Fundamentals of Biology Laboratory Manual PDF

Fundamentals of Biology BIO 1101 Department of Biology– Faculty of Science  1-A: To study about laboratory safety rules Introduction* Laboratory is a place where scientific research and development is conducted, and analyses performed. Most laboratories are characterized b...

Fundamentals of Biology BIO 1101 Department of Biology– Faculty of Science  1-A: To study about laboratory safety rules Introduction* Laboratory is a place where scientific research and development is conducted, and analyses performed. Most laboratories are characterized by controlled uniformity of conditions (constant temperature, humidity, cleanliness). It is important that the correct procedures are used in various situations, when handling hazardous or biological materials, when preparing, executing or cleaning up an experiment. It is also essential that you understand how to identify and use emergency equipment and protective gear. Goal* To study about laboratory safety rules Apparatus and tools: * i. Different types of chemicals ii. Acid and Base iii. Models iv. Equipment’s. v. Visit of lab Theory* The laboratory gives students first-hand experience and offers better opportunities for learning. A laboratory is not a contest whose object is to get the “right answer”, but the purpose is to learn how to gain knowledge, how to observe and to learn the meaning of what happens. Therefore, while working in lab students must follow the lab safety rules and instructions of the instructor. Example: Procedure: * 1. Never work alone in the laboratory without permission and prior knowledge of the instructor. 2. Do not engage in rowdy, playful, or unprofessional activities in the laboratory. This includes not being disrespectful of your instructor or classmates. 3. Students should wash hands thoroughly after first entering the lab. 4. They should never ever eat or drink anything in the laboratory without explicit permission from the instructor.  5. Wear appropriate clothing at all times in the laboratory. Wear closed-toe shoes that cover the top of the foot, unless permission otherwise is given by the instructor. 6. Wear examination gloves and safety glasses when dissecting or handling cadavers, caustic chemicals, bacterial broth cultures, or as otherwise advised by your instructor. 7. Wear gloves when handling any microorganisms. Wear lab aprons or lab coats as advised by your instructor. 8. Keep hands away from your face, eyes, and mouth when working with cadavers, chemicals, preserved specimens, microorganisms, or body fluids. This includes not applying cosmetics, not adjusting contact lenses, and not biting your finger nails. 9. If any chemicals or other agents splash into your eyes, immediately go to the nearest sink and flush your eyes with water. 10. Report ANY and ALL accidents, spills, BREAKAGES, or injuries to the instructor, no matter how trivial they appear. 11. Scalpels and other sharp objects can be used only if authorized by the instructor and only after given proper handling instructions. Use small trays to carry all sharp objects. When handling sharp objects, point their tips down and away from other people. 12. While wearing examination gloves, students must not leave the laboratory and must not touch any equipment such as microscopes, any personal items such as cell phones, or any door knobs. 13. Do not use any lab equipment without instruction and authorization from the instructor. Report any damaged or broken equipment to your instructor immediately. 14. Lab benches should be kept free of extraneous items while conducting experiments. This includes unnecessary books, backpacks, cell phones, and other personal items. 15. Any pregnant or immunocompromised student must notify the instructor of the course. Pregnant students will not be allowed to do dissections or work with any body fluids without having a doctor’s note for permission. A pregnant student is required to wear safety glasses and 2 sets of examination gloves when handling any bacterial broths or cultures. Results: * Lab safety rules were learned by students. * Required  1-B: To be able to write scientific reports. Introduction* A scientific report documents all aspects of an experimental investigation. Scientific reports allow their readers to understand the experiment without doing it themselves. In addition, scientific reports give others the opportunity to check the methodology of the experiment to ensure the validity of the results. Goal* To be able to write scientific reports. Apparatus and tools: * Theory* A scientific report is written in several stages. We write the introduction, aim/goal, and theory/hypothesis before performing the experiment. Further, we write the procedure and record the results during the experiment and complete the discussion and conclusions after the experiment. Example: Procedure: * General rules about writing scientific reports Learning how to write a scientific report is different from writing English essays or speeches! You have to use: Passive voice (which you should avoid when writing for other subjects like English!) Past-tense language Headings and subheadings A pencil to draw scientific diagrams and graphs Simple and clear lines for scientific diagrams Tables and graphs where necessary Results: *  1- C: To study the microscope Introduction* A microscope is a laboratory instrument used to examine objects that are too small to be seen by the naked eye. Microscopy is the science of investigating small objects and structures using a microscope. Microscopic means being invisible to the eye unless aided by a microscope. Goal* To study the microscope Apparatus and tools: * Microscope Theory* It is one of the most important instruments in a biology laboratory. It comes to be called as 'The primary instrument of the biologists'. It helps to increase the resolving power of human eye which fails to recognize objects lying closer between 0.01 to 0.25 mm. (Resolving Power: Property to distinguish objects lying very close as separate bodies) Example: Procedure:* Show the various parts and components of microscope to students. 1. The microscope is built around a strong base and a vertical frame (arm). 2. The base supports the vertical frame. 3. A round, rectangular or square stage is fixed to the frame. 