A sugar without a Hemiacetal is a non-reducing sugar. Primary importance to the structure and function of cells. Basic unit is amino acid. ACETAL ○ Two amino acids are joined together Are “locked” and not in equilibrium with a via peptide bonds. ring-opened form with an aldehyde. ○ Has a C-backbone. Will not react as a reducing agent. ○ Has both the carboxylic acid and amine groups. ○ Has the R-group and H on opposite poles Polypeptide ○ Chain of amino acids joined by peptide bonds. Final shape of a protein determines its function in the cell. WHY IS THIS IMPORTANT? Has four levels of structure. An important test for quantifying blood ○ Primary glucose (Benedict's solution) relies on a ○ Secondary reaction where Cu2+ is reduced by an ○ Tertiary aldehyde, resulting in a color change. ○ Quaternary Presence of protein can be detected using BARFOED’S TEST the Biuret Test. Test to detect the presence of reducing monosaccharides. ○ Disaccharides will react slower due to it being more complex - having two monosaccharides bonded by glycosidic linkage, thus needing to be hydrolyzed to break it. Similar to Benedict's Test. RedOx Reaction ○ The addition of Copper, Acetate, and Acetic Acid reacts faster to monosaccharides. ○ Copper will react with reducing sugars such as cupric ions. ○ Acetate and acetic acid provide acidic pH to the solution. Brick red precipitate means a large amount of reducing monosaccharides. IODINE TEST Test for starches. ○ Starch is a polysaccharide that contains hundreds of glucose molecules in either occasionally branched chains (amylopectin) or unbranched chains (amylose). Has polyiodide chains. ○ Iodine molecules assemble as long polyiodide chains. ○ If there's starch in the solution, this polyiodide chain will insert itself in the amylose and form amylose iodine complex that causes intense blue or black in color. Not all proteins have the four levels of structure. Amylose, as unbranched chains, may sometimes form helices. Sir John Paul Domingo BOTANY LABORATORY LBBBION | Term 1 | 2024 - 2025 BIURET TEST Test to detect polypeptides. Composed of Alkaline CuSO4. ○ The alkaline strengthens the peptide proper complex. Reacts to peptide bonds. ○ The more peptide bonds, the stronger the color reaction from blue to purple. Process ○ Copper will bind to the nitrogen FORMATION OF TRANSLUCENT SPOT atoms. Lipids have a characteristic greasy feel. ○ The nitrogen atoms donate lone When brought in contact with a substance pairs to form coordinate covalent like paper, it penetrates through it bonds with the cupric ions, forming producing a translucent spot. a peptide proper complex. Fats are non-volatile. The spot of grease can never absorb enough heat to vaporize. When the liquid is inside the sheet of paper, it di racts light - translucent phenomenon. SUDAN DYE TEST Lipid soluble dye. Insoluble to water. Positive shows a remnant of red spots. Negative results will only exhibit one layer instead of two. PARAMETERS EXPECTED IN POSITIVE RESULTS OF SUDAN DYE TEST Layers ○ You should see two layers. ○ Two layers indicate the presence of water-insoluble substances. Colors ○ You should see red on the top layer. LIPIDS ○ The Sudan IV will migrate to the top Basic units are fatty acids and glycerol. layer and color it red. This indicates ○ Bounded by ester bonds. that the top layer is fat. Functions as a membrane. It is immiscible to water. Forms such as fat, oil, and waxes. Test to determine the presence of lipids ○ Grease spot ○ Sudan dye GREASE SPOT TEST Simple test for lipids. Lipids have a higher boiling point than water. Positive shows a translucent mark on paper. Sir John Paul Domingo BOTANY LABORATORY LBBBION | Term 1 | 2024 - 2025 THE PLANT CELL ANIMAL CELL VS PLANT CELL VACUOLE Fluid filled spaces not occupied by cytoplasm. In young cells, these are often small or scattered. In older cells, they are often large and fill most of the cell. 90% or more of the volume of a plant cell may be taken up by one or two central vacuoles. Bounded by vacuolar membrane or tonoplast. The main function is to store numerous cell metabolites and waste products. Other functions include: ○ Cell growth ○ Cell pressure and pH maintenance ○ Helps in organelle breakdown and digestion PLASTIDS ○ Recycle certain materials A group of membrane-bound organelles Contains ergastic substances like crystals. occurring in photosynthetic eukaryotic cells. TONICITY All types of plastids develop from Proplastid. Ability of a surrounding solution to a ect ○ Small, green or colorless organelles. the movement of water into and out of the ○ Are unspecialised. cell. CHLOROPLASTS ISOTONIC