General Biology Lab Manual 2024-2025 PDF

GENERAL BIOLOGY LAB MANUAL 503171 Prepared by: Dr. Mais Krishan 2024-2025  Contents Lab 1: Introduction to Laboratory Safety Rules...... 3 Laboratory Equipment’s............................................... 4 Lab 2: The Micr...

GENERAL BIOLOGY LAB MANUAL 503171 Prepared by: Dr. Mais Krishan 2024-2025  Contents Lab 1: Introduction to Laboratory Safety Rules...... 3 Laboratory Equipment’s............................................... 4 Lab 2: The Microscope........................................................ 9 Lab 3: The cell.................................................................... 21 Lab 4: Biological Macromolecules I.............................. 35 Lab 5: Biological macromolecules part II................... 53 Lap 6: Physical properties of the cell......................... 67 Lab 7: Cell division........................................................... 78 Lab 8: Genetics and blood grouping............................. 96 Lab 9: Animal tissues...................................................... 108 Page | 2  Lab 1: Introduction to Laboratory Safety Rules Laboratory safety: Is a careful process, with the goal of preventing injuries and disease from occurring among students, scientists, lab.staff, and community. Laboratory Safety Rules: ✓ Wear laboratory coat ✓ Know the location of the firere extinguisher & first aid kit ✓ NEVER taste or smell chemicals ✓ If any chemicals touch your skin >> flood the area with water ✓ Discard solids in the waste jar NOT in the sink, only water soluble ✓ waste wash down in the sink ✓ Always read the labels ✓ Clean off your bench top, return material back ✓ All fires & water turned off ✓ Report all accidents to your instructor Page | 3  Laboratory Equipment’s Watch glass Wire gauze Test tube Test tube holder Test tube rack Dropper Glass rod Page | 4 Reagent bottle Erylnmeyer flask Graduated cylinder Funnel Buchner funnel Wash bottle Extension Clamp Lab. Stand Page | 5  Burette Separatory Funnel volumetric flask Page | 6 Filter paper Petri dish Water bath Bunsen Burner Ice-Water Bath Fume Hood Page | 7 Analytical Balance Autoclave incubator Inculcating loop Inculcating needle Forceps Spatula Page | 8  Lab 2: The Microscope The Microscope is an instrument that contains at least one lens, and used to view a specimens like cells, tissues, microorganisms, which are too small to be seen with the naked eye. ✓ Specimens size: between 0.01 mm and 0.1 mm Microscopy involves three basic concepts: 1. Magnification: is the process of enlarging the appearance of an object. Magnification usually written by a number followed by (X). (e.g.10X) (Total magnification= magnification of ocular x magnification of objective) 2. Resolution: the ability to Distinguish two points as separate ones. It measures the clarity of the image. 3. Contrast: Increasing differences between the lightest and darkest parts of an image and thus. The Light Microscope Page | 9 The Light Microscope ✓ Field of view (FOV): the maximum visible are through the lenses of a microscope, and it is represented by a diameter. ✓ FOV is inversely related to the magnification level. Low magnification bigger FOV Page | 10 There are three types of microscopes: I. Light microscope II. Electron microscope III. Research microscope Light microscope: utilizes visible light that passes through a specimen then through the glass lens and the resulting image is then viewed by human eye ✓ Compound light microscope: magnifies objects to about 1000 times, and is useful viewing specimen at the cellular level, the image is inverted. (2-D view) ✓ Dissecting or stereoscopic microscope: magnifies 10X to 50X times , and is useful in dissection because it allows three dimensional view of specimens. (3-D view) Compound Microscope Page | 11 Page | 12 ✓ Parts of Compound Microscope 1. Light source 2. Arm 3. Base 4. Ocular or eye piece lens: magnifies 10 times (10x) 5. Objective lenses: magnifies the object by the factor marked on the particular lens. Scanning power (4x), low power (10x), high power (40x) and oil immersion which gives the largest image (100x) 6. Noise piece: the revolving part to which objective lens are attached. Hold’s multiple lenses allowing the user to various levels of magnification (4,10, 40, 100x) 7. Body tube 8. Stage: supports the slide that is held on it by two clips, and has a hole so that light can shine up through the specimen. Always center the specimen over this hole. 9. Coarse adjustment knob: moves the stage up and down to view an focus the specimen. ( use this knob only with scanning objective lens) 10. Fine adjustment knob : moves the stage up and down 11. Iris diaphragm: regulates how much light go through the specimen 12. Condenser: a lens located above the diaphragm, which concentrate the light before it passes through the specimen Page | 13 ✓ Dissecting Microscope ✓ Dissecting Microscope with a large working distance between the specimen and the objective lens. ✓ Image under Dissecting Microscope Page | 14  The Electron microscope The Electron microscope: utilizes a beam of electron that pass through a specimen then through a set of of magnetic lenses, and the resulting image is projected onto an fluorescent screen or photographic film. Magnification is about 400,000 times ✓ Transmission electron microscope (TEM): used to study the internal ultra structure of cells ✓ Scanning electron microscope (SEM): used mainly to study the details of the surface of specimen Transmission Electron Microscope (TEM) Page | 15 Scanning Electron (SEM) Page | 16 The Research Microscope: as a fluorescent, phase-contrast and dark field Microscope. ✓ The Phase Contrast Microscope: The ONLY type that can examine living cells & tissues activities (Growth, Division & Repair) without killing them. The Wet mount : is the technique of placing the specimen in a droplet of water for viewing with the compound microscope. ✓ A drop of water is used to suspend the specimen between the slide and cover slip Page | 17 To prepare a wet mount: 1. Ask your instructor to give you a clean slide, cover slip, a teasing needle, letter e 2. Place a single drop of water on the slide 3. Place letter e on the drop of water 4. Hold the cover slip between the thumb and the fore finger of one 5. Touch the drop of water with the slide of cover slip and the water will run in a line along the cover slip 6. Gently lower the cover slip over the specimen using a teasing needle and observe under the microscope. 