EXERCISE NO. 1 - Cell Molecular Biology PDF

**EXERCISE NO. 1** **Introduction to Laboratory Tools** **Using this Module** Read carefully this section. It provides you an overview of the overall approach used for the development and implementation of learning and teaching activities. All sessions are self-directed which requires you to tak...

**EXERCISE NO. 1** **Introduction to Laboratory Tools** **Using this Module** Read carefully this section. It provides you an overview of the overall approach used for the development and implementation of learning and teaching activities. All sessions are self-directed which requires you to take full control of the learning process. You need to take initiative, with or without the assistance of your teacher, to complete all learning and assessment activities. You have to develop and sustain your motivation to succeed in this course. To become successful in your learning, you need to do the following: 1. Read the learning outcomes for each session. They articulate the knowledge you need to acquire and skills to develop. 2. Assess your prior knowledge by identifying which learning outcomes you already know, and which learning outcomes you need to focus on. 3. Develop your learning goals based on the results in \#2. Keep those learning outcomes in mind while you engage in the learning activities. 4. After completing all learning activities, reflect which learning outcomes/learning goals you have achieved. 5. Address those learning outcomes which you have not achieved yet. **Introduction** Cell biology studies depend on the laboratory method and tools that can be used to investigate cell structures and function. The following are some basic experimental tools available. Familiarization and knowledge on the proper methods of using these are very significant. 1. Identify the different coomon tools used in cell and molecular biology. 2. *Describe the different common tools used in cell and molecular biology.* 3. *Compare and contrast the parts and functions of these tools.* 4. *Understand the basic functional principles involved in using the equipment or device.* **Activating Prior Knowledge** Reflect on the seven learning outcomes above. Complete the Table below: Learning Outcomes What do you know? Any questions/clarifications in relation to learning outcomes ------------------- ------------------- --------------------------------------------------------------- 1 2 3 4  **Learning Activity 1** Read the materials below while keeping in mind the learning outcomes. While reading, be guided by the following questions and answer them after. A. **The Micropipette** 1. Keeping the micropipette upright is a must when in use, why is this so? 2. Give 3 advantages of using P1000 micropipette vs. 1ml glass pipette. 3. When adding liquid to an existing liquid, how is it done? Why? B. **The Autoclave** 1. Give 5 items in the laboratory that should not be autoclaved. 2. Why is there a need to fill the reservoir with water? 3. Differentiate sterilization and decontamination. C. **The Centrifuge Machine** 1. Give 3 importance of using the centrifuge machine in cell biology research. 2. How does temperature affect the efficiency of the centrifuge machine? 3. Why must 2 tubes of the same quantity or mass be placed opposite each other in the centrifuge machine? D. **The Spectrophotometer** 1. Give 3 importance of using the spectrophotometer in cell biology research. 2. Briefly discuss the principle involved in operation of the spectrophotometer. 3. Give 2 important steps on how to properly handle cuvettes. **Reading Material** A. **The Micropipette** Micropipettes, also known as micropipettors, are one of the standard laboratory equipment used to measure and transfer small volumes of liquids. These air displacement piston pipettes use disposable tips and allow accurate, precise and rapid dispensing of various volumes. Figure 1.1 Parts of a micropipette **Sizes of micropipettes** Micropipettes have different sizes that also measures different range of volumes. These sizes are noted on the top of the plunger button. The sizes are P 10, P20, P100, P200, and P1000. P10 is for transferring liquids 0.5-10µl, P20 is used to measure 2-20µl, P100 for 10-100µl, P200 for 20-200µl, and P1000 for measuring volumes from 100-1000µl volumes. **Pipette tips** Liquids are never drawn directly into the shaft of the pipette. Instead, disposable plastic tips are attached to the shaft. Tips are color coded. In general, blue pipette tips are used on micropipettors with capacities of 100-1000μl, yellow tips for 2-200μl, and clear ("natural") tips are used for the smallest micropipette, dispensing 0.5 to 10μl volumes. B. **The Autoclave** An autoclave is an instrument used to sterilize equipment and supplies by subjecting them to high pressure saturated steam at 121 °C for around 15--20 minutes depending on the size of the load and the contents. It was invented by Charles Chamberland in 1879 and its precursor, the steam digester was earlier created by Denis Papin in 1679. Autoclave literally means \"self-locking\" referring to the mechanism that keeps the lid in place as the pressure is increased, creating steam. As pressure increases, the boiling point of water also increases, allowing for superheating of water without boiling. Autoclaves are used when decontaminating biohazardous or potentially biohazardous material, or sterilizing solids and liquids. Biohazardous items are autoclaved to inactivate pathogens like bacteria, viruses, fungi, and spores. Autoclaves available in the laboratory are digital and stove top, pressure-cooker type autoclaves with detached pressure regulator - the simplest of autoclaves. Note: Digital autoclaves and those with built-in stove are used for sterilization and stove top autoclaves are for decontamination purposes in the Department. Figure 1.2 Autoclave types. (A) Stove top pressure cooker type; (B) Pressure cooker type with built-in stove; (C) Digital autoclave. A. **The Centrifuge Machine** A centrifuge is the name for any device that spin at a high speed to press objects outward with centrifugal force. They are used to apply specific forces onto a subject in a controlled environment. Centrifuges are used in many different scientific fields, aerospace to chemistry. They are often used to separate items in suspension. The centrifuge works using the sedimentation principle where the centripetal acceleration causes more dense substances to separate out along the radial direction (the bottom of the tube). By the same token, lighter objects will tend to move to the top (of the tube). B. **The