Biology Lab Manual.pdf

University of Sharjah College of Health Sciences Medical Laboratory Sciences Department Biology Laboratory Manual Fall 2023-2024 Compiled by: Omar Chebbo 1 Tentative Biology Practical Topics: practical...

University of Sharjah College of Health Sciences Medical Laboratory Sciences Department Biology Laboratory Manual Fall 2023-2024 Compiled by: Omar Chebbo 1 Tentative Biology Practical Topics: practical Topic 1 Lab Safety and Scientific Measurements 2 Microscopy I – Parts and function, letter e 3 Microscopy II - Wet Mount preparations (Animal and Plant cells), 4 Chemical Composition of Cells 5, 6 Tissues, Epithelial, Connective, Muscular and Nervous tissues 7 Cell Structure and Function. Osmosis and Diffusion 8 Blood and Blood type 9 Cell Cycle of Eukaryotic cells, Mitosis 10 Energy Requirement and Ideal Body weight 2  Chapter 1 Practical 1 Lab Safety and the Metric System Objectives: 1. Identify and apply the correct lab safety procedures to follow for a variety of scenarios. 2. Learning the metric system and conversion factors in the metric system. 3. Conversion between Fahrenheit scale and Celsius scale. Lab Safety Each laboratory is a restricted area. Enrolled students may work in a lab only when there are authorized personnel present. Friends of students in lab classes will not be allowed to “visit” inside the laboratory. Students are not permitted into the storage rooms or prep areas unless given specific permission by their instructor or lab personnel. Ensuring safety in the laboratory is the responsibility of everyone working in the lab. Please follow these guidelines carefully. General Safety Rules and Procedures 1. Know all emergency exit and know how to use fire extinguisher. 2. Use personal protective equipment. 3. No food or drinks permitted in the laboratory at any time. 4. Only closed-toe shoes are to be worn in the laboratory. 5. Keep hand and other objects away from the face, nose, eyes, ears and mouth. 6. Work areas and surfaces must be disinfected before and after use. 7. Laboratory coat must be worn and buttoned up during the laboratory. 8. Never smell or taste any chemical to identify it. 9. If skin contact with any chemical or body fluid occurs, wash immediately. 10. Hands must be washed before leaving the laboratory. 11. All unnecessary books, bags, and any other item must be kept in the drawers. 12. Dispose of the waste in the proper containers. 3  13. Contact lenses are not permitted to be worn in the laboratories. 14. Return all chemicals, reagents, cultures and glassware to their designated places. 15. Report any broken items in the lab. 16. Immediately contact the course instructor and/or laboratory supervisor in case of injury. 17. Follow all instructions given by the course instructor and/or laboratory supervisor. 18. Wipe clean your microscope lenses before leaving the laboratory. Carry the microscope safely. The Metric System: The metric system is based on units of 10. The base units of measure in the metric system are meter (m) for length, the liter (L) for volume, the gram (g) for mass and Kelvin (K) for temperature. More common for temperature, however, are degrees Celsius (oC). Whether measuring length, volume, or mass, the prefixes listed below are used to designate the relationship of a unit of measure to the base unit (i.e., m, L, or g). Prefixes for metric system Kilo Hecto Deca Basic unit meter, liter, or gram X Deci Centi Milli 1 kilo is equal to 10 hecto and 1 hecto is equal to 10 deca. So, 1 kilo is equal to 10x10=100 deca. 1 kilometer is equal to 100 decameters. 4 There 2 more units in the metric system that are commonly used in biology: - The micro (μ) which is 1/1000,000 or 10-6of the basic unit. - The Nano (n) which is 1/1000,000,000 or 10-9 of the basic unit. Example conversion 1- If you are asked to convert 2 kL to mL, begin by considering the conversion factors that you know. From kilo to hecto is 10 times, from hect to deca is 10 times and from deca to liter is another 10 times. So from Kilo to liter you must multiply by 10x10x10=1000 (going from up to down). So, 2 kiloliters = 2 x 1000 = 2000 liters. 2- if you are asked to convert 2500 millimeters to decameters - begin by considering the conversion factors that you know. From milli to centi divide by 10, from centi to deci divide by 10, from deci to meter divide by 10, and from meter to deca divide by 10. So from milli to deca you must devide by 10x10x10x10=10000. (going up) 2500 millimeters = 2500/10000 = 0.25 decameter Other examples 1 km = 1 x 1,000 = 1000 meters 25 meter (m) = 25x10 = 250 decimeters (dm) 1 meter (m) = 100 centimeters (cm) 1 meter (m) = 1,000 millimeters (mm) 1 liter (m) = 1,000,000 microliters (μm) 250 milligrams = 250 /1000 = 0.250 grams Celsius vs. Fahrenheit The Celsius scale, previously known as the centigrade scale, is a temperature scale used by the International System of Units (SI). This scale is used by all countries in the world, except the United States. It is named after the Swedish astronomer Anders Celsius (1701–1744), who developed the temperature scale. The Celsius scale is based on 0 °C for the freezing point of water and 100 °C for the boiling point of water at 1 atmospheric pressure. 5 The Fahrenheit scale is a temperature scale named after the Dutch-German-Polish physicist Daniel Gabriel Fahrenheit (1686–1736). The Fahrenheit scale is used in the U.S. On this scale, 32° is the freezing point of water and 212° is the boiling point. Conversion between Fahrenheit and Celsius: A- Celsius to Fahrenheit 5 C= (F - 32) 9 B- Fahrenheit to Celsius 9 F = C + 32 5 Examples: 1- Conversion from Celsius to Fahrenheit 79 oC in o F is → F= 9/5C +32 = 9/5x79 + 32 = 174.2 o F 2- Conversion from Fahrenheit to Celsius 180 F in C is → C = 5/9(F-32) = 82.2 oC 6 Recommended background reading (optional): 1. Baruch’s Biology Lab Safety Tutorial http://www.baruch.cuny.edu/tutorials/weissman/biolab/ 2. Science Made Simple http://www.sciencemadesimple.com/metric_system.html 7  Chapter 2 Practical 2 and 3 THE MICROSCOPE Objectives and learning outcomes: 1. To identify the parts of the microscope and the function of each. 2. To describe and demonstrate the proper care of the microscope 3. To define total magnification and resolution. 