7. Draw (or take a photo) and label your preparation using 4x, 10x, 40x objectives and study the characteristics of image seen with your microscope Wet mount preparation Page | 18  The image under the Compound Microscope The image under the compound microscope is inverted Page | 19 The Laboratory Report Page | 20  Lab 3: The cell ✓ The first scientist to look at cells under a microscope was the English scientist Robert Hooke. ✓ He viewed and described the appearance of cork under the microscope and decide to name the tiny box-like structures that he observed “Cells”. ✓ The cell is the functional & structural There are two types of cells: 1. Prokaryotic cells 2. Eukaryotic cells Page | 21 All cells have the following in common: 1. Plasma membrane: ✓ Marks the border of thecell. ✓ Regulates the passage of materials into and out the cell 2. The genetic material for the cell (DNA) 3. Cytoplasm: the components enclosed by the plasma membrane. 4. Ribosomes Page | 22 Page | 23 Prokaryotic cells Prokaryotes are the oldest kind of organisms on earth. Only bacteria & Cyanobacteria have prokaryotic cells. Prokaryotic cells differ from eukaryotic cells in that they lack a membrane bound nucleus and organelles. Prokaryotic cells much smaller Bacteria are the most numerous organisms on earth Bacteria can be classified by shape into: 1. Cocci 2. Bacilli 3. Spirilla Bacterial shapes Or can be classified by the structure of their cell wall into: 1. Gram positive bacteria 2. Gram negative bacteria Page | 24 Eukaryotic cells Eukaryotic cells occurs in human, animals, plants, fungi, insects Have membrane bound nucleolus (nuclear envelope). DNA is stored in the nucleous. Eukaryotic cells contain membrane bound structures called organelles, including mitochondria and chloroplast Page | 25 Procedure 1-Prokaryotic cell (Bacterium): bacteria lack a nucleus or other internal organelles, and generally very small Examine the prepared slide of different bacterial cell shapes under various magnification Observe the three basic shapes of bacteria: bacilli, cocci, spirilli 2- Eukaryotic cell: Onion epidermal cells Human cheek cells Page | 26 Onion epidermal cells The onion epidermal cells you will observe in this exercise come from a thin, translucent tissue which lines the scales’ outer curve. Page | 27 ✓ Procedure: 1. Get a clean slide. Place a drop of water on the slide 2. Take a piece of onion and fold it so that doesn’t completely break 3. Peel back one half of the onion so that you are able to obtain one layer 4. Place the layer of tissue on a slide over a drop of water and then add a small drop of Lugol solution (iodine solution used to stain) to the slide 5. Place a cover slip on the slide slowly. Lowering it over the sample to avoid creating air bubbles Page | 28 Page | 29 Onion plant cells Page | 30  Human cheek cells The lining inside your mouth is composed of many cell layers that serve to protect your body. ✓ You will examine the top layer of these cells in a wet mount of ✓ cells from the inside surface of your cheek Page | 31 ✓ Procedure: 1. Get a flat-edged toothpick and a clean glass slide 2. Put one drop of methylene blue on your slide 3. Rub the inside of your cheek lightly with the toothpick (you don’t have to scrape) 4. Transfer the material adhering to the toothpick to the glass slide by lightly rubbing the toothpick onto the methylene blue solution on the slide. 5. Dispose of your toothpick with care 6. Cover slip your slide & examine under the microscope 7. Look for single cells or, small groups of cells. The cells will appear as pentagons, the nucleus will be blue, and the cytoplasm will stain more lightly than the nucleous Page | 32  The laboratory Report ✓ Document your observations about human cheek cells & onion epidermal cells, by drawing the cells or by using your Cheek cell investigation worksheet Page | 33 Onion epidermal cell investigation worksheet Page | 34  Lab 4: Biological Macromolecules I Carbohydrates Lab ❖ All living organisms are composed of four classes of Macromolecules: 1. Carbohydrates (CHO) 2. Proteins 3. Lipids 4. Nucleic Acids ❖ The Chemical Properties of different classes depend on the presence of specific Functional Groups Page | 35 ❖ Dehydration reaction ✓ Dehydration reaction: occurs when two monomers bond together through the loss of a water molecule to become a polymer ❖ Hydrolysis reaction ✓ Hydrolysis reaction: the addition of water molecule breaks a polymer into it’s monomers a reaction that is essentially the reverse of the dehydration reaction Page | 36  Carbohydrates Carbohydrates (CHO) functions in living organisms: ✓ Energy-Storage molecules(e.g,. Starch in plants) ✓ Structural function(e.g. Cellulose) The building blocks of CHO are small molecules called sugars CHO are classified according to the number of sugar molecules they contain. Page | 37  ❖Monosaccharides Monosaccharides: the simplest sugar are monomer, these are taken into cells and used immediately for energy. have the molecular formula (CH2O)n They contain hydroxyl groups and carbonyl groups; either a ketone or an aldehyde. ✓ THESE POLAR FUNCTIONAL GROUPS MAKE SUGARS VERY SOLUBLE IN WATER Monosaccharides Glucose vs Fructose Page | 38 Disaccharides Disaccharides: are formed when a dehydration reaction joins two monosaccharides This covalent bond is called a glycosidic linkage Maltose (malt sugar): glucose+ glucose Sucrose (cane sugar): glucose+ fructose Lactose (milk sugar): glucose+ galactose All have the same molecular formula but slightly different formula and slightly different chemical properties. Polysaccharides Polysaccharides are used as:  energy storage compounds by both plants and animals.  