4. It is provided with mechanical clips to hold the slide in position. 5. Stage is vertically movable with the help of coarse and fine adjustment screws on the frame. 6. Coarse adjustment moves the stage rapidly 7. Fine adjustment screw makes image clear. 8. Below the stage, an iris diaphragm and condenser lens are present. 9. Iris diaphragm, regulate the aperture, through which light rays reach the condenser and are passed to an object. 10. Condenser is a system of two or more lenses under the stage which receives parallel light rays from light source and converge them at the level of stage. 11. Below the condenser, a light source is present with a switch and brightness adjustment 12. Body of the microscope is composed of a head. 13. At the upper end of the head, is an ocular (eye piece) 14. At the lower end of this tube is a revolving nose-piece with about three objectives viz. low power, high power and oil immersion.  15. These magnifications range from 4X to l00X. Precautions: 1. The object should always be observed with both eyes open. 2. Before and after the use, all the lenses and metal parts including stage should be cleaned with soft tissue paper. 3. Microscope is kept covered when not in use. 4. Objectives should not be ordinarily removed from the nose-piece. 5. Operating screws, condenser, iris diaphragm, mirror and stage or stage clips should always be handled carefully. 6. First, use dim light and as per requirement, increase the light. Results:* Activity No 1: Here’s a simple activity where students either prepare their own slides or use prepared slides to examine under a light microscope. This activity will help them understand the use of microscopes and learn how to observe and document microscopic details. -The name of slid: ………………………………………………………………………… -How are you seeing slide under microscope? ………………………………………………………………………………………………… …………………………………………………………………………………………….. 2: To be able to differentiate between biological molecules practically Introduction* Biomolecule, also called biological molecule, are substances that are produced by cells and living organisms. Biomolecules have a wide range of sizes and structures and perform a vast array of functions. The four major types of biomolecules are carbohydrates, lipids, nucleic acids, and proteins. Goal* To be able to differentiate between biological molecules practically Apparatus and tools: * Test tubes, Test tube holder, Test tube brushes, Pipettes, Water bath, UV spectrophotometer, Vortex, Different biological samples Chemical Required: - Benedict’s reagent: Benedict’s reagent is prepared by adding 17.3 gm of sodium citrate, 10 gm of sodium carbonate and 17.3 gm of sodium pentahydrate to 100 ml of water in a beaker. Lugol’s iodine: 5% elemental iodine is mixed with 10% potassium iodide to form the Lugol’s iodine. Ethanol and Water Biuret reagent: 0.3 g of CuSO4 and 0.9 g of sodium-potassium tartrate are added to 50 ml of 0.2N NaOH. To this, 0.5 g of KI added and the volume is made up to 100 ml by adding 0.2N NaOH. Theory* Benedict's test for reducing sugars Poly Benedict’s test is a biochemical test performed to distinguish reducing sugars (monosaccharides and some disaccharides) from non-reducing sugars. saccharides The carbohydrates having a free or potentially free, aldehyde or ketone group can act as a reducing agent. In order to detect the reducing agent, Benedict’s reagent is used. It appears deep blue in colour and consists of copper sulfate mixed with sodium citrate and a weak alkali, sodium carbonate. When reducing sugars are heated in the presence of alkali, they get converted to enediols, which are powerful reducing agents. Enediols reduce the cupric ions (Cu2+) present in the benedict’s reagent to cuprous ions (Cu+), which get precipitated as insoluble red-colored cuprous oxide.  Iodine test for starch. Blue black Iodine test is based on the fact that polyiodide ions form colored adsorption complex with helical chains of glucose residue of amylase (blue-black), dextrin (black), or glycogen (reddish-brown). Monosaccharides, disaccharides, and branched polysaccharides like cellulose remain colorless. Amylopectin produces an orange-yellow hue. Emulsion test for Lipids. This test is based on the fact that lipids dissolve in ethanol (due to hydrophobic interaction), but on the addition of water, lipids spontaneously disperse to form micelles (small droplets). These droplets form the top layer as these are less dense than water and ethanol, and also appear cloudy white as they diffract light. Biuret test for proteins The biuret reagent contains sodium hydroxide, copper (II) sulfate, and potassium sodium tartrate. Under alkaline conditions of the biuret reaction (pH 14), deprotonation of the amide nitrogen occurs which leads to high electron density at the nitrogen atom, Further, copper (II) ion complexes with four peptide nitrogen’s to yield a tetradentate violet colored coordination complex. At high pH, Cu2+ bonding with OH– ion leads to an insoluble precipitate, which is minimized by the addition of potassium sodium tartrate, which stabilizes the cupric ions. Example: Procedure: * Benedict's test for reducing sugars 1. About 1 ml of the test sample is added to a test tube along with 2 ml of benedict’s reagent. 2. The test tubes are then placed in the test tube stand, which is kept in boiling water for 4-10 minutes. 3. The color in the test tubes is observed and noted down. Iodine test for starch 1. 1 ml of the test sample is taken in a test tube, to which 2-3 drops of Lugol’s reagent is added. 2. The solution is then mixed in a vortex.  3. The color of the solution is observed. The test tubes are then placed in the boiling water bath until the color disappears. 4. The tubes are then cooled, and the color of the solution is observed and noted down Emulsion test for Lipids 1. Few drops of fat or lipid sample are added to a test tube. 2 ml of ethanol is added to the same test tube. 2. To this solution, 2 ml of water is added, and the test tube is shaken well. 3. The appearance of cloudy suspension is observed. Biuret test for proteins 1. 1 ml of the sample is taken in a test tube to which few drops of Biuret reagent is added. 