4. To demonstrate proper focusing technique. 5. To estimate the size of the objects in the field. 6. To prepare and focus a wet mount slide. Key terms: Materials: Compound microscope Stereomicroscope (Binocular Dissecting Microscope) Prepared slide of the letter e Prepared slides: Human blood smear Immersion oil and Xylene Lens paper Toothpick Alcohol Slides box Cove slips Methylene Blue Scalpel Onion Iodine solution 8 Introduction: The compound microscope is an expensive precision instrument that requires special care and handling. In this activity, you will learn the parts of a compound microscope, the function of each part, and the proper care and use of the microscope. Care and Structure of the Microscope Follow the directions of the instructor. Observe the microscope parts, Fig. (2.1), and recognize all the parts of the microscope. You should observe the following rules when handling and using the microscope: 1. Hold the microscope in an upright position with one hand supporting the base and the other hand holding its arm. 2. Report to the instructor if there were any damage or broken parts of the microscope. 3. Clean all the lenses before and after use. Always use lens paper with a drop of Xylene to clean the lenses. Use circular motion to wipe the lenses. 4. Adjust the ocular lenses, according to the suitable position for your eyes. 5. Begin the focusing process with the lowest-power objective lens, changing to the higher – power lenses as needed. 6. Use the coarse adjustment knob only with the lowest- power lens. And use the fine adjustment knob with the higher –power lenses. 7. Always switch off the light bulb, when you don’t need to use the microscope. 8. At the end of the observation, al remember to remove the slide from the stage, lower down the stage, rotate the lowest- power objective lens into position, switch off the light bulb and un-plug the wire from the socket. 9. Inform your instructor if any technical problem arises. Activity 1 Identify the parts of the microscope. 1- Obtain a microscope and identify the following parts of the microscope Base: Supports the microscope, houses the illuminator and the light intensity control. Mechanical Stage: Table of the microscope, where the microscope slide is placed. Condenser: Small sub stage lens that condense the light on the specimen. 9  Iris diaphragm: Wheel-shaped device that regulates the amount of light passing through the specimen. Head or body tube: Houses the prisms, eyepiece tube, and lenses Arm: Vertical portion of the microscope connecting the base to the head. Nosepiece: Revolving piece that carries the objective lenses Objectives: Component that magnifies the images of the specimen to form an enlarged image Ocular (eyepiece): Upper optical component that further magnifies the primary image and brings the light rays into focus Coarse adjustment knob: Used for larger focusing. Fine adjustment knob: Used for precision focusing. Stage Control knob: moves the stage left and right and also back and forth 10  2- Record the magnification of each objective lens of your microscope in report part, 2.1. 3- Rotate the revolving piece, till it clicks into position. Move the adjustment knobs, noting the distance that the stage moves. Magnification and Resolution: In the compound microscope, magnification is achieved through the multiplication of the magnifying power of the ocular lens (10 X) and the selected objective lens (4X, 10X, 410X or 100X). The objective lens magnifies the specimen to produce a real image that is projected to the ocular. This real image is magnified by the ocular lens to produce the virtual image. The compound microscope has certain limitations. The resolution (or resolving power), that is the ability to discriminate two close objects as separate, is actually limited. The compound microscope has a resolution of 0.2m. Objects closer than of 0.2 m are seen as a single fused image. Activity 1: Viewing objects through the microscope: 1- Obtain a prepared slide of the letter e. Adjust the condenser to the highest position and switch the light source of the microscope. 2- Place the prepared slide on the stage and place the spring clips on the two sides of the slide. Make sure that you can see the light beam passing through the specimen. 3- Turn the (scanning) or lowest- power objective lens in position over the slide. Use the coarse adjustment knob to focus slowly, in order to bring the objective lens as close as possible to the slide. 4- Look through the ocular lens. Adjust the space between the two ocular lenses, as it suits your eyes. Use the coarse adjustment knob to focus slowly the letter e. Complete the focusing with the fine adjustment knob, if needed. 5- Calculate the total magnification. …….. x ………. =................. X 6- Describe the apparent orientation of the letter e. …………………………………………… 11  7- Change the objective lens. Without touching the focusing knob, rotate the high power lens out of position, till you hear a “click” like sound. Start with 10X objective lens, then 40X. Calculate the total magnification and the working distance each time………… 8- Remove the letter e slide. Be careful when removing the slide. Always remember to lower the stage to the lowest level before removing the slide from the stage. 9- Bring any other prepared slide form the working area, (Human blood smear…etc.). Follow all the previous steps, from 1 to 9. Activity 2: Preparing and observing wet mount When a specimen must be prepared for observation, the object should always be viewed as a wet mount. A wet mount is prepared by placing a drop of liquid on a slide or, if the material is dry, by placing it directly on the slide and adding a drop of water or stain. The mount is then covered with a cover slip (Figure 2.2). The following procedures demonstrate how to make a wet mount of an animal cell and a plant cell. Figure 2.2 Procedure for preparation of a wet mount. A- HUMAN EPITHELIAL CELLS Epithelial cells cover the body's surface and line its cavities. 1. Obtain toothpick and sanitize it with alcohol. 2. Gently scrape the inside of your cheek with the toothpick. 3. Place the scrapings on a clean, dry slide. 