Structural components Page | 39 ✓ Identifying carbohydrates You will conduct two tests specific to two important classes of carbohydrates 1) Benedict’s test: to detect reducing sugar 2) Lugol’s test (Iodine test): to detect starch only The Benedict’s test: The Benedict’s test is used to detect reducing sugar (sugars with free aldehyde or ketone group) All monosaccharides are reducing sugars All disaccharides are reducing sugars Except Sucrose When Benedict’s reagent (solution) is heated with the reducing sugar. The sugar is transformed into strong reducing agents by alkaline sodium carbonate. Reduction reaction takes place, copper ions in the blue coloured benedict’s solution is reduced by the sugar and forms a yellowish to red- coloured precipitate. Page | 40 The Benedict’s test Page | 41 The Iodine Test (Lugol’s Solution) The Iodine test is used to detect the presence of starch by staining with iodine (iodine-potassium iodide). ✓ Iodine interacts with coiled polymers only (starch is a coiled polymer of glucose) and gives a blue – dark blue colour. ✓ Other polymers lack then precise coiled structure of starch and do not give the blue colour Page | 42 Iodine test Page | 43 Proteins: Proteins are specialized molecules composed of monomers called amino acids. There are 20 different amino acids, all have a similar basic structure. Proteins are composed of one or more polypeptides The amino acids are linked together by peptide bond (carboxyl group of one amino acid reacts the amino group of the next amino acid in a dehydration synthesis reaction) Page | 44  Identifying Proteins and amino acids 1) Biuret test: to detect proteins 2) Ninhydrin test : to detect amino acid The Biuret test: The Biuret test used to detect polypeptide Biuret Reagent is a 1% solution of CuSO4 (copper sulfate). The reagent reveals the presence of peptide bond, by Cu2+ complexes with the peptide bond of amino acids to give violet colour. Page | 45 The Biuret test ✓ The violet color is a positive result ✓ The intensity of color is related to the number of peptide bonds that react Page | 46 The Ninhydrin test: Ninhydrin reagent reacts with free amino group on an amino acid to form a purple- violet colored substance. Because ninhydrin needs a free amino group, proteins should be heated or denaturated to hydrolyzed into free amino acids. Page | 47 Page | 48 Procedure: In each of the following tests you will include a substance that does not react in the test to serve as a negative control. The Benedict’s test 1. Label four test tubes (1 to 4) 2. Place 1 ml of the following solutions in the corresponding tubes ( water, glucose, sucrose, starch) 3. Add an equal amount of Benedict’s solution to each tube 4. Place the tubes in a boiling water bath for 3 mins 5. Record your results **(Note the formation of precipitate and any colour changes, interpret the result of each test regarding the presence or absence of reducing sugar) Test Solution Result Conclusion tube 1 Glucose 2 Water 3 Starch 4 Sucrose Page | 49 Procedure: The Iodine test ( Lugol’s test ) 1. Label three test tubes (1 to 3) 2. Place 1ml of the following solutions in the corresponding tube (water, starch, glucose) 3. Add an equal amount of lugol’s solution to each tube and shake the tubes. 4. Record your results (Note the colour change and explain why it did or did not occur) Test Solution Result Conclusion tube 1 Glucose 2 Water 3 Starch Page | 50  Procedure: The Biuret test 1. label four test tubes (1 to 4) 2. Place 1 ml of the following solutions ( water, egg albumin solution, glycine, milk) 3. Add 3 drops of Biuret reagent and 1 drop of 10% NaOH to each test tube and mix 4. Note the colour of each tube & record the results Test tube Solution Result Conclusion 1 Glycine 2 Water 3 Milk 4 Egg albumin Page | 51 Procedure: The Ninhydrin test 1. label four test tubes (1 to 4) 2. Place 1 ml of the following solutions ( water, egg albumin solution, glycine, milk) 3. Add 5 drops of Ninhydrin reagent to each test tube 4. heat the test tubes in a boiling water bath for about 5 mins (so the proteins hydrolysed into free amino acid) 5. Note the colour of each tube & record the results Test tube Solution Result Conclusion 1 Glycine 2 Water 3 Milk 4 Egg albumin Page | 52  Lab 5: Biological macromolecules part II Lipids are the class of macromolecules that mostly serve as long-term energy storage, they serve as signaling molecules, and insulation. Lipids are hydrophobic because they consist mostly of hydrocarbons, which form non-polar covalent bonds Lipids are insoluble in polar solvents such as water and are soluble in non-polar solvents such as ether and acetone. Three major classes of lipids are: Triglycerides, Phospholipids, and the Steroids Fats or triglycerides are made of glycerol and three fatty acid chains. They form through 3 dehydration synthesis reactions between a hydroxyl of the glycerol and the carboxyl group of the fatty acid. Fatty acids vary in length (number of carbons) and in the number and locations of double bonds Saturated fatty acids have the maximum number of hydrogen atoms possible and no double bonds, The molecule takes up little space in three dimensions. Unsaturated fatty acids have one or more double bonds, A kink from the double