SOLUTION Green. Water inside and outside the cell are equal. Site of photosynthesis. There is no net movement of water into the cell. LEUCOPLASTS Cell becomes flaccid (limp). Colorless. HYPOTONIC SOLUTION Storage of starch or oil (e.g. amyloplast). Types Water concentration outside the cell is ○ Amyloplast - stores starch greater than inside the cell. ○ Elaioplast - stores oil Water will move inside the cell until it ○ Proteinoplast - site of enzyme swells and the wall opposes uptake. activity The cell becomes turgid (firm). CHROMOPLAST HYPERTONIC SOLUTION Colored or pigmented. Water concentration is higher inside the ○ Yellow, orange or red due to cell than outside the cell. carotenoid pigments Water will move outside the cell and the Produce and store pigments. solute concentration inside the cell will become more concentrated. The cell membrane will shrink and the cytoplasm will clump in the middle. Causes plasmolysis. ○ Process where cells lose water. Sir John Paul Domingo BOTANY LABORATORY LBBBION | Term 1 | 2024 - 2025 TYPES OF PLANT CELLS PARENCHYMA With thin cell walls and protoplast is alive at maturity Chlorenchyma ○ Photosynthetic parenchyma. Storage parenchyma ○ Contain lots of amyloplasts. Aerenchyma ○ Parenchyma cells with large air spaces. Stellate Parenchyma ○ Highly branched parenchyma cells connected to each other by means of the branches. Plant cells don't burst under a hypotonic solution since they have a cell wall. COLLENCHYMA CRYSTALS Sides of the young stems and in the stalk or Since vacuoles store numerous cell midrib of leaves. metabolites and waste products such as With unevenly thickened cell walls. salt, sugar, and organic acids, sometimes Protoplast is alive at maturity. crystallization occurs inside the vacuoles. Cells are somewhat elongated. Varies in terms of: Mainly for support and strength. ○ Shape ○ Size ○ Number May be composed of calcium oxalate or calcium carbonate. Common shapes ○ Prismatic (diamond shaped) ○ Druse (star-like) ○ Rosette (star with many points) ○ Raphide (needle-like) ○ Cystolith (grape cluster) SCLERENCHYMA With a thick secondary wall with lignin. Cells die at maturity, protoplast is dead. Cell walls absorb stains. Functions for transport of water and support. Most common: Sclereids and Fibers Sclereids ○ Short cells that are variable in shape, are common in the shells of nuts and IDIOBLAST the stones of fruits, such as cherries Crystal forming cells in vacuoles. and peaches. Isolated plant cell that di ers from ○ Pears owe their slightly gritty neighboring tissues. texture to the presence of clusters of sclereids. Sir John Paul Domingo BOTANY LABORATORY LBBBION | Term 1 | 2024 - 2025 Fibers DIFFUSION ○ Long, tapered cells that often occur Passive transport. in groups or clumps, are particularly Movement of molecules from a region of abundant in the wood, inner bark, high concentration to a low concentration. and leaf ribs (veins) of flowering The molecules spread out into the available plants. space. Function for protection, water transport For solutes that are readily permeable such and support. as nonpolar and small polar molecules. OSMOSIS Movement of water through a semi-permeable plasma membrane. Region of high water concentration to a low water concentration. Refers to the di usion of water. Does not require energy. WATER POTENTIAL (ΨW) MATERIAL TRANSPORT IN PLANTS Free energy of water. ○ Capacity of water. CELL MEMBRANE ○ Free to di use. Outer boundary of the living part of the cell. Measure of the potential energy in water, or It regulates the movement of substances the di erence in potential energy between into and out to the cell. a given water sample and pure water. Functions as the maintenance of a steady living state or equilibrium in the midst of an Water's capacity to do work can be changed in ever changing environment. other ways as well. It is semi-permeable. When water adheres to a substance, these water molecules form hydrogen bonds to the material and are not as free to di use as are other water molecules: their capacity to do work has decreased. Consider a small beaker of water: if it is pure water, it can flow, move. dissolve material, and hydrate substances, but if a sponge is added, water molecules adhere to the sponge material and can no longer flow or easily dissolve things. TRANSPORT The potential of pure water (Ψwpure H2O) is Across the membrane can either be passive designated a value of zero. or active depending on the need for energy ○ Even though pure water contains input. plenty of potential energy, that energy is ignored. Water potential is a ected by the