12 4. Add a drop of very weak methylene blue stain and cover with a cover slip. 5. Observe under the microscope. Start with the scanning power objective to find some cells, and then observe under both low and high power. 6. Locate the cell membrane, the cytoplasm, and the nucleus. 7. Make a drawing of what you see. In the report part 2.4. Be sure to label the drawing with what it is, and what power you were using when you made your drawing. B- PLANT CELLS 1. With a scalpel or your fingers, strip a thin, transparent layer of cells from a piece of onion. 2. Place it gently on a clean, dry slide. 3. Add a drop of Iodine (Logol’s solution) stain and cover with a cover slip. 4. Observe under the microscope and draw what you see in the report part 2.4 be sure to label your drawing. 5. Locate the cell wall. Is a nucleus visible? …………………………………………….. 6. Count and record the number of cells across the diameter of the high power field, both lengthwise and side to side. …………………………………………………………………………………………… 7. Using the number you calculated in activity 3, calculate and record the length and width of an onion cell in micrometers. …………………………………………............................................................................ 8. Record some obvious differences and any similarities between the human cheek cells and the onion cells, in the report part 2.4. 13  Chapter 3 Practical 4 Chemical Composition of Cells Learning Outcomes: 1- Proteins. Testing for the presence of proteins 2- Carbohydrate. Monosaccharide, disaccharide, and polysaccharide. Testing for the presence of carbohydrates. 3- Lipids, testing for the presence of lipids Introduction In this laboratory, you will be studying proteins, carbohydrates (monosaccharide, disaccharides, polysaccharides), and lipids. Large organic compounds form during dehydration reaction when smaller molecules bond as water given off. During hydrolysis bonds are broken as water is added. Fat (triglyceride) contains one glycerol and three fatty acids. Proteins and carbohydrates are polymers because they are made up of large numbers of smaller molecules called the subunits. Proteins contains a large number of amino acids (the subunit) joined together by a peptides bond. A polysaccharide such as starch contains a large number of glucose molecules joined together by glycosidic bond. 1- Proteins Proteins are polymers of amino acids joined together by peptide bonds. Proteins have numerous functions in cells and they are divided into: a- Functional proteins: antibodies are functional protein that combine with disease causing pathogens as part of body’s immune response. Enzymes, such as amylase, lipase, and pepsin, are also functional proteins that speed up the biological reactions. b- Transport proteins: Hemoglobin, in the red blood cells, transport oxygen throughout the body. Albumin is another transport protein in the blood that transport fatty acids. c- Regulatory proteins: control cellular metabolism like insulin regulates the amount of glucose in the blood. 14 d- structural proteins: include keratin, which is found in hair and nails, and myosin, which is found in muscle. Dipeptide Amino acid Test for Proteins using Biuret reangent Biuret reagent: Biuret reagent is a blue solution made of copper sulfate and sodium hydroxide. This reagent is used to test for the presence of protein in the sample. The reagent changes color to purple in the presence of proteins. Procedure: A- Label 5 test tubes as follow:. 1- label tube 1 as water and add to it 2 ml of water. 2- Label tube 2 as protein and add to it 2 ml of protein solution (Albumin) 3- Label tube 3 as unknown 1 and add to it 2 ml of unknown 1 solution 4- Label tube 4 as unknown 2 and add to it 2 ml of unknown 2 solution 5- Label tube 5 as unknown 3 and add to it 2 ml of unknown 3 solution B- To each tube add 3 drops of Biuret reagent Conclusions: Observe the color change in each tube and draw a conclusion of the content of the unknown solutions. 15 2- Carbohydrates Carbohydrates includes sugars and molecules that are polymers of sugars. Glucose, which has only one sugar unit, is a monosaccharide. Fructose and galactose are also monosaccharides. Sucrose (glucose-fructose), lactose (galactose-glucose), and maltose (glucose-Glucose) are disaccharides. Starch, glycogen, and cellulose are polysaccharides, which are polymers made up of chains of glucose units. Glucose is used by all organisms as an energy source. Animals store glucose as glycogen and plants store glucose as starch. The plant cell walls are made of cellulose. Glucose Test for starch or glycogen using iodine solution Iodine Solution: iodine solution is used to test for starch. Iodine solution is brown in color. When adding iodine solution to a solution containing starch the color shifts from brown to a deep purple to black. Procedure: A- Label 5 test tubes as follow:. 1- label tube 1 as water and add to it 2 ml of water. 2- Label tube 2 as starch and add to it 2 ml of starch solution 3- Label tube 3 as unknown 1 and add to it 2 ml of unknown 1 solution 4- Label tube 4 as unknown 2 and add to it 2 ml of unknown 2 solution 5- Label tube 5 as unknown 3 and add to it 2 ml of unknown 3 solution B- To each tube add 3 drops of Iodine solution 16 B- Conclusions: Observe the color change in each tube and draw a conclusion of the content of the unknown solutions. The left tube is negative for starch, brown is the color Of iodine. The right tube is positive for starch the color turns into deep purple or black. Testing for Glucose using Benedict Reagent Benedict reagent: Benedict reagent, blue in color, is a chemical mixture of sodium carbonate, sodium citrate and copper(II) sulfate. It is used to test for the presence of glucose in a solution. When Benedict reagent is added to a solution containing glucose, the color change can range from green to orange red after heating the tube in boiling water. The color change depends on the concentration of glucose present in the solution. See table below Chemical Benedict reagent color after heating 1 Water Blue (no Change) 2 Glucose Varies with concentration: Very low Green Low yellow Moderate yellow orange High orange Very high orange red 3 Starch Blue (no change) 17 Procedure A- Label 5 test tubes from as follow:. 