bond increases the amount of three dimensional space that the molecule fills. Page | 53  Saturated fats versus Unsaturated fats Fats made from saturated fatty acids are called saturated fats, and are solid at room temperature Most animal fats are saturated Fats made from unsaturated fatty acids are called unsaturated fats or oils, and are liquid at room temperature Plant fats and fish fats are usually unsaturated Page | 54 Page | 55  A trans fatty acid. Despite an unsaturated bond, the molecule fills as much space as a saturated fatty acid and is solid at room temperature. Trans fats usually arise from artificial saturation techniques (hydrogenation) Hydrogenation is the process of converting unsaturated fats to saturated fats by adding hydrogen These trans fats may contribute more than saturated fats to cardiovascular disease Phospholipids, two fatty acids and a phosphate group are attached to glycerol The two fatty acid tails are hydrophobic tail, but the phosphate group and its attachments form a hydrophilic head Phospholipids are the major component of all cell membranes (phospholipids bilayer) Page | 56 Steroids are lipids characterized by a carbon skeleton consisting of four fused rings Cholesterol, an important steroid, is a component in animal cell membranes Page | 57  ❖ Practical Testing for lipids Lipid solubility test: Lipids are insoluble in polar solvents and soluble in non-polar solvents. For this test the polar solvent is water, the non-polar solvent is mineral oil (mixture of hydrocarbons) ❖ Procedure: 1. Label four test tubes 1-4 2. Fill two test tubes (1&2) with 2 ml of water 3. Fill two test tubes (3&4) with 2 ml of mineral oil ( flower oil) 4. Add 2ml of ( corn oil, starch ) to the test tubes 1&2 5. Add 2ml of ( corn oil, starch ) to the test tubes 3&4 6. Mix the contents of each tube 7. Wait 2 mins 8. Record your observations Page | 58  ❖Practical Sudan red test for fats Sudan red is lipid soluble dye. When Sudan red is added to a mixture of lipids and water, the dye will move into the lipid layer colouring it with red color. ❖ Procedure: 1. Label two test tubes 1 & 2 2. Place 1 ml of oil and 5 ml of water in tube 1 3. Place 5 ml of water in tube 2 4. Add few drops of Sudan IV solution to each tube and shake vigorously 5. Examine each tube carefully, where is the red color formed? A red color indicates the presence of lipids. 6. Record your results and conclusions Page | 59  ❖ Practical The grease spot test A simpler test for lipids is based on their ability to produce translucent grease-marks on unglazed paper. ❖ Procedure: 1. Obtain a piece of brown wrapping paper from your lab instructor. 2. Use a dropper to add a drop of olive oil or com oil on the piece of paper. 3. Add a drop of water on another piece of paper. 4. Let the fluids evaporate. 5. Examine the paper by holding it up to a light source. 6.. Record your results and remember that lipids make unglazed paper translucent (clear) 7. Note the spread of oil on a paper sheet to give a transparent spot (A) while in (B) water did not spread. Page | 60 Nucleic acid DNA and RNA are nucleic acids and make up the genetic instructions of an organism. Their monomers are called nucleotides, which are made up of individual subunits. Nucleotides consist of pentose sugar , a charged phosphate group and nitrogenous base (Adenine, Guanine, Thymine, Cytosine or Uracil) One major difference between DNA and RNA is that DNA contains deoxyribose, and RNA contains ribose sugar DNA DNA is a double helical molecule. Two anti-parallel strands are bound together by hydrogen bonds. Adenine forms 2 H-bonds with Thymine, Guanine forms 3 H-bonds with Cytosine. This AT & GC matching is referred to as complementarity. While the nitrogenous bases are found on the interior of the double helix (like rungs on a ladder), the repeating backbone of pentose sugar and phosphate form the backbone of the molecule. Notice that phosphate has a negative charge. This makes DNA and RNA, overall negatively charged. Page | 61  ❖ Practical DNA extraction from fruit: ❖ Procedure: 1. Mash about 10 gum or 3cm of overripe banana 2. Add 7 ml of salt solution 3. Add 7 ml of liquid detergent and mix 4. Place a filter paper over a beaker 5. Pour about 5 ml of filtrate into a test tube 6. Slowly pour 5 ml of cold ethanol down the side of tube to form a layer on top of the fruit fluid 7. Spool the DNA use a loop Page | 62  Step1: Mash about 10 gum or 3cm of overripe banana Step 2: Add 7ml of salt solution : The salt solution helps the DNA to aggregate (clump together). Step 3: Add 7 ml of liquid detergent and mix The detergent dissolves the lipids in the cell and nuclear membranes and releases DNA into the salt solution Step 4: Place a coffee filter over a cup or beaker and fasten with an elastic band Page | 63  Step 5: Pour about 5 ml of filtrate into a test tube Step 6: Slowly pour 5 ml of cold ethanol down the side of tube to form a layer on top of the fruit fluid. ✓ carefully run the alcohol down the side to form a separate layer on top of the fruit solution ✓ Do not mix the alcohol and banana solution. ✓ Ice-cold 100% ethanol works better Page | 64  Step 7:Spool the DNA: use a plastic loop or glass rod to gently swirl at the interface of the two solutions Step8: The interface is where the two solutions meet. DNA is not soluble in alcohol. bubbles may form around a wooly substance (this is the DNA) Page | 65  ❑ What was the purpose of using the detergent? The plasma membrane around the outside of the cell, and the nuclear envelope, are made of lipids and proteins. The detergent will destroy the cell nuclear membrane to allow DNA to get out. It does this by dissolving lipids and proteins that holds the membranes together. ❑ What was the purpose of using the salt? The salt enables the DNA strands