Spectrophotometer** The Spectrophotometer is an analytical instrument used to measure the intensity of light as a function of its wavelength. It measures transmission or apparent reflectance of light as a function of wavelength. Thus it allows precise analysis of colour or accurate comparison of luminous intensities of two sources or specific wavelengths. According to Beer\'s law, the amount of light absorbed by a medium is proportional to the concentration of the absorbing material or solute present. Thus the concentration of a colored solute in a solution may be determined in the lab by measuring the absorbency of light at a given wavelength. Wavelength (often abbreviated as lambda) is measured in nm. The spectrophotometer allows selection of a wavelength pass through the solution. Usually, the wavelength chosen which corresponds to the absorption maximum of the solute. When using the spectrophotometer, you should have two cuvettes in a plastic rack. Solutions which are to be read are poured into cuvettes which are inserted into the machine. One should be marked \"B\" for the blank and one \"S\" for your sample. A wipette should be available to polish them before insertion into the cuvette chamber. Cuvettes are carefully manufactured for their optical uniformity and are quite expensive. They should be handled with care so that they do not get scratched, and stored separate from standard test tubes. Try not to touch them except at the top of the tube to prevent finger smudges which alter the reading. Figure 1.4 The Spectrophotometer. II. **Learning Objectives** 1. demonstrate the proper use of the micropipettor, autoclave, spectrophotometer, and the centrifuge machine 2. be familiar with the parts and functions of the laboratory tools. 3. understand the basic functional principles involved in using the equipment or device. III. **Materials** A. Micropipette in different sizes/ volume range, pipette tips (blue, yellow, white), 50 ml beakers or Eppendorf tubes B. Autoclave, regulator C. Centrifuge machine, test tubes, Eppendorf tubes D. Spectrophotometer, cuvettes IV. **Procedure** A. **The Micropipette** Pipette tips are racked in plastic boxes with covers. When you receive a box, it will be sterile. Please be careful when touching box or tips not to contaminate them. The box should be closed when not in use to prevent airborne contamination. Note: for practice on dispensing volumes the plastic tips used in the laboratory are not sterile. Inserting the Tip: 1\. Select the correct pipette tip size/ volume. 2\. Open the box without touching the tips with your hands. 3\. Insert the micropipette shaft into the tip, press down firmly and twist slightly to ensure an airtight seal. This will attach the tip to the shaft. Do not press the tip with your fingers. 4\. Remove the micropipette with the tip attached. 5\. Close the box without touching the tips with your hands. Be careful not to allow the tips to touch any surface or material other than its intended solution. Measuring and transferring liquid: 1. 2. 3. 4. 5. 6. 7. 8. Micropipette Rules: Each micropipette is expensive. To keep these pipettes functioning properly it is important that they be handled with care. Please follow these rules to keep from breaking the micropipettes: 1\. Never adjust the volume beyond the range of the micropipette. No micropipette should be adjusted below zero µl. The P20 micropipette should never be adjusted above 20µl, the P200 over 200 µl and the P1000 over 1ml. 2\. Never force the volume adjustment knob. If the knob becomes difficult to adjust, it probably means that you are exceeding the limits for the pipette or the pipette is damaged. 3\. Do not drop the micropipettes. 4\. Always use a smooth motion when using the pipettes. This will help give you accurate measurements and also prevent breakage of pipettes. There should be no \"snapping\" noises. 5\. Always keep pipettes upright. Place the micropipettes on the mounted rack on your bench when not in use. Always check labels and make sure that the micropipettes are stored in its appropriate box. 6\. Always choose the appropriate pipette size for the volume you are measuring. 7\. Always dispose tips in appropriate waste containers. Never leave tips on benchtops or in glasswares and chemical solution containers. B. **The Autoclave** Simple measures can be used to ease the flow of heat and steam thorough and into the contents of the containers. Here are some fundamental rules: 1\. Load the material in such a manner to present the least resistant passage of air exchange through the load, from the top of the chamber to the bottom. 2\. Avoid crowding or stacking. 3\. Place packages on their edges to enhance steam penetration, place a rack or other item against these items to prevent them from slipping. 4\. For loads which are mixed (fabric and hard goods) place the hard goods on the bottom to prevent possible condensation from dripping on to the fabric. 5\. Place empty flasks, test tubes or other non-porous containers on their sides with loose covers. This provides a horizontal pathway and prevents trapping air pockets. 6\. Containers should not touch each other, this will ensure all surfaces are sterilized. 7\. No items should touch the top or sides of the autoclave container 8\. Liquids and dry goods should be processed separately as they require different cycle selections. 9\. A load of liquid-filled containers should be of similar size, shape, content and volume; because exposure time is based on these characteristics. 10\. Materials to be sterilized should be processed separately from those to be decontaminated. Guidelines for Removing Sterilized and Decontaminated Items: 1\. Wear all necessary personal protective equipment. 2\. The chamber pressure gauge of the autoclave should be zero before opening the autoclave\'s lid or door. 3\. For digital autoclaves, crack the door slightly and stand back to allow steam to escape, for stove top autoclaves, unlock the opposing clamps together and lift slightly. To minimize the risk of accidents caused by steam escape, the person who opens the autoclave door should stand directly behind it. 4\. Slowly open the autoclave door. Opening the autoclave door too quickly may result in glassware breakage and/or steam burns to the skin. 5\. If boiling or bubbling is present, wait until it subsides. Assess the risk of super-heated liquids. 6\. Overexposure of saline or water is not a critical factor (as it is with media), so these liquids may be allowed to cool (for 10 min.) in the autoclave after cracking the door to release the steam. This is also recommended for all other items. 