1. label tube 1 as water and add to it 2 ml of water. 2. Label tube 2 as glucose and add to it 2 ml of glucose solution 3. Label tube 3 as unknown 1 and add to it 2 ml of unknown 1 solution 4. Label tube 4 as unknown 2 and add to it 2 ml of unknown 2 solution 5. Label tube 5 as unknown 3 and add to it 2 ml of unknown 3 solution B- To each tube add 5 drops of Benedict reagent C-Heat all the 5 tubes in a boiling water bath of 5 to 10 minutes Conclusions: Observe the color change in each tube and draw a conclusion of the content of the unknown solution. 3- Lipids Lipids are compounds that are not soluble in water and soluble is solvents, such as alcohol and ether. Lipids include fats, oils, phospholipids, and cholesterol. Typically fats and oils are composed of three molecules of fatty acid bonded to one molecule of glycerol. Phospholipids have the same structure as fats except in place if the third fatty acid there is a phosphate group. Steroids are derived from cholesterol. 18 Fats is long term stored energy in human body found in adipose tissue. Phospholipids are found in the plasma membrane of cells. Cholesterol are imbedded in the cell membranes and steroids are very important compound in the body; for example the sex hormones are steroids. Glycerol + 3 Fatty acids → triglyceride 19  Chapter 4 Practical 5 and 6 Body tissues Introduction: Cells are the building blocks of life. They are usually specialized in structure and function. A group of cells that are similar in structure and function are called tissues. The four primary tissue types-epithelial, connectives, nervous and muscular- have distinct structures, patterns and functions. To perform specific body functions, the tissues are organized into organs such as heart, kidneys, and lungs. Most organs contain several types of tissues, and the arrangement of these tissues determines the organs structure and function. Out comes: The students will be able to observe, identify, and draw cells and tissues listed within the lab as well as identify tissue slides under the microscope Objectives: ▪ To familiarize the student with the major similarities and dissimilarities of the primary tissue. ▪ To understand the basic structure, function, and location of the four tissue types found in human body. 1- Tissue types ▪ Epithelial Tissue 1- Simple squamous epithelium. - Simple cuboidal epithelium - Simple columnar epithelium - Stratified squamous epithelium - Pseudo stratified columnar epithelium ▪ Connective Tissue - Loose connective tissue - Dense connective tissue - Hyaline Cartilage - Adipose tissue - Compact bone - Blood 20  ▪ Muscular tissue - Skeletal muscle - Cardiac muscle - Smooth muscle ▪ Nervous tissue 1- Epithelial Tissue Epithelial tissue forms a continuous layer over the entire body surface and most of the body’s inner cavity. Therefore, epithelial tissue has various functions including protection against anybody injury and bacterial invasion, protection against drying out, absorb nutrients, produces and release secretions. The epithelial tissue is classified according to: 1. Cell shape; Squamous (scale like), cuboidal (cube like) and columnar (column-shaped). 2. Cell arrangement (layers); - Simple epithelium, which consist of one layer of cells attached to the basement membrane. - Stratified epithelium, which consist of more than one layer of cells a- Simple Squamous Epithelium: Is a single layer of thin, flat, many-sided cells each with a central nucleus. Lining the internal cavities of the: Heart & lungs, Urinary and the blood vessels Figure 1: Squamous epithelium under a microscope 21 b- Simple Cuboidal Epithelium: Is a single layer of cubic shaped cells each with central nucleus lining the: - kidney tubules and ducts of various glands - Function: absorption and secretion Figure 2: cuboidal epithelium looks like under a microscope c- Simple Columnar Epithelium Is a single layer of tall, cylindrical cells each with a nucleus near the base lining the: Digestive tract from the stomach to the anus. This tissue contains mucus-secreting cells called goblet cell; it has a goblet- shaped and a clear interior. The goblet cells contain mucus, which may be stained a light blue. -Function: Absorption of nutrients, secretion of mucous, enzymes and other substances. Figure 3: Simple columnar Epithelium under microscope 22 d- Stratified Squamous Epithelium (Non keratinized) Consist of many layers, the inner most layers produce cells that pushes the other cells towards the surface and become flattened. The inner most layer; basil cells are cuboidal or columnar; the outer most layer of flattened surface cells are squamous; this layer of cells remain soft and moist. They are lining the mouth, the throat Function: protects underlying tissues in areas subjected to abrasion Figure 4: Stratified Squamous Epithelium (Non-keratinized) under a microscope. e- Stratified Squamous Epithelium (keratinized) As the cells move towards the surface, they begin to flatten and accumulate a protein called keratin and eventually die. This keratin makes the outer layer of epidermis tough, protective and able to repel water. This type is found lining the: - Epidermis of the skin Function: protects underlying tissues in areas subjected to abrasion Figure 5: Stratified Squamous Epithelium (keratinized) under a microscope 23 f- Pseudo stratified ciliated columnar epithelium They appear to be layered but they consist of a single layer of cells of differing heights, some are not reaching the free surface; nuclei seen at different levels; may contain goblet cells & bear cilia. The cilia waves back and forth, moving mucus and debris up towards the throat. They are lining the: Trachea Function: sweeps impurities towards the throat. Figure 6: Pseudostratified ciliated columnar epithelium under microscope. 