to stick together or "precipitates The positive charge of the sodium interacts with the negatively charged phosphate groups at the 5 'ends of DNA stands to neutralize the molecule. This helps to make the DNA less soluble in the water/alcohol mixture. ❑ What happens when the cold ethanol is added? DNA is not soluble in alcohol. the other components of the mixture stay dissolved in solution. The DNA will become apparent as white mucous like strands that can be spooled with a glass pipette or rod ❑ Would it make a difference. If the ethanol was warm? The colder that DNA solution is. the more likely it will precipitate or solidify Cooling the ethanol increases the amount of DNA precipitated. Page | 66  Lap 6: Physical properties of the cell The plasma membrane is the boundary that separates the living cell from its surroundings The cell membrane consists primarily of phospholipids bilayer. Phospholipids are amphipathic with a polar head and a hydrophobic tail The lipid tails interact with each other and the phosphate heads face the external water environment or the internal cytoplasm of the cell. The plasma membrane exhibits selective permeability, allowing some substances to cross it more easily than others Page | 67  The Small uncharged molecules diffuse through the phospholipids bilayer freely. Polar, charged or large molecules have great difficulty to diffuse through the membrane and require the aid of transmembrane proteins. An example of a transmembrane protein that facilitates movement of a polar substance is aquaporin, which permits the free movement of water. Page | 68 Diffusion: is the net movement of substance from region of high concentration to region of low concentration (down it’s concentration gradient) The movement continues until equilibrium is reached This movement does not require any external energy Page | 69 Factors that affect diffusion: Temperature: Increasing the temperature increases the rate of diffusion. This is because the particles have more kinetic energy, so they move faster Molecular weight: The rate of diffusion is inversely proportional to the size of the molecules. This means that the smaller is the size of the molecule, the higher is the rate of diffusion. Methylene blue: large molecular weight Slower diffusion Methylene blue: large molecular weight Slower diffusion Page | 70 Osmosis is a special case of diffusion. The diffusion of water molecules only from a region of high water concentration (low solute concentration), to a region of low water concentration (High solute concentration) across a selectively semi- permeable membrane until isotonicity is reached. The net movement of solvent. Water moves freely across the plasma membrane of a cell. Page | 71 There are three different types of osmotic solutions: 1. An isotonic solution is one that has the same concentration of solutes both inside and outside the cell. 2. A hypertonic solution is one that has a higher solute concentration outside the cell than inside. 3. A hypotonic solution is the one that has a higher solute concentration inside the cell than outside. Cell walls of a plant retain the shape of the cell despite the state of external tonicity. Page | 72 Plants have rigid cell walls composed of cellulose. ✓ These cell walls permit for maintenance of cellular integrity when the external environment is hypotonic (less dissolved substances). In this situation, the water moves into the cell. Without the cell wall, the cell would burst open from the excessive water pressure entering the cell. This state of swelling is referred to as turgid, resulting from turgor pressure. ( the best situation for plant cell ) ✓ When the exterior environment is hypertonic , (greater amount of dissolved substances), the reverse condition occurs whereby the cellular fluid exiting the cell reduces the size of the cytoplasm. This condition is referred to as plasmolysis Page | 73 Animal cell lacking a cell wall is affected greatly by the tonicity of the environment. ✓ In a hypertonic solution where the concentration of dissolved solute is higher then the interior of the cell, water will be drawn out of the cell. ✓ In a hypotonic solution where the concentration of dissolved solute is lower than the interior of the cell, the cell will be under great osmotic pressure from the environmental water moving in and can rupture the cell. (Lack cell wall) Page | 74 ❖ Procedure of Diffusion 1. Obtain a beaker filled with water. 2. Add 2-3 drops of India ink and observe the motion of the dye inside the beaker. 3. Place the beaker on tripod stand and start the Bunsen burner under the beaker for 5 minutes and observe the rate of movement. Note: Do not boil the water. (The effect of temperature on diffusion). Page | 75 An ink drop gradually dissolves in a glass of water by diffusion Thistle (Osmometer) Demonstration Page | 76 In this experiment, a thistle funnel whose mouth is covered with a semipermeable membrane, is filled with different concentration of egg albumin solution. It is then kept inverted in a beaker containing fixed concentration of egg albumin. The water diffuses across the membrane due to osmosis. Thistle funnel (osmometer): This device consists of a hollow glass bell attached to a long tube. The tube is filled with molasses or sugar solution, and the large opening of the tube is covered with dialysis tubing and secured with a rubber band. The thistle tube osmometer is placed in a beaker of water and clamped to a ring stand. The level of the molasses or sugar solution is indicated with a marker. Page | 77  Lab 7: Cell division Cell division: The process by which a parent cell divides into two or more daughter cells. Cell division occurs as a part of a larger cell cycle Cell division in Prokaryotic cells : Prokaryotes undergo a vegetative cell division known