7\. Bring the autoclave trolley/ deep plastic tray to the chamber. 8\. Using heat resistant gloves, carefully transfer the containers (pans) to the trolley/plastic tray. Be careful not to jolt the containers as it could result in breakage. 9\. Move the containers to a draft free area. 10\. If not yet cool, wait for 10 minutes before storing sterilized materials. Never store hot materials or media immediately in the refrigerator. 11\. After every use, it is advised to close the autoclave door but do not seal the door as this will shorten the life span of the rubber gaskets on the door. C. **The Centrifuge Machine** General operating precautions: - Operate centrifuges in designated research space only to ensure adequate ventilation. - Check tubes for cracks before each use. Inspect the inside of the cups for rough walls caused by erosion. Remove any adhered matter. - Use metal or plastic tubes (other than nitrocellulose) whenever possible. - Make sure that two test tubes/ Eppendorf tubes have the equal volume or weight placed oppositely in the well. - Do not scratch or damage the aluminum oxide layer that protects the machine\'s underlying metal. - Observe speed reductions specified in the manual for running high- density solutions, plastic adapters, or stainless steel tubes. - Set and confirm the proper run speed every time to prevent over-speeding. D. **The Spectrophotometer** The sequence of events in a spectrophotometer can be listed as follows: - The light source enters the sample against a blank sample. - The sample absorbs light. - Then the detector determines how much light is absorbed by the sample (equivalent to the amount of light that reached the detector relative to blank or no absorbed light by blank substance). - The detector then converts the amount of light absorbed by the sample into a number. - The numbers are plotted straight away, or are transmitted to a computer to be further manipulated **V. References** Amsco/ American Sterilizer 613R. Retrieved from: COE College. Parts of a Micropipette. Retrieved from Cooper, G.M. 2000. **The Cell**, **2nd edition**. **A Molecular Approach**. Boston University. Sunderland (MA): Sinauer Associates. ISBN-10: 0-87893-106-6. FAO Corporate Document Repository. Regional Office for Asia and the Pacific. A basic laboratory manual for the small-scale production and testing of I-2. Retrieved from Laboratory equipment. Retrieved from: http://www.laboratoryequipmentworld.com/ spectrophotometer.html Molecular Biology Lab. Retrieved from Use of micropipettes. Retrieved from: **Exercise No. 1** **Introduction to Laboratory Tools** **Answer Sheets** Name: \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ Rating: \_\_\_\_\_\_\_\_\_\_ Laboratory Section: \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ Date: \_\_\_\_\_\_\_\_\_\_\_\_ A. E. **The Micropipette** 4. Keeping the micropipette upright is a must when in use, why is this so? 5. Give 3 advantages of using P1000 micropipette vs. 1ml glass pipette. 1\. disposable tips, which minimizes the risk of cross-contamination between samples. 2\. easier to use, especially for small volumes. 3\. precise sample volume adjustments and accurate measurements, 6. When adding liquid to an existing liquid, how is it done? Why? Slowly Immerse the tip into the liquid when adding more liquid tramsfer by not touching the side of the container. Avoid or minimize too much bubbles before transferring the sample in order for precise measurement. F. **The Autoclave** 4. Give 5 items in the laboratory that should not be autoclaved. 5. Why is there a need to fill the reservoir with water? water generates steam, which is necessary for effective sterilization by providing the required pressure and temperature. 6. Differentiate sterilization and decontamination. Sterilization completely eliminates all microorganisms present in a splid or liquid sample including apparatuses, while decontamination reduces pontially pathogens. G. **The Centrifuge Machine** 4. Give 3 importance of using the centrifuge machine in cell biology research. 1.Separation of Cell Components (e.g. organelles) for data analysis 2.Separation of contaminants or unwanted cells to purify sample 3\. Decontamination of pathogens that may interfere in having accurate measurement. 5. How does temperature affect the efficiency of the centrifuge machine? Temperature influences centrifuge efficiency by affecting fluid viscosity, with higher temperatures improving separation and lower temperatures hindering it. Maintaining optimal temperatures is crucial for preserving the integrity of biological samples, as elevated heat can lead to degradation. Additionally, operating outside the recommended temperature range can impair centrifuge performance and affect the sedimentation rate of particles. 6. Why must 2 tubes of the same quantity or mass be placed opposite each other in the centrifuge machine? Two tubes of the same quantity or mass must be placed opposite each other in a centrifuge to ensure balance and to have ccurate results during centrifugation. H. **The Spectrophotometer** 4. Give 3 importance of using the spectrophotometer in cell biology research. 5\. Briefly discuss the principle involved in operation of the spectrophotometer. Beer\'s principle, also known as Beer-Lambert law, states that the absorbance of light by a substance is directly proportional to its concentration and the path length of the light through the sample. In a spectrophotometer, as light passes through a solution, some of it is absorbed by the molecules present. The greater the concentration of the absorbing species and the longer the path length, the more light is absorbed. This relationship allows for the quantification of the concentration of substances in a solution by measuring the amount of light that passes through it. 6\. Give 2 important steps on how to properly handle cuvettes. 1\. maintain cleanliness on the optical lenses for accurate readings. 2\. properly placed cuvetted iin the spectrophotometer **EXERCISE NO. 2** **Microscopy** I. **Introduction** II. **Learning Objectives** 4. demonstrate the proper procedure in using the compound light microscope. 5. determine the total magnification and field of view using the different objectives of the microscope. 6. describe cells through microscopic observation and make proper biological drawings/ documentation. 