2. The test tube is then mixed well by shaking the test tube well. 3. The change in color of the solution is then observed and noted down. Results: * Benedict's test for reducing sugars Iodine test for starch Emulsion test for Lipids. Biuret test for proteins Activity No 2: Students are divided to do experiments. Each group writes the name of the experiment they did and the result they obtained. Experiment name: ……………………………………………………………………….. Materials used and procedure: ……………….……………………………………………………………………………………... ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… Result: ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… Conclusion: ……………………………………………………………………………………………………… ……………………………………………………………………………………………………… 3: To differentiate between prokaryotic & eukaryotic cells, in addition to plant and animal cells. Introduction* A cell is defined as the smallest, basic unit of life that is responsible for all of life’s processes. Cells are the structural, functional, and biological units of all living beings. A cell can replicate itself independently. Hence, they are known as the building blocks of life. Each cell contains a fluid called the cytoplasm, which is enclosed by a membrane. In addition, present in the cytoplasm are several biomolecules like proteins, nucleic acids and lipids. Moreover, cellular structures called cell organelles are suspended in the cytoplasm. Goal* To differentiate between prokaryotic & eukaryotic cells, in addition to plant and animal cells. Apparatus and tools:* Permanent Slides, Model of Prokaryotic and Eukaryotic cell, Microscope, Electron Micrograph Theory* A cell is the structural and fundamental unit of life. The study of cells from its basic structure to the functions of every cell organelle is called Cell Biology. Robert Hooke was the first Biologist who discovered cells. All organisms are made up of cells. They may be made up of a single cell (unicellular), or many cells (multicellular). Mycoplasmas are the smallest known cells. Cells are the building blocks of all living beings. They provide structure to the body and convert the nutrients taken from the food into energy. Cells are the lowest level of organisation in every life form. From organism to organism, the count of cells may vary. Humans have more number of cells compared to that of bacteria. Cells comprise several cell organelles that perform specialised functions to carry out life processes. Every organelle has a specific structure. The hereditary material of the organisms is also present in the cells. Example: Prokaryotic Cell: Bacteria Eukaryotic Cell: Plant, Animal, Fungi Procedure:* Observe the permanent slides and electron micrograph Results:* Prokaryotic Cell: Bacteria Observation: 1. The first organisms to inhabit Earth were prokaryotes 2. Most prokaryotes are unicellular. 3. Prokaryotic cells have a variety of shapes 4. The outermost layer is the cell wall, which maintains cell shape and protects the cell. 5. Cell wall is made up of peptidoglycan. 6. Cell wall is followed by plasma membrane. 7. Ribosomes(70S) are present in the cytoplasm. 8. DNA is present in nucleoid, a region of cytoplasm that is not enclosed by a membrane. 9. Flagella may be present over the entire surface of the cell or concentrated at one or both ends. 10. Pili are appendages that pull two cells together prior to DNA transfer from one cell to the other. A comparative study of electron microscope of bacteria with the help of a diagram Eukaryotic Cell: Plant Observations There are a large number of regularly shaped cells lying side by side and each cell has a distinct cell wall. A distinct nucleus is present on the periphery of each cell. Lightly stained cytoplasm is observed in each cell.  Cytoplasm Nucleus Cell wall W.M. Onion Epidermal Peel Eukaryotic Cell: Animal Observations A large number of flat and irregular -shaped cells are observed. The cells do not have a cell wall. However, each cell has a thin cell membrane. A deeply stained nucleus is observed at the centre of each cell. No prominent vacuoles are observed in the cells. W.M Human Cheek epithelial cell Activity No 3: What are four differences between Prokaryotic and Eukaryotic cells? Comparison of features of Prokaryotic and Eukaryotic cells Characteristic Prokaryotic Eukaryotic Size of cell Typical organism (Example) Nucleus Cell division 4: To reveal the presence of enzymes in some vegetables and fruits. Introduction* There are thousands of enzymes are found in living cells where they act as catalysts or the thousands of chemical reactions which occur. In addition to making life possible, many enzymes have numerous applications that affect our daily lives in other ways such as food processing, clinical diagnoses, sewage treatment, and the textile industry Goal* To reveal the presence of enzymes in some vegetables and fruits. Apparatus and tools:* For Amylase: Starch-agar plates (0.2% soluble starch, 2% agar), Wax pencil, Distilled water (in wash bottle), Plant samples as in procedure. For Catalase: Test tubes (one for each material to be tested plus extra for control) Hydrogen peroxide (H2O2) (3% solution) Assorted living tissue: sliced raw potato, ground meat, liver, yeast cells, ground young leaves Assorted non-living material: piece of baked potato or cooked liver, etc. (Use caution with rocks or sand, some will “bubble.”) For Papain: Gelatin (Knox, Jello), Beaker (150 ml), Balance or teaspoon, Stirring rods (3) Test tubes (2), Test tube rack, Beaker of ice water, Hot plate, Distilled water (100 ml) Theory* Enzyme experiments are ideal for “hands on” opportunities and since several factors affect the rate at which enzymatic reactions proceed, an enzyme experiment presents many opportunities in the biology laboratory. The experiments presented are selected on the basis that they: (1) are simple to prepare, (2) use materials which are familiar to the student, and (3) can be used as a base from which to construct additional experiment. For Amylase: Amylase is an enzyme that catalyses the hydrolysis of the polysaccharide starch (amylose) to the disaccharide maltose. It is readily abundant in saliva, but somewhat unpleasant to obtain in large quantities. It is widely distributed in plant tissues, but is most abundant in seeds, where it apparently functions in initiating the breakdown of stored starch to glucose which is needed in large amounts during germination. For Catalase: Hydrogen peroxide (H2O2) is naturally formed in living organisms, however it is very harmful and is broken down immediately by several enzymes including catalase. This enzyme catalyses the breakdown of hydrogen peroxide to water and oxygen. For Papain: Papain, from the latex of the papaya plant, is one of a family of plant enzymes that includes bromelin (from pineapple) and ficin (from fig), all of which break down proteins. This is why the directions on a box of Jello remind you never to use fresh or frozen pineapple in your gelatin, since gelatin is the protein responsible for the “gel.” A convenient source of papain is fresh pineapple juice or meat tenderizer. Example: Procedure:* For Amylase 1. Prepare starch-agar plates (do not have to be sterile if used within a day or two). Allow to solidify and cool. 2. Use a wax pencil to label the bottom of the plate: “soaked seeds”, “boiled seeds”, “dry seeds”, etc. (You might want to include a few drops of saliva from your mouth for comparison.) 