2- Connective tissue Connective tissue is found in all parts of the body, they join different parts of the body together, and they are made up of fibers forming a framework and support structure for body tissues and organs. Connective tissue protects, support and bind together other tissues of the body. All types of connective tissue consist of cells that produces the fibers; fibroblast surrounded by matrix that contains fibers. The two most common types of fibers are Elastic fibers; composed of a protein called elastin and Collagenous fibers; composed of protein called collagen. Collagen fibers are for strength while the elastic ones are for elasticity of the tissue. Types of connective tissue: 1. Loose fibrous connective tissue 2. Dense fibrous connective tissue 4. Adipose tissue 3. Compact bone 5. Hyaline cartilage 6. Blood 24 a- Loose fibrous connective tissue Loose connective tissue is also called Areolar connective tissue. The fibroblasts produce fibers and other intercellular materials. In the loose or areolar connective tissue, the thick pink bands are the protein collagen, while the thin dark threads are the protein elastin. This type of tissue occurs beneath the skin and most epithelial layers. Fig. 7: Loose Connective Tissues b- Dense fibrous connective tissue Dense connective tissue is characterized by having the collagenous fibers closely packed together in a regular, parallel pattern. As in tendons, which connect muscles to bones, and in ligaments which connects bones to other bones at the joints: Fig. 8: Dense Connective tissues c- Adipose tissue The cells of the adipose tissue, adipocytes, aner are characterized by having a large internal fat droplet so that the cytoplasm is reduced to a thin layer and the nucleus is displaced to the edge of the cell. The adipose 25  serves as a storage site for fats (lipids), also protects certain organs and forms an insulating layer under the skin which helps regulate body temperature. Location: Beneath the skin, around heart and other organs. Fig. 9: Adipose Tissue d- Compact bone Compact bone is found in the bones that makeup the skeleton. It consists of osteons, with central canal and concentric rings of spaces called lacunae at which are connected by canaliculi. The central canal contains; nerve and blood vessels and the lacunae contain; bone cells called osteocytes. Separating the lacunae is a matrix that is hard because it contains minerals as Calcium salts. Fig. 10: Compact Bone Under Microscope e- Hyaline cartilage There is three basic types of cartilage in the human body: hyaline cartilage, elastic cartilage and fibrocartilage. In this laboratory, you will examine the most common 26  type of cartilage, the hyaline cartilage. The cells of the hyaline cartilage are known as chondrocytes are embedded within lacunae. The lacunae are surrounded by a flexible matrix contains weak collagenous fibers. The function of the hyaline cartilage is to provide slightly flexible support and reduce friction within joints.. Fig 11: Hyaline Cartilage f- Blood It is a connective tissue in which the matrix is intracellular fluid called plasma. In circulating blood two different cell types are found: enucleated erythrocytes or red blood cells (RBCs) and nucleated leukocytes or white blood cells (WBCs). RBCs are small (7 um) cells lacking a nucleus. They stain red with eosin and due to their concave shape have a lighter staining center. WBCs are divided into two groups: (1) granular leukocytes, containing distinctive cytoplasmic granules, including neutrophils, eosinophils and basophils and (2) agranular leukocytes, without granules, including monocytes and lymphocytes. Basophil Monocyte 27  Lymphocytes Neutrophil Eosinophil 3- Muscular tissue Muscles are composed of cells called muscle fibers. The contraction of muscular tissue results in movement of the body. There are three types of muscular tissue, skeletal muscle, cardiac muscles and smooth muscles. Fig. 12: The three types of muscles a- Skeletal muscle Skeletal muscle is the muscle attached to the skeletal bones. The cells are tubular in shape with multiple nuclei located peripherally. The skeletal muscle is voluntary because they are under the control of an 28 individual and striated; it contains light and dark bands. The striation is caused by the arrangement of the contractile filaments (actin and myosin filaments). Fig. 13: Skeletal muscle b- Cardiac muscle It is found only in the wall of the heart as they function in the pumping of blood. Cardiac muscle is involuntary and striated with a single nucleus. Cardiac muscle fiber cells are branched and bound together at intercalated disks that appear unstained (pale) under the microscope. Fig. 14: Cardiac muscle under the microscope 29 c- Smooth muscle Is found in the wall of the internal organs, example is intestine and blood vessels. Smooth muscle cell is spindle in shape with a single nucleus. Cells are non-striation and are involuntary; they function in the movement of substance in lumens of body. Under the microscope smooth muscle forms neat, parallel lines. Fig. 15: Smooth muscles under the microscope 4- Nervous tissue Nervous tissue is the main component of the nervous system, it is composed of neurons, which transmit impulses, and the neuroglia cells, which assist propagation of the nerve impulse as well as provide nutrients to the neuron. Neurons have several dendrites, processes that take signals to a cell body, where the nucleus is located, and an axon that takes nerve impulses away from the cell body. Fig. 16: Nervous tissue under the microscope 30  Fig. 17: Neuron References: ▪ http://www.haspi.org/curriculum-library/A-P-Core- Labs/03%20Histology/Labs%20&%20Activities/Lab%20-%20Tissue.pdf. ▪ http://www.biocasts.com/mctc/1128/lab4.htm ▪ http://www.bio.davidson.edu/people/kabernd/BerndCV/Lab/EpithelialInfoWeb/Simple% 20Squamous%20Epithelium.html ▪ http://www.uoguelph.ca/zoology/devobio/210labs/muscle1.html ▪ Body tissue for biology lab students. 31  Chapter 5 Practical 7 Cell Structure and Function Learning Outcomes 5.1 Human Cell Structure 5.2 Crossing the Plasma Membrane 5.3 Osmosis and Tonicity 5.4 The Enzyme Catalase 5.1 Human Cell Structure Cells are an important means of organizing living things. Simple cells, like bacteria, lack internal complexity and are barely visible with light microscopes. More complex cells are larger and enclose specialized processes inside membrane enclosures, or organelles. All cells are surrounded by a plasma membrane, which regulates the movement of molecules into and out of cytoplasm. We will study how concentration affects the movement of solutes (dissolved particles) or Solvent (which dissolve solute into or out of cells. The biological solvent is water. Fig 1: Illustration of an animal cell 32 5.2 Crossing the Plasma Membrane The plasma membrane regulates the passage of molecules into and out of cells. It is said that the plasma membrane is selective, because certain small molecules can freely cross the membrane. 