7. familiarize the use of the hemacytometer and the interpretation of results. III. **Materials** A. compound light microscopes (ideally, one for each student) ocular/ eyepiece micrometers stage micrometers transparent millimeter rulers B. compound light microscopes with oil immersion objective (OIO) ocular/ eyepiece micrometers C. compound light microscope for serial dilution: hemacytometer 20 mL test tubes (3) Pasteur pipette 10 mL graduated cylinder (1) cover slip test tube rack (1) test tube brush 50 mL beaker with distilled water IV. **Procedure** A. **Measuring the field of view (FOV) and calibrating the microscope** 1. With the 10x objective engaged, place a short, transparent ruler over the opening in the center of the stage so that the lines are visible through the microscope. 2. Move the ruler so that a vertical millimeter mark is just visible at the left edge of the circular field of view. 3. Count the number of millimeters from the left side to the right. If the right side of the field does not line up with one of the vertical markings, estimate the fraction of a millimeter. This is the diameter of the low power field of view. Record your measurement in millimeters (mm) and in micrometers (um). 4. Carefully move the 40x objective into place. Note that the diameter of the field is less than 1 mm. Rather than measuring the field directly, we can calculate the diameter based on the direct measurement of the low field diameter and the following equation: [diameter of the high power field] [low power magnification] diameter of the low power field high power magnification 5. To properly calibrate your eyepiece with a stage micrometer using the low power objective (LPO), align the zero line (beginning) of the stage micrometer with the zero line (beginning) of the eyepiece micrometer (Figure 1). Carefully scan over until you see the lines line up again. Determine the value that each line represents in your eyepiece. https://www.microscopeworld.com/images/reticle-grid.jpg Figure 2.1 The eyepiece and stage micrometers (www.microscopeworld.com/t-calibration.aspx). B. **Microscopic observation of cells** 1. 2. (Note: due to the availability of the eyepiece micrometers, some may estimate the cell measurements using the computed FOV). 3. C. **Counting cells using the hemacytometer** 1. 2.  Figure 2.2 The hemacytometer showing: (a) side view, (b) top view and grids. 3. - - - - - - - - 4. 5. 6. 7. 8. 9. 10. **V. References** Afshar, G. Basics of Cell Culture A student laboratory manual. Retrieved from http://www.bio-link.org/home/ sites/files/students\_manualv7.pdf on August 2015. Basics of Using the Hemocytometer. 2010. BS111L. Retrieved from **http://www.smccd.edu/ accounts/case/biol230/algae/hemocytometer.pdf** on August 2015. Campbell, N.A., J.B. Reece and E.J. Simon. 2004. Essential Biology. 2^nd^ ed. Benjamin Cummings: San Francisco CA. ISBN 0-8053-7495-7. Chieco, P., Jonker, A., and C.J.F. van Nooren. 2001. Image Cytometry. BIOS Scientific Publisher Limited: Magdalen Road, Oxford, 1RE, UK. ISBN 0-387-91618-0 Springer -- Verlag New York Berlin Heidelberg SPIN 10761200. **Department of Biology. Bates College. Use of Light Microscopes. Online Resources for Biology. Retrieved from http://abacus.bates.edu/\~ganderso/biology/resources/microscopy.html on August 2015.** Exercise 8: Manual Leukocyte Count. MLAB 1315 Hematology. Retrieved from **http://www.austincc. edu/mlt/hem/Lab8Manual WBC\_08.doc on August 2015.** **Microscope world. Calibrating a Microscope. Retrieved from www.microscopeworld.com/t-calibration.aspx on August 2015.** Snyder, J. (compiler). 1992. Exercises in Cell Biology for the Undergraduate Laboratory. A project of the American Society for Cell Biology Education Committee. Ledbetter, M.L. (ed). Retrieved from http://www. ascb. org/newsfiles/ exercises cell\_bio.pdf on August 2015. **Exercise No. 2** **Microscopy** **Answer Sheets** Name: \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ Rating: \_\_\_\_\_\_\_\_\_\_ Laboratory Section: \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ Date: \_\_\_\_\_\_\_\_\_\_\_\_ A. Objective Magnification Field of View (mm) Field of View (µm) ----------- --------------- -------------------- -------------------- 4x 40 10x 100 B. +-----------------------------------+-----------------------------------+ | Illustration | Description | +===================================+===================================+ |  | Under a microscope, a Paramecium | | | appears as an elongated, | | Fig. | slipper-shaped organism. Its most | | 2.3\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ | distinctive feature is the | | \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\ | numerous tiny hair-like | | _\_\_\_\_\_\_\_\_\_ | structures called cilia that | | | cover its entire surface. These | | | cilia beat rhythmically, | | | propelling the Paramecium through | | | the water and helping it capture | | | food. | | | | | | Dark nucleus | | | | | | Clear vacuole | +-----------------------------------+-----------------------------------+ | Fig. | Amoebas are shapeless, | | 2.4\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ | single-celled organisms that move | | \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\ | using pseudopods. | | _\_\_\_\_\_\_\_\_\_ | | | | An amoeba is a single-celled | | | organism that looks like a tiny | | | blob of jelly with a dark spot | | | inside. The jelly is cytoplasm, | | | and the dark spot is the nucleus. | | | | | | They have a simple structure with | | | a nucleus and a contractile | | | vacuole. | +-----------------------------------+-----------------------------------+ +-----------------------------------+-----------------------------------+ | Illustration | Description | +===================================+===================================+ |  | Human squamous cells under a | | | microscope typically appear as | | Fig. 2.5 | flat, scale-like cells. They are | | \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\ | often arranged in layers, forming | | _\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ | a protective barrier. The nucleus | | \_\_\_\_\_\_\_\_ | of these cells is usually | | | flattened and located near the | | | center of the cell. | +-----------------------------------+-----------------------------------+ | Fig. | In dicot stems, the vascular | | 2.6.