3. Use a sharp razor blade to cut the corn grain longitudinally and place, cut surface down, onto the agar surface. (You may wish to dissect out the embryo.) Be sure to space corn grains at least 2 cm from each other. 4. Incubate for 30 minutes. 5. Remove corn and rinse plate gently with distilled water. 6. Flood plate with iodine solution, swish around as color develops, rinse with distilled water, record results. (Any clear areas of agar can be removed and tested for sugars.) For Catalase: 1. Fill each labelled test tube approximately 1/3 full with fresh hydrogen peroxide. 2. Add a small amount of material to be tested. 3. Note whether or not bubbles are produced. For Papain: 1. Prepare a gelatin solution by heating 1 teaspoon (3.0 g) of gelatin in 100 ml distilled water until dissolved. (Gently mix, do not boil.) Cool to room temperature. 2. Pour meat tenderizer into one of the two test tubes until it fills approximately 0.5 cm of the tube. Label this tube as P. Do not put meat tenderizer in the other tube. 3. Fill each test tube 1/3 full (5 ml) with the gelatin solution. Mix gently. 4. Place tubes in ice water for 10 minutes. 5. Remove from ice bath and note the degree of gelatinization. Results:* For Amylase: After flooding the plates with iodine solution, the agar will stain a deep purple in all areas where starch remains. Areas of agar where dead seeds were placed will be purple, likewise for dry seeds (unless the incubation period is much longer) since dormant seeds produce very little amylase. Areas of the agar covered by saliva, or by a living embryo, will appear clear since the starch has been broken down. For Catalase: Any fresh, living material will normally have enough catalase present to produce bubbles of gas (oxygen) upon exposure to the hydrogen peroxide. Dry yeast and liver are most impressive when used in this experiment For Papain: The tube without meat tenderizer (papain) will contain firm gelatin. Tube P which contains papain will be almost liquid. Activity No 4: Describe how an enzyme works, and illustrate the process with a diagram? 1-……………………………………………………… 2-……………………………………. 3-……………………………………. 4-……………………………………. 5: To study the effect of osmosis on different types of cells through experiments on plant and animal cells. Introduction* All cells are enclosed by a cell membrane. This structure has two layers. Each layer has two main components, phospholipids and proteins. The phospholipid molecules are able to move around within the layers and give the cell membrane flexibility. Protein molecules are found embedded in the two layers of phospholipids. Membrane proteins have a wide variety of functions. Some allow a cell to respond to specific chemical signals from other cells, others are enzymes and some proteins are involved in the transport of substances across the cell membrane. The cell membrane is selectively permeable. It lets some substances pass through rapidly and some substances pass through more slowly, but prevents other substances passing through it at all. Some small molecules such as water, oxygen and carbon dioxide can pass directly through the phospholipids in the cell membrane. Larger molecules such as glucose require a specific transport protein to facilitate their movement across the cell membrane. Very large molecules such as proteins are too big to move through the cell membrane which is said to be impermeable to them. Goal* To study the effect of osmosis on different types of cells through experiments on plant and animal cells. Apparatus and tools:* Blood Sample, Test tube, Glass slide, coverslip, Microscope, Salt solution (0.85%), Rhoeo leaf, petriplates, Sugar solution (5%), Microscope Theory* Cells can gain or lose water by the process of osmosis. This depends on the water concentration of the solution inside the cell compared to water concentration of the solution outside the cell. The water concentration can be thought of as the proportion of a solution that is water. Solutions with a high concentration of solute molecules, such as sugars or salts, have a low concentration of water molecules and vice versa. The net movement of water from a region of high water concentration to a region of low water concentration through a selectively permeable membrane. Example: Procedure:*  Animal Cell Take blood sample and place in different concentration of salt solution a) Red blood cells (RBC) placed in a solution with the same water concentration as their cytoplasm (0.85 per cent salt solution). b) Red blood cells placed in a solution with a higher water concentration compared to their contents (eg pure water) c) Red blood cells placed in a solution with a lower water concentration compared to their contents (eg 1.7 per cent salt solution) Observe all under microscope after 15 minutes. Plant Cell Repeat the process with the Rhoeo leaf, it has coloured cell sap, which can be examined clearly under a compound microscope. a) Rhoeo epidermal peel placed in a solution with an equal water concentration to the contents of their cytoplasm and vacuole (dilute sugar solution) b) Rhoeo epidermal peel placed in a solution with a high water concentration compared to their contents (eg pure water) c) Rhoeo epidermal peel placed in a solution with a low water concentration compared to their contents (concentrated sugar solution) Results:* Blood Sample a) RBC will not experience an overall change in volume. No osmosis occurs. b) RBC will gain water by osmosis, swell up and burst. c) RBC will lose water by osmosis and shrink. Rhoeo Leaf a) Rhoeo epidermal peel will not experience an overall change in volume. No osmosis occurs. b) Rhoeo epidermal peel will gain water by osmosis and swell up until their cytoplasm and cell membrane are pushing against their cell wall. They are said to be turgid. c) Rhoeo epidermal peel will lose water by osmosis. Their cell membranes will peel away from their cell walls and they are said to be plasmolysed. 