as Binary fission, where their genetic material is segregated equally into two daughter cells. Cell division in Eukaryotic cells, There are two types of cell division: 1) Meiosis: a reproductive cell division, whereby the diploid number (2N) of of chromosomes in daughter cells is reduced by half to produced haploid gametes (1N) 2) Mitosis: a nuclear division in conjunction with cytokinesis (cytoplasmic division), whereby each daughter cell is genetically identical to the parent cell. Page | 78 The Cell cycle: Phases of the Cell Cycle There are two primary phases in the cell cycle: 1. Interphase: This phase was thought to represent the stage between subsequent cell divisions 2. M Phase (Mitosis phase): This is where the actual cell division occurs. There are two key steps in this phase, nuclear division & cytokinesis Page | 79 Cell cycle The interphase further comprises three phases: 1. G1 Phase (Gap 1): The cell is metabolically active and grows continuously during this phase. 2. S phase (Synthesis): The DNA replication or synthesis occurs during this stage. 3. G2 phase (Gap 2): Protein synthesis happens in this phase. The cell division: Mitosis is part of the cell cycle of cell division, associated with growth and repair of tissues, the chromosomes of a cell are copied to make two identical sets of chromosomes, and the cell nucleolus divides into two identical nucle. There are four stages in the Mitosis Phase 1. Prophase 2. Metaphase 3. Anaphase 4. Telophase Page | 80 Mitosis Page | 81 Page | 82 Page | 83 Page | 84 Meiosis (Reductional division): is a process of nuclear division that reduces the number of chromosomes in the resulting cells by half. The resulting cells become specialized “sex cells” or gametes. In organisms that reproduce sexually, chromosomes are typically diploid (2N) or occur as double sets (homologous pairs) in each nucleus Each homolog of a pair has the same sites or loci for the same genes. Meiosis reduces the number of chromosomes to a haploid (1N) or single set. As in Mitosis, Meiosis is preceded by replication of each chromosome to form two chromatids attached at a centromere. First, meiosis includes two rounds of chromosome separation. Chromosomes are replicated before the first round, but not before the second round. The genetic material is replicated once and divided twice. This produces half the original number of chromosomes. (1N) Second, during an early stage of meiosis each chromosome (comprised of two chromatids) pairs along its length with its homolog. This pairing of homologous chromosomes results in a physical touching called synapsis, during which the four chromatids (a tetrad) exchange various segments of genetic material. This exchange of genetic material is called crossing-over and produces new genetic combinations. each chromatid of the chromosomes contains different segments (alleles) that is exchanged with other chromatids. Page | 85  Crossing over between chromatids of homologous chromosomes increases genetic diversity during meiosis I. Synapsis occurs during prophase I as the homologous chromosomes begin to pair up ✓ In Meiosis, the chromosome duplicate and homologous chromosome exchange genetic information (chromosomal crossover) during the first division called meiosis I. ✓ The daughter cells divide again in meiosis II, splitting up sister chromatids to form haploid gametes. ✓ Two gametes fuse during fertilization, creating a diploid cell with a complete set of paired chromosome. Page | 86  Stages and Events of Meiosis Meiosis I Page | 87 Meiosis II Page | 88 Cell division in animal cell versus plant cell A major difference between cell division in plants & animals occurs during cytoplasmic division. Page | 89  ❖Practice: ✓ Mitotic stages in the onion root cell ✓ Interphase: DNA is uncondensed and in the form of chromatin. Individual chromosomes are not visible. The nuclear membrane is intact. ✓ The nucleolus is visible. Page | 90  ✓ Prophase:Chromatin begins to condense into distinguishable chromosomes. These “puffy” structures are seen throughout the nucleus. Nucleoli begin to disappear. ✓ In late prophase (often called prometaphase) the nuclear membrane is no longer visible. ✓ Metaphase: The chromosomes line up in the middle of the cell. Spindle fibers attach to kinetochores at the centromere and extend to the poles of the cell. Page | 91 ✓ Anaphase: Centromeres split, separating each former chromatid into two individual chromosomes. The chromosomes move toward opposite poles. Telophase and Cytokinesis: Chromosomes reach the poles. The nuclear envelopes begin to reform. The formation of a cell plate forms between the two cells to carry out cytokinesis. Page | 92 ✓ The formation of a cell plate forms between the two cells to carry out cytokinesis. Page | 93 Laboratory Report Page | 94 Page | 95  Lab 8: Genetics and blood grouping Genetics: is the study of how genes are transmitted from one generation to the next and how they are expressed to produce specific characteristics. Genes: are units of DNA determines traits, which are located in linear order on the chromosomes. Genes governing variations of the same trait are termed Alleles. Alleles: the alternative forms or versions of a gene (one from each parent) Multiple alleles: three or more alleles for a particular gene (example: ABO blood group) Page | 96 An individual can have a pair of alleles for a particular gene, which may be homozygotes, heterozygotes, dominant, or recessive. Homozygous: when you inherited two identical alleles (versions) of a particular gene from each of your parents Heterozygous: when inherited two different alleles of a particular gene Recessive allele will produce its effect only in homozygous individual, only show their effect if the individual has two copies of the allele. (aa) Dominant allele