\_\_\_\_\_\_\_\_\_\_\_\_\_\_\ | bundles are arranged in a ring. | | _\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ | Like dicot roots, dicot stems are | | \_\_\_\_\_\_\_\_\_\_ | protected by an outer layer of | | | dermal tissue called the | | | epidermis. Then, also similar to | | | dicot roots, dicot stems have a | | | layer of ground tissue called the | | | cortex beneath the epidermis. | +-----------------------------------+-----------------------------------+ +-----------------------------------+-----------------------------------+ | Illustration | Description | +===================================+===================================+ |  | | | | | | Fig. | | | 2.7\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ | | | \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\ | | | _\_\_\_\_\_\_\_\_\_ | | +-----------------------------------+-----------------------------------+ | Fig. | | | 2.8\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ | | | \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\ | | | _\_\_\_\_\_\_\_\_\_ | | +-----------------------------------+-----------------------------------+ C. Square number Cell count Square number Cell count --------------- ------------ --------------- ------------ Area of each square: \_\_\_\_\_\_\_\_mm x 0.1mm depth = volume of each square Volume of each square = \_\_\_\_\_\_\_\_\_\_ mm^3^ Average number of cells per mm^3^ = \_\_\_\_\_\_\_\_\_\_\_\_\_\_ Number of cells per cm^3^ = \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ (Note: Number of cells per cm^3^ is also the number of cells per ml.) Number of cells per ml = \_\_\_\_\_\_\_ x dilution factor \_\_\_\_\_\_\_\_ 7. What is the difference between magnification and resolution? How are these two properties of the microscope related? Explain your answer. \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ 8. The development of microscopes has been very useful in scientific research. Give examples of its importance in the survival of organisms. \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ **EXERCISE NO. 3** **Staining Techniques** I. **Introduction** Staining is a technique that can be used to enhance visualization of structures in biological tissues under a microscope. By using different stains, one can preferentially stain certain tissues, cell components, such as a nucleus or a cell wall, or the entire cell. Bacteria for instance are nearly colorless, so their features are difficult to distinguish when they are suspended in a fluid and viewed directly under a microscope. Stains then are salts that color particular ions in the bacterial cell, and make more visible distinctions. The chemical composition of the cell determines whichstain is absorbed. Acidic parts of a cell absorb stains that are positively charged; alkaline parts of a cell combine with stains that are acidic or negatively charged. Staining can also be carried out to highlight metabolic processes or to differentiate between live and dead cells in a sample. Before tissues are stained, a thin layer of cells that have been sliced from the specimen (a 1. smear) is prepared by fixing. 2. Fixing a specimen that has been placed on a slide is done by either allowing it to dry at room temperature, or by passing the specimen quickly over a flame. Next the specimen is 3.stained either one of the three categories: 1) Morphological stains to provide information about the cell size, shape, and arrangement. Examples of morphological stains include the **simple stain** and the **negative stain**. 2) Differential stains to differentiate bacteria based on the chemical composition of the cell wall. The differential stains require two stains (primary stain and counterstain) be used; one for the positive bacteria, the other for the negative bacteria. The differential stain that will be used in this activity is the **Gram stain**. Many staining techniques require you to start by carrying out a smear preparation first. A smear is done to fix a **thin** layer of cells to the microscope slide prior to staining. Cells must adhere to the slide so they do not wash off during the staining and washing processes. There must only be a thin layer of cells so the morphology and arrangement of individual cells can be visualized. The procedure outlined below should be followed each time a smear preparation is called for. II. **Learning Objectives** 1. determine the structures of a cell. 2. determine the morphology and arrangements of microbial cells. 3. determine the cell wall composition of a bacterium. 4. differentiate the importance of different staining techniques. III. **Materials** A. Glass slide, toothpick, dropper, a pair of forceps, alcohol lamp, match, tissue paper, wash bottle with distilled water B. C. IV. **Procedure** A. **Smear preparation** 1. Put a drop of water at the center of a clean glass slide. Note: the more liquid you add to the slide, the longer it will take to dry. 2. Scrape the inside region of your cheek gently using a clean toothpick. 3. Suspend your specimen in the drop of water and spread. 4. Allow the smear to dry in air. 5. Hold the slide using a pair of forceps and fix it by briefly passing the slide over a flame, side with smear up. 6. Use your prepared smear for the next activity. B. **Simple staining** 1. Add a few drops of methylene blue to cover the whole smear prepared earlier. Let the stain stay for one (1) minute. 2. Wash off the stain by rinsing it with water. Do not apply water directly above the slide, just let it flow into your smear. Blot dry with tissue at the slide. 3. Examine under the microscope from the **low power** up to the **high power** objectives. 4. Record your observations through drawings or photo-documentation. Label the visible parts properly in each drawing/ figure. Measure the size of one cell in micrometers (μm). Indicate the size of the **nucleus** as well. C. **Special staining: Gram Staining** 1. Prepare smear as previously done (A), but this time obtain sample of materials lodged between your teeth or on the surface of your teeth at the gum line. 2. Add a few drops of Gram I (crystal violet) into your smear. Do not drop it directly into your smear, just let it flow towards the slide. 3. Let the stain stay for 1 minute. Then wash all the stain off with water. 4. Add a few drops of Gram II (Gram's iodine) by letting it flow into your smear and then wait for 1 minute. Wash off with water. 5. Decolorize your smear with Gram III (ethyl alcohol) by letting it flow briefly for 30 seconds into your slide. Quickly wash with water thereafter. \[Note: ethyl alcohol is not a stain, it is just a decolorizing agent\] 6. Lastly, stain the smear with Gram IV (safranin) for 1 minute. 7. Wash all stain off with water and blot dry. 8. Observe under the **oil immersion objective** lens of the microscope. Determine the shape, arrangement of the cells, and Gram reaction. Record your observations through photo-documentation. \[**Note**: Gram positive cells are colored violet, blue or purple while Gram negative cells are colored pink or reddish.