6a: To study the different stages of Mitosis in onion root tips. Introduction* Somatic growth in plants and animals takes place by the increase in the number of cells. A cell divides mitotically to form two daughter cells wherein the number of chromosomes remains the same (i.e., unchanged) as in the mother cell. In plants, such divisions rapidly take place in meristematic tissues of root and shoot apices, where the stages of mitosis can be easily observed. In animals, mitotically dividing cells can be easily viewed in the bone marrow tissue of a vertebrate, epithelial cells from gills in fishes and the tail of growing tadpole larvae of frog. Goal* Preparation and study of mitosis in onion root tips. Apparatus and tools:* Onion bulbs, wide mouth glass tubes/jar/bottle, glacial acetic acid, ethanol 2-4% acetocarmine/acetoorcein stain, N/10 HCl, spirit lamp/hot plate, slide, cover slips, blotting paper, molten wax/nail polish and compound microscope. Theory* For entities to mature, grow, maintain tissues, repair and synthesize new cells, cell division is required. Cell division is of two types: 1. Mitosis; 2. Meiosis In mitosis, the nucleus of the Eukaryotic cells divides into two, subsequently resulting in the splitting of the parent cells into two daughter cells. Hence, every cell division involves two chief stages: Cytokinesis – Cytoplasm division Karyokinesis – Nucleus division Example: Procedure:* 1. Cut 2–3 cm long freshly grown roots and transfer them to freshly prepared fixative, i.e., aceto-alcohol (1:3: glacial acetic acid : ethanol). 2. Keep the root tips in the fixative for 24 hours and then transfer them to 70% ethanol. 3. Take one or two preserved roots, wash them in water on a clean and grease free slide. 4. Place one drop of N/10 HCl on the root tip followed by 2–3 drops of aceto-carmine or aceto-orcein stain on it. 5. Leave the slide for 5–10 minutes on a hot plate. 6. Carefully blot the excess stain using blotting paper. 7. Now cut the comparatively more stained (2–3 mm) tip portion of the root and retain it on the slide and discard the remaining portion. 8. After (10–20 seconds) put one or two drops of water and blot them carefully using blotting paper. Again, put a drop of water on the root tip and mount a cover slip on it avoiding air bubbles.  9. Place the slide in between the folds of blotting paper using the fingers in such a way that the cover slip mounted on the slide is properly held. 10. Now slowly tap the cover slip using the blunt end of a pencil so that the meristematic tissue of the root tip below the cover slip is properly squashed and spread as a thin layer of cells. 11. Carefully seal the margins of the cover slip using molten paraffin wax or nail polish. This preparation of onion root tips cells is now ready for the study of mitosis. 12. Place the slide on the stage of a good quality compound microscope. First observe it under the lower magnification (10 X objective) to search for the area having a few dividing cells. Examine the dividing cells under higher magnification of the microscope to observe the detailed features of mitosis. Results:* The cells are mostly rectangular, oval or even circular in shape, with almost centrally situated densely stained nucleus. The chromatic (coloured) material of the nucleus is homogeneous and looks granular. The boundary of the nucleus is distinct. One or few nucleoli (sing: nucleolus) can also be observed inside the nucleus. Stages of Mitosis (a) Prophase Intact nuclear outline is seen (Fig 6a). The chromatin (seen as a homogeneous material in the nucleus at interphase) appears as a network of fine threads (chromosomes). Nucleoli may or may not be visible If the cell under observation is in the early stage of prophase then the chromatin fibres (chromosomes) are very thin. However, in the cells at late prophase, comparatively thicker chromatin fibres would be visible. Besides this, in the late prophase the nuclear membrane may not be noticed. (b) Metaphase The nuclear membrane disappears. Chromosomes are thick and are seen arranged at the equatorial plane of the cell. Each chromosome at this stage has two chromatids joined together at the centromere, which can be seen by changing the resolution of the microscope. Nucleolus is not observed during metaphase. (c) Anaphase This stage shows the separation of the chromatids of each chromosome. The chromatids separate due to the splitting of the centromere. Each chromatid now represents a separate chromosome as it has its own centromere. The chromosomes are found as if they have moved towards the two poles of the cell. The chromosomes at this stage may look like the shape of alphabets 'V', 'J' or 'I' depending upon the position of centromere in them. Different anaphase cells show different stages of movement of chromosomes to opposite poles, and they are designated to represent early, mid and late anaphase (d) Telophase Chromosomes reach the opposite poles, lose their individuality, and look like a mass of chromatin. Nuclear membrane appears to form the nuclei of the two future daughter cells Different Stages of Mitosis Cytokinesis: In plants, a cell plate is formed in the middle after telophase. The plate can be seen to extend outwards to ultimately reach the margin of the cell and divide the cell into two. Such cell plates are characteristic of plant cells. However, in an animal cell, the two sides of the cell show inpushings or constrictions formed from the peripheral region in the middle of the cell, which grow inward and meet to divide the cell into two daughter cells (Fig 6b). Cytokinesis in Plants Discussion Mitotic index (MI) is defined as a ratio of the total number of dividing cells (n) and the total number of cells (N) in a particular focus chosen randomly under the