will produce its effect in either a homozygous or a heterozygous individual. (AA, Aa) Page | 97  The genotype of an organism is its genetic constitution, the pair of alleles for specific characteristics is represented by letter symbol. (Genetic code: AA, Aa, aa ) The phenotype is the observable result of the genotype, ie, what the organism looks like because of its genotype. (Appearance: brown color) Page | 98  ❖ Procedure ❖ Mendelian genetics: This exercise deals with the action of human genes on discrete, easily observable traits. You will make observations of these traits in yourself and your classmate to gain an understanding of how particular genes are expressed in the human organism. Human Genetics A Some observable Human/traits Most of our traits are complex, involving many genes or interactions between genes. A number of traits listed in Table 1, exhibit Mendelian inheritance, and are known to be single-gene traits (expressions of two alleles at one gene locus). 1. Examine your phenotype for each trait listed in Table 1 and fill in Table 2. 2. List your possible genotype (s) for each trait. Page | 99 Table 1: Single gene human genetic traits and their alleles Page | 100 Page | 101  ❖ Sex influenced characters: Pattern baldness appears in both sexes, but it appears more clearly in males than in females Non-Mendelian Genetics ✓ Co-Dominance and multiple alleles ✓ Co-dominance is said to occur when there is an expression of two dominant alleles. The prototypical case for this is the human ABO blood grouping. ✓ Three alleles exist in the ABO system: A, B and O. This results in four blood types: A, B, O and the blended AB. Page | 102 The Rh-groups Blood type Genotype Antigen Antibodies (phenotype) present present in the blood Rh+ DD or Dd Rh None Rh- dd None None Page | 103 ❖Procedure 1. Obtain a clean glass slide and divide it using a pencil marker into three equal sections. Label them as: Anti-A, Anti-B, and Anti-D. 2. Add one drop of the three types of sera provided to you by your instructor to the proper sections 3. Wipe the tip of your finger with a piece of cotton saturated with alcohol to sterilize it. 4. Puncture the sterile area of your fingertip with a sterile lancet to obtain a good drop of blood. 5. Add one drop of the blood to each anti-sera on the slide, and then wipe your finger sterile. 6. Use the tips of toothpicks to mix the blood with the sera. 7. Wait about 2 minutes then examine for the signs of agglutination. You may use a microscope at a low magnification to see the agglutination. (Agglutination appears when the antibody combines with its respective antigen present on the surface of the red blood cell) Page | 104 Page | 105  Laboratory Report I Table 2: Mention if you have the following phenotype what is your possible genotype? Trait Your Phenotype Your possible Parents phenotype Genotype if known Bent pinky (B,b) Pattern baldness (P,p) Hair colour (H,h) Eye colour (E,e) Tongue rolling (R,r) Widow's peak W,w Mid-digital hair (M,m) Attached ear lobe(A,a) Page | 106  Laboratory Report II: My blood type ABO,Rh antigens ABO, Rh antibodies My possible My possible (Phenotype) present in my blood present in my blood genotypes for ABO genotype for Rh blood group group Page | 107  Lab 9: Animal tissues Multicellular organisms consist of groups of cells with similar structure and function. (Such groups of cells make Tissues) Different types of tissues make Organs (stomach, nose, heart) Different organs comprise an Organ System (digestive system) Various organ systems work in harmony to form an Organism Histology: the science that study the normal tissues There are four types of tissues in our body 1. Epithelial tissue 2. Connective tissue 3. Muscular tissue 4. Nervous tissue A.Each kind of tissue is composed of cells with characteristic size, shape and arrangement Study the type (name) of the tissue; the location; the composition ; and the function of each studied tissue. You will examine prepared slides of variety of tissues, most slides have section of tissues usually 6-10 μm thick Sections are stained with hematoxylin dye which gives nuclei dark blue color, and eosin which stains cytoplasm in light red color Page | 108 I. Epithelial tissue: cover body surface (as in epidermis of skin) or line tubular organs ( as in small intestine) The Functions of epithelial tissue: protection , secretion, absorption, sensation There are different types of epithelia, but all share the following features: have one free space, lack blood vessels and attach to basement membrane Epithelial tissue classified based on: 1. Cell shape ( Squamous, Cuboidal, Columnar) 2. Number of cell layers (Simple, Stratified) 3. Special type op epithelia is called Pseudostratified Page | 109 A. Simple squamous epithelium: comprised of flat cells with wide central part which contains a nucleolus ✓ Function: to exchange materials ✓ Location: alveoli of lungs and capillaries B. Simple cuboidal epithelium: consist of cells with almost equal sides ✓ Function: secretion or absorption ✓ Location: lining kidney tubules Page | 110 C. Simple columnar epithelium: consist of elongated cells with basal nuclei ,some of theses cells are goblet cells ( secret mucus) ✓ Function: secretion & absorption ✓ Location: lining of the small intestine Page | 111 D. Pseudostratified epithelium: is a type of epithelium which comprise only a single layer of cells, all cells reach the basement membrane. They are found in the linings of the trachea as well as the upper respiratory tract. ✓ Function: absorption and secretion of mucus, protection from foreign particles ✓ Location: lining of trachea ✓ Example: Ciliated pseudo stratified columnar epithelium between these cells there are goblet cells ( mucus secreting cells ) this type of epithelium present in the respiratory