\] V. **References** Bruckner, M. Z. Microbial life educational researchers: Basic Cellular Staining. Montana State University, Bozeman, USA. Retrieved from:http://serc.carleton. edu/microbelife/research\_methods/microscopy/cellstain.html Chan, E.C.S., Pelczar, M. Jr. and Kreig, N.R. 1993. Laboratory exercises in Microbiology. 6th ed. McGraw --Hill, Inc. Printed in the USA. p.71 http://science.jrank.org/pages/1328/Cell-Staining-Stainingtechniques.html Devlin, K. R. Microbiology Laboratory Exercises. College of Natural Sciences, California State University San Bernardo, CA, USA. http://kdevlin.com/ **Exercise No. 3** **Staining Techniques** **Answer Sheets** Name: \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ Rating: \_\_\_\_\_\_\_\_\_\_ Laboratory Section: \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ Date: \_\_\_\_\_\_\_\_\_\_\_\_ A. Observations: Illustration Description -------------------------------------------------------------------------------------------- ------------- Fig. 3.1\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ Fig. 3.2\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ Illustration Description Fig. 3.3\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ Fig. 3.4\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ 1. What is the difference between basic and acidic stains? How are these applicable in studying the cell? Basic stains are attracted to negatively charged molecules in the cell including nucleic acids (DNA and RNA) and some proteins.Acidic stains can be positive stains (stain the cells) or they can be negative stains (stain the background and not the cells). Acidic stains can stain positively charged molecules in cells including some proteins. 2. Identify the importance of each step in Gram staining. Gram 1 (crystal violet)- give them the initial color. Both Gram-positive and Gram-negative bacteria become directly stained. Gram\'s iodine solution- llows the stain to be retained better by forming an insoluble crystal violet-iodine complex. Both Gram-positive and Gram-negative bacteria remain purple after this step. Gram 3 (ethyl alcohol)- Gram-positive bacteria retain the crystal violet-iodine complex while Gram-negative are decolorized. Gram 4 (safranin)- Since the Gram-positive bacteria are already stained purple, they are not affected by the counterstain. Gram-negative bacteria, which are now colorless, become directly stained by th e safranin. Thus, Gram-positive appear purple, and Gram-negative appear pink. 3. What are the differences between simple staining and differential staining? Simple staining Involves the use of only sngle dye (e.g., methylene blue) to have observations of cell size, shape and arrangement. While differential. staining involves multiple dyes or reagents. A common example is the Gram stain, which differentiates between Gram-positive and Gram-negative bacteria based on their cell wall composition. **EXERCISE NO. 4** **DNA Extraction** I. **Introduction** II. **Learning Objectives** 1. extract DNA from own cheek cells. 2. describe the properties of DNA. 3. discuss the importance of DNA. III. **Materials** +-----------------------------------+-----------------------------------+ | sports drink | | | | | | liquid dishwashing soap | | | | | | meat tenderizer (enzyme) | | | | | | gloves | | | | | | disposable cup (1) | | | | | | a piece of string or yarn | | +-----------------------------------+-----------------------------------+ IV. **Procedure** To get the DNA out of your cheek cells you need to break open both the cell membranes and the nuclear membranes. Cell membranes and nuclear membranes consist primarily of lipids. Dishwashing detergent, like all soaps, breaks up lipids. This is why you use detergents to remove fats (which are lipids) from dirty dishes. Adding the detergent to your cheek cell solution will break open the cell membranes and nuclear membranes and release your DNA into the solution. The nucleus of each of your cells contains multiple long strands of DNA with all the instructions to make your entire body. If you stretched out the DNA found in one of your cells, it would be 2-3 meters long. To fit this entire DNA inside a tiny cell nucleus, the DNA is wrapped tightly around proteins. The enzyme in meat tenderizer is a protease, which is an enzyme that cuts proteins into small pieces. As this enzyme cuts up the proteins, the DNA will unwind and separate from the proteins. The cold alcohol reduces the solubility of DNA. When cold alcohol is poured on top of the solution, the DNA precipitates out into the alcohol layer, while the lipids and proteins stay in the solution. V. **References** Bagley, M. LiveScience Contributor. Science Experiment: Seeing Your DNA. June 06, 2013. Retrieved from http://www.livescience.com/37252-dna-science-experiment.html Biotechnology Online. What is DNA? **Retrieved from** www.biotechnologyonline.gov.au/biotec/whatisdna.html Campbell, N.A., J.B. Reece, L.A. Urry, M.L. Cain, S.A. Wasserman, P.V. Minorsky, and R.B. Jackson. 2008. Biology. 8^th^ ed. Pearson Benjamin Cummings: San Francisco CA. Doherty, J. and I. Waldron. 2007. BioRad's "Genes in a bottle. Department of Biology, University of Pennsylvania. Retrieved from http://www.bio- rad.com/cmc\_upload/Literature/ 54133/4110034B.pdf Genetic Science Learning Center. "DNA Extraction." Learn Genetics. 12 September 2015. Retrieved from http://learn.genetics.utah.edu/content/labs/extraction/ Genetic Science Learning Center. "How to Extract DNA from Any Living Thing." Learn Genetics. 12 September 2015. Retrieved from http://learn.genetics.utah.edu/units/activities/extraction/ **Exercise No. 4** **DNA Extraction** **Answer Sheets** Name: \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ Rating: \_\_\_\_\_\_\_\_\_\_ Laboratory Section: \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ Date: \_\_\_\_\_\_\_\_\_\_\_\_ **Guide Questions:** 1\. The protease in meat tenderizer actually comes from plants, but animals also make proteases. Where in your body do you think you make protein-cutting enzymes? 2\. The drawings below show a very small section of the DNA double helix from three very different organisms: a plant, a mammal, and a bacterium. Each strand of DNA shown contains five nucleotides, each with a: 3\. Does a liver cell contain the same chromosomes as a cheek cell? 4\. Why is DNA so important in biology? What is the function of DNA? **EXERCISE NO. 5** **Enzymes** I. **Introduction** II. **Learning Objectives** III. **Materials** 17 test tubes (10ml) thermometer fresh liver (chicken) IV. **Procedure** **Factors Affecting the Activity of the Enzyme Catalase.