microscope and is calculated as Number of dividing cells/ total number of cells x 100 Mitosis Features Interphase Cytokinesis Prophase Metaphase Anaphase Telophase Cell Morphology Nuclear Morphology Chromosomes 6b: To study the different stages of Meiosis using permanent slides Introduction* Meiosis is a type of cell division in which the number of chromosomes is halved (from diploid to haploid) in the daughter cells, i.e., the gametes. The division is completed in two phases, meiosis I and meiosis II. Meiosis I is a reductional division in which the chromosomes of homologous pairs separate from each other. Meiosis II is equational division resulting in the formation of four daughter cells. Stages of meiosis can be observed in a cytological preparation of the cells of testis tubules or in the pollen mother cells of the anthers of flower buds. Goal* To study the different stages of meiosis using permanent slides. Apparatus and tools:* Permanent slides of meiosis and compound microscope Theory* Meiosis is the process in which a single cell divides twice to form four haploid daughter cells. These cells are the gametes – sperms in males and egg in females. The process of meiosis is divided into 2 stages. Each stage is subdivided into several phases. Meiosis I Meiosis II Example: Procedure:* Place the slide on the stage of the microscope and search for the dividing cells using lower magnification. When dividing cells are located observe them under higher magnification. Results:* Observe various stages of meiosis and identify them on the basis of the specific features given below. A significant number of cells will be in the Interphase. These cells have a centrally positioned densely stained nucleus. In case of slide of animal tissue a few mitotically dividing spermatogonial cells may also be seen. Different stages of meiosis and their features Meiosis I Leptotene: The nuclear membrane and nucleolus are not distinctly observable. Fine network of thin threads are seen uniformly distributed in the nucleus.These are chromatin threads, which may be observed as more prominent structures in the later stages Zygotene: This stage is characterised by the pairing of the homologous chromosomes, which can be seen as paired chromatin threads Pachytene: The chromatin threads get condensed and appear shortened and thick. Pairs of homologous chromosomes can be seen. Each chromosome has two chromatids and thus each bivalent consists of four chromatids. This configuration is called tetrad. Diplotene: The homologous chromosomes (each made up of two chromatids) show distinct separation from each other except at few regions where attachments are seen figure. These are chiasmata (sing. chiasma) representing the site of exchange of the parts between two homologous chromosomes. Diakinesis: The homologous pair of chromosomes appear more shortened, thick and prominent. Chiasmata can be still observed, All the homologous pairs appear scattered in the cell. Metaphase I: Homologous chromosomes are still in pairs and are arranged along the equatorial plane of the cell. At this stage, the number of bivalents can be counted. Chiasmata may still be seen in a few bivalents. Anaphase I: The chromosome pairs appear to have moved towards the two opposite poles of the cell. At the later stage, the anaphase - I may show the assembly of chromosomes at two poles. This results into the reduction of number of chromosomes to half. This stage can be identified by the presence of two chromatids in each chromosome. Telophase I: The chromosomes present at the two poles appear decondensed and form two distinct nuclei. The chromosomes are completely pulled apart and new nuclear envelope forms. Meiosis II Prophase II: Distinct thread- like chromatin fibres or rod- shaped chromosome are seen. Metaphase II: The chromosomes having two chromatids attached at the centromere are observed arranged at the equatorial plane of the cell. Each chromosome of metaphase II has two chromatid whereas in metaphase I these are paired homologous chromosomes each having two chromatids thus forming tetrad. In the metaphase I of meiosis, a few chiasmata are observed, where as no chiasmata are observed during metaphase II Anaphase II: The two chromatids of each chromosome after separation appear to lie at the two poles of the cell. Telophase II: The separated chromosomes appear decondensed and form nuclei. Figure (A–E) show prophase I which is represented by (A) zygotene, (B) pachytene, (C) diplotene and (D) diakinesis. (E –G) represent the remaining stages of the first meiotic division with (E) illustrating metaphase I, (F) anaphase I nd a (G) telophase I. The second meiotic division is shown in panels (H –L) with (H) representative of prophase II, (I ) being metaphase II, (J ) anaphase II, (K) telophase II and (L) tetrad formation. 7: Investigating Anaerobic Respiration in seeds Introduction* One of the basic and fundamental life processes that are carried out by living entities is respiration. It is a catabolic process wherein complex organic molecules are broken down into simpler molecules. The process releases energy either in the absence or presence of oxygen, and hence respiration can be of two kinds: Aerobic respiration: This kind of respiration takes place in the presence of oxygen, hence it results in the complete glucose oxidation with the release of energy. It includes three stages – namely, Krebs cycle, ETS and Glycolysis. All events relating to ETS take place inside mitochondria while stages connected with glycolysis take place in the cytoplasm. Anaerobic respiration: In this type of respiration, oxidation of food takes place in an environment lacking oxygen supply. Less energy is released as a result of incomplete oxidation of glucose. Goal* Study of Anerobic Respiration in Germinating seeds. Apparatus and tools:* Germinating seeds (gram/urad/moong), flower buds, a small test tube/ glass vial, petridish, a plastic tray slightly bigger than the size of petridish, mercury, forceps, KOH pellets, burrette stand with clamp. Theory* Breakdown of food substances to yield energy in the absence of oxygen is called anaerobic respiration. It is observed in several soil anaerobic microorganisms, yeast and certain types of tissues in human body. Anaerobic respiration yields much less energy per mole of glucose as compared with aerobic respiration Glucose (C6H12O6) → CO2 + C6H5OH Example: Procedure:* 1. Take a test tube and completely fill it with mercury. Invert it over a petridish which is also filled with mercury. There must be a continuous column of mercury in the test tube. 2. Tilt the test tube slightly and with the help of forceps introduce 3 - 4 healthy germinating gram seeds. 