1- Diffusion Diffusion is the movement of molecules from an area of high concentration to an area of low concentration or down concentration gradient. Diffusion is an important process for living things; it is how substances move in and out of cells. Small molecules can diffuse across the lipid bilayer of the cell membrane, while larger molecules require special carriers. The Carriers are proteins that are embedded in the membrane. When a solvent molecule diffuses across the plasma membrane, the process in called osmosis. For a solute molecule diffusing across the plasma membrane, the process is called dialysis. 2- Passage of molecule across plasma membrane Figure 2: The 3 forms of transport across the cell membrane 33 Molecules diffuse across the cell membrane by: a- Simple diffusion: small lipid soluble molecules can diffuse freely through the lipid bilayer of the plasma membrane without requiring any carrier proteins. Alcohols, oxygen, and carbon dioxide simply diffuse down concentration gradient across the plasma membranes, no energy is required (ATP). b- Facilitated diffusion or transport: molecules like amino acids and glucose can diffuse across plasma membranes from the side of higher concentration to the side of lower concentration. This type of diffusion requires carrier protein but does not require for the cell to expend energy. c- Active transport: when molecules move from an area of lower concentration to an area of higher concentration (against concentration gradient). In this case a carrier protein is required and also the cell needs to expend energy (APT). Example is when the cell needs to pump Na+ from inside the cells to outside the cell. 5.3- Osmosis and Tonicity Osmosis is the diffusion of solvent, such as water, across selectively permeable membrane. Just like other molecule, water follows its concentration gradient and moves from region of higher concentration to a region of lower concentration. Tonicity is the relative concentration of solute and solvent (water) outside the cell compared to inside the cell. An isotonic solution has the same concentration of solute and water as the cell. When cells are placed in isotonic solution, there is not net movement of water inside and outside the cells and the cells maintains normal appearance. A hypertonic solution has a higher solute (therefore lower water) concentration than the cell. When cells are placed in hypertonic solution, water moves from inside the cells into the solution and the cells shrivel up (crenate). A hypotonic solution has a lower solute (therefore higher water) concentration than the cell. When cells are placed in a hypotonic solution, water move from the solution into the cell and the cells swell or burst depends on how hypotonic the solution is. Effect of Tonicity in Red Blood Cells Three test tubes have the following contents: 1- Tube 1: 3 ml of 0.6 % NaCl. Tube 2: 3 ml of 0.85% NaCl Tube 3: 3 ml of 10% NaCl 2- Record the tonicity of each tube 3- Add to each tube 100 microliter of human blood 4- Mix the tube and let them sit for 10 minutes 5- Make a wet mount from each tube and observe them under microscope and draw a conclusion. 34 5.4- Experiment with the Enzyme Catalaze Catalase is a common enzyme found in nearly all living organisms exposed to oxygen (such as bacteria, plants, and animals). It catalyzes the decomposition (break down) of hydrogen peroxide to water and oxygen. It is a very important enzyme in protecting the cell from oxidative damage. Experiment procedure: Catalase Activity 1- Label 3 test tubes as tube 1 , tube2 and tube 3. 2- To each tube add 2 milliliter of hydrogen peroxide. 3- To a pinch of sand to tube 1 4- Add small cube of potato to tube 2 5- Add small amount of macerated potato to tube number 3. Conclusion a- Record the amount of bubbling in each tube. Hydrogen peroxide and potato b- Give an explanation for the difference of amount of bulling in each tube. 35  Chapter 6 Practical 8 Blood and Blood Types Objective: By the end of this lab, students should be able to: 1- Define white blood count, WBC, and red blood cell count, RBC. 2- Know the normal range of WBC and RBC. 3- Know the clinical significance of the highs and lows of WBC and RBC. 4- Determination of Hematocrit 5- Determination of blood type Introduction: Blood is a specialized bodily fluid that delvers necessary substances such as nutrients and oxygen to the cells and transports metabolic waste products away from those same cells. Blood is composed of blood cells suspended in a liquid called blood plasma. Plasma, which constitutes about 55% of blood fluid. The plasma is 92% water and contains proteins, glucose, mineral ions, hormones, carbon dioxide ect. The blood cells are white blood cells (also called leukocytes or WBC), red blood cells (also called erythrocytes or RBC), and platelets. A- The red blood cell count (RBC count): is the number of red blood cells in mm3 or µl of whole blood. The RBC normal or reference range: 4.2 – 5.8 106/mm3 for men 3.6 – 5.6 106/mm3 in women 5.0 – 6.5 106/mm3 for newborn infants Higher than normal RBC count, erythrocytosis, is found in case of: 1- Polycythemia vera 2- Dehydration. 3- Living in higher elevation (low oxygen at higher elevation) 36 Lower than normal RBC Count (erythrocytopenia) or anemia, found: 1- Decrease of iron intake or iron deficiency. 2- Decrease of vitamin B12, and follicle acid 3- Leukemia 4- Kidney disease 5- Pregnancy (because of water retention and the fetus demand of RBC) Definition of Hematocrit: when anticoagulated whole blood is centrifuged, the space occupied by the packed red blood cells is called hematocrit and it is expressed as the % of the red blood cells in a volume of whole blood. It is also known as PCV (packed red cell volume). The values of hematocrit parallel the values of Hgb and RBC. Normal Range: Women: 37 – 47% Men: 40 – 54% B) Manual hematocrit 1- Allow well-mixed anticoagulated whole blood to enter two microhematocrit tubes until they are approximately two-third filled with blood. 