** A. **Normal catalase reaction** 1\. Place 2 mL of the 3% hydrogen peroxide (H~2~O~2~) solution into a clean test tube. B. **Is catalase reusable?** What is the reaction rate? C. **What tissues contain catalase?** You will now test for the presence of catalase in tissues other than liver. D. **Effect of temperature on catalase activity** E. **Effect of pH on catalase activity** F. **Effect of an inhibitor to the rate of activity of the enzyme catalase** **V. References** Bairoch, A. 2000. The ENZYME database in 2000. Nucleic Acids Res 28 (1): 304--5. doi:10.1093/nar/28.1.304. PMC 102465. PMID 10592255. Biology Corner. Retrieved from www.biologycorner.com/worksheets/enzyme\_lab.html Grisham, C.M. and R.H. Garrett.1999. Biochemistry. Philadelphia: Saunders College Pub. pp. 426--7. ISBN 0-03- 022318-0. Haak, M. Alberta Ingenuity Centre for Carbohydrate Science. Retrieved from www.uofaweb.ualberta.ca/\.../AICCSLabInvEnzymeActivity.doc http://faculty.montvilleschools.org/departments/Science/LabReports/CatalaseLabSample.pdf Smith, A.L. 1997. Oxford dictionary of biochemistry and molecular biology. Oxford \[Oxfordshire\]: Oxford University Press. ISBN 0-19-854768-4. **Exercise No. 5** **Enzymes** **Answer Sheets** Name: \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ Rating: \_\_\_\_\_\_\_\_\_\_ Laboratory Section: \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ Date: \_\_\_\_\_\_\_\_\_\_\_\_ **Factors affecting the activity of the enzyme catalase.** 1. What gas is being released in procedure \#2? [The catalase of the chicken liver when in contact with hydrogen peroxide produce air bubbles which is the oxygen gas being released, as an evident of chemical reaction.] 2. Has the test tube become warmer or colder? Is the reaction endothermic or exothermic? [The reaction is exothermic because it releases heat into the surroundings which is the test tube. As a result, the test tube became warmer,indicating an increase of temperature in its system.] 3. What is the component of the liquid poured into a second test tube in procedure \#5? [In procedure 5, the liquid component poured into the second test tube is primarily composed of water, oxygen gas, and some residue of hydrogen peroxide that wasn\'t decomposed during the reactiion. ] What do you think would happen if you added more liver to this liquid? [After adding more liver to this liquid, we observed that there is a reduced chemical reaction. This may due to the reason that the hydrogen peroxide has been used up, so there is nothing left for the catalase enzyme in the liver to work on] 4. Is catalase reusable? Explain. [Catalase is an enzyme (biological catalysts) which speeds up a reaction of decomposing hydrogen peroxide, a toxic chemical, into 2 harmless substances--wpater and oxygen. Without being consumed or altered in the process, catalase is reusable].\_Hence, the same catalase can be used to break down multiple hydrogen peroxide molecules. 5. In the three tissues tested (in procedure \#7), which tissues contained catalase? \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ Do some contain more catalase than others? Discuss. \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ 6. What will boiling do to an enzyme (procedure \#8)? \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ 7. 8. In procedure \#18: height of the O~2~ bubbles: test tube \#1 \_\_\_\_\_\_\_\_\_ 9. Write all your results on the reaction rates below. **Tests** **Rate of Reaction (0-5)** --------------------------- ---------------------------- procedure \#3 procedure \#5 procedure \#6 procedure \#7: potato apple chicken liver boiled liver and peroxide cold liver and peroxide warm liver and peroxide liver in strong acid liver in diluted acid liver in strong base liver in diluted base liver in water (neutral) **Guide Questions:** 1. 2\. How would you determine the optimal temperature and pH of an enzyme? 3\. Give one practical application of this experiment. **EXERCISE NO. 6** **Cell Membrane** **I. Introduction** All prokaryotic and eukaryotic cells are separated from the external environment by a cell membrane. In the latter part of the nineteenth century, scientists discovered that the cell membrane is selectively permeable, meaning that some molecules are able to cross the cell membrane while others are prevented from crossing the membrane. However, most cells are freely permeable to water molecules, which can move into and out of a cell by diffusion. **Diffusion** is defined as the movement of solute molecules from an area of higher concentration to an area of lower concentration. Specifically, water moves across the cell membrane by **osmosis**, a membrane process in which water molecules move across the membrane in response to a concentration gradient. Membranes are lipid--protein assemblies in which the components are held together in a thin sheet by non-covalent bonds. Lipid molecules are insoluble in water but dissolve readily in organic solvents. It is the shape and *amphipathic* nature of the lipid molecules that cause them to form bilayers spontaneously in aqueous solution. When lipid molecules are surrounded on all sides by water, they tend to aggregate so that their hydrophobic tails are buried in the interior and their hydrophilic heads are exposed to water. The lipid bilayer has been regarded as the universal basis for the cell-membrane structure. Its structure can be attributed to the special characteristics of the lipid molecules. Lipid bilayer exhibits two important properties. First, the hydrophobic core is an impermeable barrier that prevents the diffusion of water-soluble (hydrophilic) solutes. Importantly, this simple barrier function is modulated by the presence of membrane proteins that mediate the transport of specific molecules across this otherwise impermeable bilayer. The second property of the bilayer is its stability. The bilayer structure is maintained by hydrophobic and van der Waals interactions between the lipid chains. Even though the exterior aqueous environment can vary widely in ionic strength and pH, the bilayer has the strength to retain its characteristic architecture. II. **Learning Objectives** At the end of this exercise, students will be able to: 1. 2. 3. 4. III. **Materials and Chemicals** A. **Cell Membrane: Exploring Its Hydrophobic and Hydrophilic Properties** B. **Lipid Solubility of Membrane** **IV. Procedure** A. **Cell Membrane: Exploring Its Hydrophobic and Hydrophilic Properties** 1\. Assemble your yarn thread and straw to make a square. Insert the yarn thread into the straw. Make sure that the two straws are parallel in position. Make sure that a perfect square is made then. 2\. Take a 1 gal container and pour 1000 ml water, 500 ml glycerol and 600 ml liquid soap into it and shake to mix. (Prepare this mixture a day before using it). 