tract Pseudo Stratified Epithelium in the Trachea Page | 112 E. Stratified squamous epithelium: two or more layers of cells ✓ Function: protection of organs which are exposed to high degree of friction with external or internal environment. ✓ Divided into two subtypes: 1. Keratinized ( skin) 2. Nonkeratinized (esophagus) Stratified squamous epithelium Page | 113 F. Transnational epithelium: it consist of many cell layers with round nuclei , cell shape vary depending on wether the bladder is relaxed (dome shape) or stretched (elongated). ✓ Location: in urinary tract & urinary bladder ✓ Function: of transitional epithelium is to allow tissue to expand and contract. Transitional Epithelium in the ureter & urinary bladder Page | 114 II. Connective tissue: this tissue made up of cells, fibers ( collagen, elastic fibers ) and ground substance (solid =bone , semisolid=cartilage , liquid= blood). ✓ This tissue is highly vascular ( except for cartilage) ✓ Functions: connects different parts of the body together, offers support, defense and protection to different organs, storage of energy ✓ According to composition & functions, connective tissue is divided into the following types: 1. Proper connective tissue ( Dense , Loose) 2. Special connective tissue ( Blood , Bone , Cartilage) 1. Proper connective tissue: A.Loose (areolar) Connective Tissue: it consists of cells ✓ Fibroblast: the most common type have elongated oval shaped nuclei, ✓ Mast cells: are oval shaped & have lots of granules contains heparin & histamine, ✓ Macrophages: appear as a round cells with irregular outlines) scattered in a matrix which contains collagen & elastic fibers ✓ Function: this tissue fills in the spaces between other tissues & its cells have important role in human body’s defense Loose (areolar) Connective Tissue: Page | 115  B. Adipose connective tissue: the main function of adipose tissue is to store energy in the form of lipids (fat) C. Dense Connective Tissue : this type has tightly packed collagen fibers which make this tissue stronger than the loose tissue. Two types of dense connective tissue : 1. Regular dense connective tissue 2. Irregular dense connective tissue Adipose connective tissue: A. Regular dense connective tissue : in which the fibers are arranged parallel B. Irregular dense connective tissue : in which the fibers are randomly arranged Page | 116  2. Special Connective tissue: includes blood, cartilage and bone Blood: considered as a a connective tissue because it is composed of cells (red, white and platelets) and matrix ( plasma) I. Red blood cells (Erythrocytes): numerous pink - stained cells which are biconcave disk without a nucleus. 2. Platelets (Thrombocytes): fragments of cells clumped together and have one small blue-stained granule. 3. White blood cells (Leukocytes): regarding the texture of cytoplasm and the shape of nucleus they maybe; Granulocytes or Agranulocytes 1. Granulocytes: have a granular cytoplasm and a lobular nucleus of different shapes: ✓ Neutrophils: the cytoplasmic granules stained by neutral dye and have a faint blue color, the nuclei are made of 3-5 lobes which are connected with thin threads of chromatin ✓ Eosinophils: contain pinkish granules which are stained by acidic dyes (eosin) and have bilobed nucleus. ✓ Basophils: contain large dark-blue granules which are stained by basic dyes (methylene blue) and have an S-shaped nucleus. 2. Agranulocytes: have nongranular cytoplasm and a large nucleus of different shapes : ✓ Lymphocytes: have a round nucleus which occupies most of the cell. ✓ Monocytes: have a hors-shoe shaped nucleus. Page | 117 A. Erythrocyte B. Large lymphocyte C. Neutrophil, segmented D. Eosinophil E. Neutrophil, segmented F. Monocyte G. thrombocyte H. Lymphocyte I. Neutrophil, band J. Basophil Page | 118 Cartilage: consist of cartilage cells called Chondrocytes which are located in the lacunae ( the small holes in the matrix) Chondrocytes are mainly responsible to produce collagen and the extracellular matrix that will lead to the maintenance of cartilaginous tissues within joints ✓ There are three types of cartilage: hyaline, elastic and fibrous Hyaline cartilage in (Trachea) provides structural support to the respiratory system Page | 119 Bone: this type of connective tissue either hard ( compact bone) or flexible (spongy bone) ✓ Bone tissue is composed of Osteocytes (bone cells ) trapped into lacunae ✓ Osteocytes communicate with one another and with capillaries by canaliculi, they are arranged around central canals (Haversian canals) in concentric layers called Lamellae. This type of arrangement is called Haversian system. Page | 120 MUSCLE TISSUE : Muscle tissue is composed of muscle cells (Myofibers) which contain many small,logitudinal, parallel contractile fibers called myofibrils. o There are three types of muscle tissue: 1. Smooth muscle 2. Skeletal muscle 3. Cardiac muscle A. Smooth muscle Composed of unstriated, spindle-shaped cells with a central nucleus. The unstriated fibers are found in the walls of the viscera, they work (involuntary). B. Skeletal muscle. This type is striated by light and dark bands, not branched, each muscle cell has man peripheral nuclei (syncytium). These muscles are connected to the skeleton and contract (voluntary) C. Cardiac muscle (muscle of the heart). The cardiac muscles are found only in the wall of the heart and contract (involuntary). *The cardiac muscle cells as the skeletal are striated but branched with a central nucleus. There are intercalated disks which join the myofibers together. Page | 121 Smooth muscle Skeletal muscle ( Striated muscle) Page | 122 Cardiac muscle Nervous tissue: is composed of specialized cells called neurons. ✓ Each neuron has a cell body, a number of processes called the dendrites, and one axon. Page | 123