2- Seal one end of the microhematocrit tube with the clay material by placing the dry end of the tube into the clay in a vertical position. The plug should be 4 to 6 mm long. Place the two microhematocrit tubes in the radial grooves of the centrifuge with the sealed end away from the center of the centrifuge. 3- Centrifuge for 5 minutes. 4- Remove the hematocrit tubes as soon as the centrifuge has stopped spinning. Obtain the results for both microhematocrits. Results should agree within 2%. Hematocrit = (X) 100 Y 37  C- The white blood count (WBC): is the number of white blood cells in 1 µl, or mm3. WBC normal range Child/adult: 4.5 – 11.0 103/mm3. Higher than normal WBC count, leukocytosis is found in cases of: 1- Appendicitis 2- Pneumonia 3- leukemia 4- Meningitis 5- Pregnancy and menstruation. Lower than normal WBC count, leukocytopenia, is found in cases of: 1- Measles 2- infection hepatitis 3- Radiation therapy 4- influenza 5- rheumatoid arthritis Fog 1: normal red blood cells, white blood cells And platelets. D- Blood Types Red blood cells have molecules on their membranes that can be different in different people. These molecules can function as antigens, also called blood group antigens, can bond to specific 38 antibodies present in the plasma of a person with a different blood type. The major blood group antigens are the antigen of the ABO system and. the D or Rh antigen The ABO antigen system Each individual inherits 2 genes, one from each parent, that control the synthesis of red blood cell antigens of the ABO system. Each gene can produce one of three antigens: antigen A, antigen B, or no antigen O. Thus, a person can have one of six possible combinations: AA, AO, BB, BO, AB, OO genes. Person who has AO genes will produce A antigen and he or she will be group A. person who has AB genes will produce A antigen and B antigen and he or she will be group AB. as for the person who has OO genes will produce no antigen and He or she will be group O. The antigens of the ABO system are actually sugars attached to the red blood cells. Fig. 2. Antigens on RBC, antibodies in plasma, and blood types. 39 The D antigen One of the antigens on the surface of red cells is called the D antigen or Rh factor. The presence of this antigen on the red blood cells is inherited as a dominant trait and it is produced by both heterozygous (RR) and the Homozygous (Rr) genotypes. Individuals who have the homozygous recessive genotype (rr) do not have this antigen on the red blood cells and are said to be Rh negative or D negative. The D antigen is protein attached to the red blood cell. Slide Test for Determination for ABO group and D on Red Cells b- Procedure 1- On a glass slide make 3 circles using black markers 2- Label the first circle as A, the second circle as B and the third circle as D. 3- Place one drop of a anti-A on circle labeled A, one drop of anti-B on circle labeled B, and one drop of anti- D on circle labeled D. 4- Add to each drop of reagent on drop of blood. 5- Mix the reagents and the blood thoroughly using clean toothpicks. 6- Gently tilt the slide continuously for 2 minutes 7- Read, interpret, and record the results. c- Interpretation 1- If the red cells are agglutinated by anti-A and anti-D, the cells are group A+. 2- If the cells are agglutinated by anti-D only the cells are group O+. 3- If the cells are agglutinated by anti-B and anti-D, the cells are group B+ 4- If the cells are agglutinated by Anti-A, anti-B, and anti-D, the cells are group AB+ 5- If the red cells are agglutinated only by anti-A, the cells are group A-. 40 6- If the cell are agglutinated by anti-B only the cells are group B- 7- If the cells are agglutinated by anti-A and anti-B only the cells are group AB- 8- If the cells are not agglutinated by any of the antibodies, the cells are group O-. 41  Chapter 7 Practical 9 Cell Cycle of Eukaryotic cells Mitosis Figure 1: Eukaryotic cell The genetic information of eukaryotic organisms resides in DNA molecules or chromosomes. All cells must replicate their DNA when dividing. Dividing cells experience nuclear division (mitosis), cytoplasmic division (cytokinesis), and a period between the division called interphase. The cell cycle results of 2 daughter cells each contains the same number and type of chromosomes as the parental cell. 42  Figure 2: Cell Cycle Interphase: During interphase, the cell grows, performs routine life processes, and prepares to divide. 3 distinct phases are included in the interphase. The 3 phases are: G (gap phase 1) – time of cell growth. Proteins produced include those needed for DNA replication. 1 S phase – DNA replication. Duplicated chromosome that consists of two sister chromatids held together at a centromere. Figure 3: one unduplicated chromosome and one duplicated chromosome with 2 chromatids G (gap phase 2) – chromosomes condense, becoming tightly coiled. 2 Centrioles (microtubule-organizing centers) also replicate 43 Mitosis: is a part of the cell cycle when replicated chromosomes are separated into two new nuclei. Mitosis is divided into 4 phases: 1. Prophase 2. Metaphase 3. Anaphase 4. Telophase 1. Prophase: -chromosomes continue to condense -centrioles start moving toward each pole of the cell -spindle apparatus is assembled -nuclear envelope dissolves Figure 4:Prophase 2. Metaphase: -chromosomes become attached to the spindle apparatus -microtubules pull the chromosomes to align them at the center of the cell 3. Anaphase: Figure 5: Metaphase - The centromeres split and the sister chromatids for each chromosome separate given rise to two daughter chromosomes - the daughter chromosomes begin to move toward Opposite poles. - Each pole receives the diploid number of daughters Chromosomes. Figure 44 6: Anaphase 4. Telophase: -spindle apparatus disassembles -nuclear envelope forms around the daughter chromosomes at the pole and each daughter nucleus contains the same number of chromosomes as the parental cell. -chromosomes begin to uncoil -nucleolus reappears in each new nucleus Figure 7: Telophase Cytokinesis – cleavage of the cell into equal halves -in animal cells – a constriction of actin filaments produces a cleavage furrow -in plant cells – plasma membrane forms a cell plate between the nuclei Procedure of Mitosis in Onion Root Tips. Materials Required 1- Compound light microscope 2- Water 3- Hydrochloric acid, 5N. 4- Filter paper 5- Glass slide and coverslip 6- Aceto alcohol (Glacial acetic acid and Ethanol in the ratio 1:3) 7- Onion root tips 8- Forceps, blade, Watch glass, and Dropper 9- Eppendorf tube Procedure of the experiment 1. Grow root tips by placing an onion in a beaker filled with water. 