3. Gently tap the test tube with your finger nail/forceps so that the seeds move upwards in the mercury column. 4. With a clamp fix the test tube to a stand and keep the setup undisturbed for two hours.  5. Observe the setup 6. Introduce 3-4 KOH pellets in the same way as seeds were introduced. Observe the changes. Results:* A space is formed at the top of the test tube due to downward displacement of mercury. When KOH pellets are introduced, the gap slowly disappears, and mercury again fills up the entire tube. The germinating seeds respire in a situation when these are completely cut off from air in the presence of a continuous column of mercury. The carbon dioxide gas released gets collected at the top of the tube and displaces mercury. The CO2 released disolves in KOH and the mercury level rises again. This establishes the fact that seed/buds have respired anaerobically Fig: Experimental setup Observation: Table: Rate of respiration of Seed S. No Seeds Rate of Respiration Trial 1 Trial 2 Trial 3 Average  Activity no.7 Differentiate between aerobic and anaerobic respiration? Characteristics Aerobic Anaerobic Definition Oxygen Requirements Products Location Energy released 8: To investigate the different types of plant tissue. Introduction* Plants are immobile and hence have been provided with tissues made up of dead cells, which provide structural strength. They must endure unfavorable environmental situations like strong winds, storms, floods etc. A tissue is a cluster of cells, that are alike in configuration and work together to attain a specific function. Goal* To study the different types of plant tissue Apparatus and tools:* Blade, tender stems of any plant, glass slides, cover slips, safranin, glycerine, dissecting needle, brush, blotting paper, watch glass and compound microscope Theory* A group of cells of the same size and shape, or of a mixed type, having a common origin and performing an identical function is called tissue. Plant tissues are of two types-meristematic and permanent. Meristematic tissue cells are capable of dividing, while permanent tissue cells are not. Parenchyma, collenchyma, and sclerenchyma are the three types of simple permanent tissues. Example: Procedure:* 1. Take a small piece of tender stem and hold it vertically. 2. Cut several thin sections using a blade. 3. Transfer the sections into a watch glass containing water. 4. Select a section which is complete, thin, and uniform, and transfer it onto a slide with a drop of water, using a brush. 5. Add one drop of safranin solution (stain) and leave it for 3 minutes. 6. Remove the excess stain by washing. 7. Add 2 drops of glycerin and put the cover slip, using needle. 8. Blot out extra glycerin and focus the slide, first under low power and then under high power. 9. Compare the slide with structure given in figure. Results:* Carefully observe the different type of plant tissues.  Figure. Two types of simple plant tissues Precaution: Select tender herbaceous stem. Sections should be kept in water Activity no. 8 Identify the following plant tissue pictures: 9: To study the different types of Animal tissues Introduction* A tissue can be defined as a group of structurally similar cells performing a specific function or functions. The study of the structure and function of the tissues is known as histology. An organ is a structural and functional unit of a living body composed of different tissues. Essentially the various organs of animals are composed of different types of tissues. In this lab students will be studying the structure of different types of animal tissues from the prepared slides and know about their function. Goal* To investigate the different types of animal tissue Apparatus and tools:* Permanent slides of muscles, epithelial, connective and nerve fibres, and compound microscope Theory* A tissue is a group of connected cells that have a similar function within an organism. There are four basic types of tissue in the body of all animals, including the human body. These make up all the organs, structures and other contents of the body. figure below shows an example of each tissue type. The four basic types of animal tissue are: Epithelial tissue is made up of layers of tightly packed cells that line the surfaces of the body for protection, secretion, and absorption. Examples of epithelial tissue include the skin, the lining of the mouth and nose, and the lining of the digestive system. Muscle tissue is made up of cells contain contractile filaments that move past each other and change the size of the cell. There are three types of muscle tissue: smooth muscle which is found in the inner linings of organs; skeletal muscle, which is attached to bone and moves the body; and cardiac muscle which is found only in the heart. Nervous tissue is made up of the nerve cells (neurons) that together form the nervous system, including the brain and spinal cord. Connective tissue is made up of many different types of cells that are all involved in structure and support of the body. Bone, blood, fat, and cartilage are all connective tissues. Connective tissue can be densely packed together, as bone cells are, or loosely packed, as adipose tissue (fat cells) are. Example: Procedure:* 1. Keep the permanent slides of striated muscles, epithelial, connective and nerve fibres one by one under low power and then under the high power of microscope. 2. Carefully study the different characteristics of each tissue. Results:* (A) Striated muscle fibres 1. The cells are long, cylindrical, non- tapering and unbranched. 2. They are multi-nucleated. 3. Light and dark bands are seen alternating with each other. (B) Nerve cell 1. Nerve cell has a cell body, a nucleus, dendrites, and axon. (C) Epithelial tissue 1. They cover the body, organs, blood vessels and all body cavities. 2. The cells are thin and lower most layer rest in a basement membrane. 