osmotic potential and pressure or turgor potential. WATER POTENTIAL EQUATION PASSIVE TRANSPORT WATER POTENTIAL (Ψw) Movement of molecules across the cell Plant cells usually have a negative water membrane without the use of ATP. potential, unless they are in equilibrium with pure water. ACTIVE TRANSPORT Such cells are fully turgid and have a Y equal Movement of molecules across the cell to zero. membrane with the use of ATP. ○ To move molecules against their concentration gradients. Sir John Paul Domingo BOTANY LABORATORY LBBBION | Term 1 | 2024 - 2025 OSMOTIC POTENTIAL (ΨS) FLACID CELL IN HYPERTONIC SOLUTION Solute or osmotic potential, the When this cell is placed in a hypertonic contribution made by dissolved solutes. solution, the water will move out of the cell It is always negative in sign. until equilibrium. The addition of solutes lowers the water Plasmolysis potential of the solution proportionally. ○ When water continuously goes out It is the e ect that solutes have on water of the cell, the water will eventually potential. die. ○ In pure water, no solutes are present and osmotic potential is given the value of 0.0 MPa. Adding solutes can only decrease water's free energy because water molecules interact with solute molecules and cannot di use easily; therefore, osmotic potential is always negative. INCIPIENT PLASMOLYSIS Initial stage of plasmolysis. TURGOR POTENTIAL (ΨP) ○ Plasmolysis is the osmotically Pressure or turgor potential is related to induced shrinkage of the cytoplasm. the counter-pressure exerted by the cell The stage wherein the protoplast has lost wall as the expanding vacuole presses the just enough water to pull slightly away plasma membrane against the cell wall if a from the wall. plant cell is placed in a hypotonic medium. ○ The cell has lost very little water, so The e ect that pressure has on water its volume change and osmotic potential. potential change have not been ○ If water is under pressure, the great. pressure potential increases and so ○ However, due to the zero potential does water potential. water potential, the equation is ○ If pressure decreases, so do the pressure potential and water potential. Pressure can be positive (when something ○ If cell has not reached equilibrium is compressed) or negative (when during incipient plasmolysis, it something is stretched). continues to lose water and Potential is measured in units of pressure, protoplast pulls completely away usually in megapascals (MPa) or bars. from the way and shrinks. ○ Water potential continues to become more negative because of the osmotic potential as solutes become more concentrated. Defined as the osmotic condition where 50% of the cells are plasmolyzed. Ψcell = Ψs = Ψs of surrounding solution WATER RELATIONS IN PLANT CELLS Distilled Water ○ Doesn't have impurities. ○ No minerals are present. ○ The pressure potential is 0 because there is no cell wall. FLACID CELL IN HYPOTONIC SOLUTION When this cell is placed in water, equilibrium will take place. When placed in a hypotonic solution, it will become turgid. Sir John Paul Domingo BOTANY LABORATORY LBBBION | Term 1 | 2024 - 2025 OSMOTIC POTENTIAL EQUATION Although plant cells cannot absorb so much water that they burst, water loss can be a serious problem. Imagine that our demonstration cell, now in pure water and with a water potential of 0.0 MPa, is placed in a strong sugar solution with a water potential of -2.0 MPa. Water moves out of the cell, osmotic potential becomes slightly more negative, and the pressure potential drops rapidly. In such a strong solution, long before the cell reaches the pressure potential at equilibrium it loses so much THE CELL CYCLE (MITOSIS) water that the protoplast shrinks in volume and no longer presses against the wall. IMBIBITION A transport mechanism that does not require a semipermeable membrane. Process results in swelling of material or enormous increase in volume. A type of di usion and unrelated to osmosis. In biology, an example would be the absorption of water by seeds and dry wood. Molecules, such as cellulose and starch, usually develop electrical charges when they are wet. The charged molecules attract water molecules, which adhere to the internal surfaces of the materials. Because water molecules are polar, they can become both highly adhesive to large organic molecules such as cellulose and cohesive with one another. CELL SAP The cell sap present in the central vacuole of a beetroot cell contains a red pigment. Bleeding (the escape of this pigment) indicates that the cell's plasma and vacuolar membranes have been damaged. Sir John Paul Domingo