3\. Pour the mixture in the container or tray. Place also the square which is made up of yarn and straw and soak it with the liquid soap mixture. Make sure when you lift the yarn with straw, a bubble is formed inside the shape. Observe what will happen if you poke the dry pencil at the center of the bubble; then wet the pencil with the soapy solution and again poke the center of the bubble; or drop dry paper clips or dice again at the center of the bubble as well as wet dice. 4\. Using the plastic dropper with water, observe what will happen if you drop water three times at the center of the bubble (analogous to pinocytosis). 5\. Also, one can demonstrate fusion (using the plastic dropper, make a big bubble and make another one near it), fission (with your finger, separate the small bubbles from one another), flexibility of the membrane (move the straw upward and downward), and phagocytosis and transport through channel protein (with a small string, place it on the bubble and drop the paper clips or dice inside the string) using this bubble laboratory activity. B. **Lipid Solubility of Membranes** 1\. Red onion cells contain a high concentration of the red pigment anthocyanin. When exposed to a compound which dissolves the cell membranes, the anthocyanin will leak out of the cells and cause a red color to occur in the surrounding media. Cut thin slices of a red onion so that they can be placed on a microscope depression slide and viewed with the lowest power (4X). 2\. While watching the edge of the sliced red onion, add approximately 1-2 drops of each of the above alcohols to the slide, until the onion section is submerged. Be careful not to allow the alcohol to flow off the slide. ***Iso-amyl alcohol has a strong, obnoxious odor and the fumes are somewhat irritating. Adequate ventilation is required.*** 3\. Immediately begin to time the **dissolution** of the onion cell membranes. Mark the time when a red color is first observed in the surrounding alcohol solution. 4\. Repeat the entire series for 1/2 and 1/4 dilutions of each of the alcohols. 5\. For each dilution of each alcohol, calculate a penetration coefficient by dividing the time of pigment appearance by the molar concentration of the alcohol. Plot this penetration coefficient against the relative miscibility of the alcohol (known as the partition or distribution coefficient). C. **Egg Experiment** Now students will experiment with osmosis and diffusion by submerging their de-shelled egg in a substance of their choice. As a measurement of change students will record whatever observations/parameters they could think of as scientists. First, students will remove the eggshell by soaking it in vinegar. Vinegar is an acid called acetic acid (CH~3~COOH).[^1^](#fn1){#fnref1.footnote-ref} White vinegar from the store is normally 4% vinegar and 96% water. Eggshells are made of calcium carbonate (CaCO~3~). The acetic acid in the vinegar reacts with the calcium carbonate in the eggshells to make calcium acetate plus water and carbon dioxide bubbles that you immediately see on the surface of the egg once it is submerged in vinegar. The egg will get slightly bigger after soaking it in vinegar because some of the water in the vinegar solution entered the egg through osmosis. Egg's membrane is making an effort to equalize the concentration of water on both sides of the membrane. Students can gently rub the sides of the egg to encourage the removal of the shell. Be careful they do not rub too hard because they will pop the egg. Once all the calcium carbonate shell is removed you are left with a white membrane. The de-shelled eggs are a good model of human cells. The white membrane of the egg is selectively permeable like cell membrane in human cells as well as the egg being a single cell. The white membrane of the egg is actually two membranes held tightly together. After the eggshell is removed they will soak it in water to "reset" the egg. Students can bring in the substance of their choice (Choose at least 5 substances). Anything too alkaline or basic will simply pop the egg. The experiment will be done by group. Each group should make their own hypothesis, objectives, method and submit their results following the scientific method. However, submission of their scientific writing will be done individually. **V. References** Alberts B., A. Johnson, J. Lewis, M. Raff, K. Roberts, and P. Walter. 2002. Molecular Biology of the Cell. 4th edition. Garland Science: New York. Experiment with Naked eggs Activity (https://www.**exploratorium**.edu/) Heidcamp, W. H. Cell Biology Manual. Biology Department, Gustavus Adolphus College, St. Peter, MN 56082 \-- cellab\@gac.edu Karp, G. Cell and Molecular Biology. 6^th^ edition (e-book). Lodish,H.,  A. Berk, P. Matsudaira, C. A. Kaiser, M. Krieger, M. P. Scott, L. Zipursky,  and J. Darnell. 2003. Molecular Cell Biology. 5th edition. WH Freeman: New York. **Exercise No. 6** **Cell Membrane** **Answer Sheets** Name: \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ Rating: \_\_\_\_\_\_\_\_\_\_ Laboratory Section: \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ Date: \_\_\_\_\_\_\_\_\_\_\_\_ **Guide Questions:** **A. Cell Membrane: Exploring Its Hydrophobic and Hydrophilic Properties** 1. **How does the soap film demonstrate the selectively permeable nature of the plasma membrane?** \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ 2. **Describe the fluid-mosaic model of the cell membrane. Relate it to their roles in cell transport.** \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ 3. **Why is it important that the cell membranes are composed of lipids?** What makes lipids soluble in organic solvent and insoluble in water? \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ 4. **Define briefly and give the significance of the following for the survival of cells:** **a. fission** **b. fusion** **c. pinocytosis** **d. phagocytosis** **e. protein channels** **B. Lipid Solubility of Membranes** 1. Why are the lipid molecules in cell membranes *amphipathic*? \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ 2. What is the relationship between penetration coefficient and relative miscibility \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ ::: {.section.footnotes} ------------------------------------------------------------------------ 1. ::: {#fn1} ::: :::