3. Basically protective. Could be secretory and absorptive in function. (D) Muscular Tissue 1. Smooth, Skeletal, and cardiac muscles Nerve fibres Uni – bi – multipolar neurons Epithelial tissue Connective Tissue Exercise Mach between Colom A And B 1-Simple Epithelial Tissue 1- C A- 2-Connective Tissue 2- D B- 3-Smooth Muscle Tissue 3- E C- 4-Cardiac Muscle Tissue 4- B D- 5-Nervous Tissue 5- A E- 1-Simple Epithelial Tissue 1- … A- 2-Connective Tissue 2- … B- 3-Smooth Muscle Tissue 3- … C- 4-Cardiac Muscle Tissue 4- … D- 5-Nervous Tissue 5- … E- Draw the nerve cell with the correct data. Write the correct data : 10: To study the different organ systems of the frog (frog dissection) Introduction* Frogs belong to the class Amphibia. Amphibians have adaptations for living in terrestrial as well as aquatic environments. There are three orders of amphibians are Anura, Apoda and Caudata (Urodela). Goal* To study the different organ systems of the frogs. Apparatus and tools:* Preserved frog, Scissors, Forceps, Probe, Pins, Dissecting Tray, Dissection kit Theory* Frog dissection describe the appearance of various organs found in the frog as well the organs that make up various systems of the from. Example: Procedure:* A. To study external Anatomy of the frog: 1. Obtain a preserved frog. Rinse the frog with water to remove excess preservative. 2. Identify the dorsal and ventral surfaces and the anterior and posterior ends of the frog. 3. Locate the forelegs and the hindlegs. Each foreleg is divided into four regions: upper arm, forearm, wrist, and hand. Each hindleg also has four regions: thigh, lower leg, ankle, and foot. 4. Locate the two large, protruding eyes and nictitating membrane. 5. Locate the tympanic membranes on both sides of the head. 6. Locate two openings called the external nares (singular, naris), or nostrils. 7. Locate Jaw by using scissors. 8. Observe tongue is the most noticeable structure in the mouth. 9. Examine gullet opening, vertical slit and the glottis at the back of the mouth region. 10. Examine the roof of the mouth. B. To study Internal Anatomy of the frog: 1. Place the preserved frog on the dissecting tray with the ventral surface up. 2. With dissecting pins, securely pin the frog’s feet and hands to the bottom of the dissecting tray. 3. Use scissors to lift the abdominal muscles away from the body cavity. Cut along the midline of the body to the forelimbs (as represented in the figure) 4. Make transverse (horizontal) cuts near the arms and legs. 5. Life the flaps of the body wall and pin back. 6. Locate each of the organs below. a. Fat Bodies: Spaghetti shaped structures that have a bright orange or yellow color, if you have a particularly fat frog. Usually they are located just on the inside of the abdominal wall. b. Peritoneum: A spider-web like membrane that covers many of the organs. c. Liver: The largest structure of the body cavity. This brown colored organ is composed of three lobes. The right lobe, the left anterior lobe, and the left posterior lobe. The liver is not primarily an organ of digestion, it does secrete a digestive juice called bile. Bile is needed for the proper digestion of fats. d. Heart: at the top of the liver, the heart is a triangular structure. The left and right atrium can be found at the top of the heart. A single ventricle located at the bottom of the heart. The large vessel extending out from the heart is the conus arteriosus. e. Lungs: Locate the lungs by looking underneath and behind the heart and liver. They are two spongy organs.  f. Gall Bladder: Lift the lobes of the liver, there will be a small green sac under the liver. This is the gallbladder, which stores bile. g. Stomach: Curving from underneath the liver is the stomach. The stomach is the first major site of chemical digestion. Frogs swallow their meals whole. Follow the stomach to where it turns into the small intestine. The pyloric sphincter valve regulates the exit of digested food from the stomach to the small intestine. h. Small Intestine: Leading from the stomach. The first straight portion of the small intestine is called the duodenum, the curled portion is the ileum. The ileum is held together by a membrane called the mesentery. Note the blood vessels running through the mesentery, they will carry absorbed nutrients away from the intestine. Absorption of digested nutrients occurs in the small intestine. i. Large Intestine: As you follow the small intestine down, it will widen into the large intestine. The large intestine leads to the cloaca, which is the last stop before solid wastes, sperm, eggs, and urine exit the frog's body. j. Spleen: Return to the folds of the mesentery, this dark red spherical object serves as a holding area for blood. k. Esophagus: Return to the stomach and follow it upward, where it gets smaller is the beginning of the esophagus. The esophagus is the tube that leads from the frogs mouth to the stomach. Open the frogs mouth and find the esophagus, poke your probe into it and see where it leads. 7. Cut the stomach out of the frog and open it up. You may find what remains of the frog's last meal in there. Look at the texture of the stomach on the inside. 8. The frog's reproductive and excretory system is combined into one system called the urogenital system. You will need to know the structures for both the male and female frog a. Kidneys - flattened bean shaped organs located at the lower back of the frog, near the spine. They are often a dark color. The kidneys filter wastes from the blood. Often the top of the kidneys have yellowish stringy fat bodies attached. b. Testes - in male frogs, these organs are located at the top of the kidneys, they are pale colored and round. c. Oviducts - females do not have testes, though you may see a curly structure around the outside of the kidney, these are the oviducts. Oviducts are where eggs are produced. Males can have structures that look similar but serve no actual purpose. In males, they are called vestigial oviducts. d. Bladder - An empty sac located at the lowest part of the body cavity. The bladder stores urine. e. Cloaca - mentioned again as part of the urogenital system - urine, sperm and eggs exit here. Results:* Locate and identify each of the organs bel ow. Internal Anatomy of a frog Exercises Data shown in the anatomical image Write the correct information on the anatomical image of the frog.