2. New roots may take 3–6 days to grow. 3. Cut off 2–3 cm of freshly grown roots and let them drop into a watch glass. 4. Using forceps, transfer them to the vial containing freshly prepared fixative of aceto-alcohol (1:3: glacial acetic acid: ethanol). This step preserves the DNA in the root. 45 5. Keep the root tips in the fixative for 24 hours. 6. Using forceps, take one root and place in an Eppendorf tube of 5N HCl for 5 minutes. This step will break down pectin that keeps the cellulose walls of plant cells together and also will allow the stain to defuse into the cells and also enable us the squashed the tissue into one cell thick layer. 7. Remove the onion tip from the HCL and place it on glass slide. 8. Using a blade, cut off the root keeping the tip only, 2-3 mm, on the slide and discard the remaining portion. 9. Add 1–2 drops of Safranin or Methylene blue stain (2%) on top of the onion tip. Allow 5 minutes to stain. 10. Carefully blot the excess stain using filter paper. 11. After that, put one drop of water on the root tip. 12. Mount a cover slip on it, avoid introducing air bubble in the wet mount. 13. Now, slowly tap the cover slip with the thumb and the tissue paper to spread the cells into a single layer and to dry the excess stain around the cover slip sides. 14. Place the slide under the compound microscope and observe the different stages of mitosis. Various stages of mitosis are prophase, metaphase, anaphase and telophase. 46 Figure 8: Stained onion tip Showing mitosis of various phase Figure 9: Stained onion tip showing mitosis of various phases 47  Practical 8 Practical 10 Energy Requirement and Ideal Body Weight Objectives: 1- Ideal Body Weight 2- Determining Average Daily Energy intake 2- Determining Average Daily energy Requirement 3- Comparison of Average Daily Energy Intake and Average Daily Energy Requirement A- Ideal Body Weight Ideal Body Weight is defined as the weight which is necessary for a person to lead a healthy life style. It is mainly based on height but changed by aspects such as gender, age, build, and degree of muscular development. Figure 10: Stained onion tip showing mitosis of various phases There are 3 different methods by which the ideal body weight can be determined. 1- Ideal Weight Based on Height and Weight 2- Ideal Weight Based on Body Mass Index (BMI) 3- Ideal Weight Based on Body Composition (percentage of body lean and percentage of body fat). *In this lab we are going to determine the ideal body weight based on BMI. So, each student is required to measure his/her weight and height at the beginning of the session. The formula for calculating the BMI is: By comparing Student’s BMI with that suggested in the table 1i it can be determined whether student should lose weight, gain weight or stay the same. 48  Table 1: Body mass index (BMI) is categorized by the International Obesity Task Force B- Average Daily Energy Intake The energy intake is the energy derived from the nutrients, fat, protein, and carbohydrate, that the individual consumes on a daily basis. *Student is required to calculate his/hers daily energy intakes taking table 2 as a guideline Table 2: The Calorie Content in some of the foods 43 C- Average Daily Energy Requirement The body needs energy for three purposes: 1- Energy required to support basal metabolic rate (BMR) 2- Energy required for physical activity 3- Energy required for Specific Dynamic Action (SDA) 1- Energy required to support Basal Metabolic Rate The basal metabolic rate is the amount of energy required by a person to keep the body functioning at rest. Example, the energy needed for breathing, blood circulation, body temperature, nerve functioning, etc… * Student is required to calculate his/her BMR using table 3ii Table 3: Formula for calculating BMR in male and female 2- Energy Required for physical activity The energy required for physical activities can be calculated using table 4iii. Student is required to calculate the daily required for physical activity using table 4. For example, a 68-kilogram person doing 30 minutes of Tennis can spend 222 kcal. 30 min. x 7.4kcal/min = 222 kcal. 44  Figure 4: Energy cost for selected sports activities. 3- Energy Required for Specific Dynamic Action (SDA) The specific dynamic action refers to the amount if energy needed to process food. For example muscles that move food along the digestive tract and gland that make digestive enzymes use energy. The amount of energy required for SDA is calculated as 10% of the sum of the energy for BMI and the amount of energy required for physical activities. Example: if the amount of energy needed for BMR is 2000 Kcal/day and the amount of energy required for physical activities is 1120 kcal/day. So, the energy needed for SDA is: (2000 + 1120 )x10 = 312 Kcal/day 100 Conclusion Depending on your current weight and ideal body weight you have to decide whether you need to increase or decrease your weight. 45 If your BMI is less than 18.5 you need to increase your weight by increasing your Daily Energy Intake (food intake). But if your BMI is over 25 than you have of decrease your food intake and increase your physical activities. The choice is yours. i Saskatchewas Health Autority. Bariatric Surgical Program: Body Mass Index. Retrieved from: http://www.rqhealth.ca/department/bariatric-surgical-program/bariatric-surgical-program-body-mass-index-bmi ii Oziva. BMR and TEE: What is all about. Retrieved from: http://blog.oziva.in/bmr-and-tee-what-is-it-all-about/ iii Ushar Panihar. Clissification of Work by Energy Expenditure. Retrieved from: https://www.slideshare.net/ushapanihar/exercise-physiology-classification-of-work-by-energy-expenditure- 46