Kadir Has University Biology I - Laboratory Guide Book PDF

KADIR HAS UNIVERSITY BIOLOGY I - LABORATORY GUIDE BOOK DEPARTMENT OF MOLECULAR BIOLOGY AND GENETICS Instructor: Dr. Yiğit Kocagöz 2024-2025 Welcome to the Biology-I Laboratory Hello and welcome to the biology laboratory at Kadir Has U...

KADIR HAS UNIVERSITY BIOLOGY I - LABORATORY GUIDE BOOK DEPARTMENT OF MOLECULAR BIOLOGY AND GENETICS Instructor: Dr. Yiğit Kocagöz 2024-2025 Welcome to the Biology-I Laboratory Hello and welcome to the biology laboratory at Kadir Has University. This laboratory manual is intended to assist you in the completion of a sequence of tasks and experiments that will enhance the knowledge of biological concepts and techniques. By actively engaging in these laboratory sessions, you will acquire practical experience that enhances your theoretical understanding and fosters the development of critical skills in scientific methodology and research. Purpose of the Laboratory The primary purpose of the biology laboratory is to provide a beginner friendly supportive environment where freshmen students can engage in the practical application of biological theories and principles. Here, you will have the opportunity to observe living organisms, conduct experiments, and analyze data to draw meaningful conclusions, by mainly using light microscopy. These experiences are very important in helping you understand the complexity and diversity of life, the intricacies of biological processes, and the scientific methods used to study them. Learning Outcomes By the end of this course, you should be able to:  Demonstrate proficiency in using basic laboratory equipment and techniques.  Design and conduct experiments, including formulating hypotheses, collecting and analyzing data, and drawing reliable conclusions.  Communicate scientific findings effectively through written reports and presentations.  Understand and apply the principles of biological safety and ethical considerations in the laboratory setting.  1- LAB SAFETY AND INTRODUCTION TO LIGHT MICROSCOPY Laboratory Safety Your first concern while working in a lab should be your safety. Working in the biology lab means handling several items and tools that, if improperly handled, might cause hazards. To guarantee a safe and effective lab environment, you should pay close attention to the following section including essential safety rules and practices. In class, we will walk over these guidelines verbally. General Safety Rules 1. Know the Location of Safety Equipment: Please look around and familiarize yourself with the location of safety showers, eye wash stations, fire extinguishers, and first aid kits. In case of an emergency, these could be very important. 2. Proper Attire: Always wear a lab coat, gloves, and closed-toe shoes. Long hair should be tied back, and loose clothing should be secured. Entering the lab with too much exposed skin is a risk. 3. Personal Protective Equipment (PPE): Depending on the experiment, additional PPE such as thicker heat protective gloves and eye googles/face shields may be required. Always check the experiment guidelines for specific PPE requirements or ask your instructor if you have any specific questions regarding to your health situation. Example: if you have asthma, you should be especially careful about working with volatile chemicals. 4. No Food or Drink: Eating, drinking, or storing food in the lab is absolutely forbidden! If you need to drink water or take your medicine, you have to take off your lab equipment, go outside of the lab and then eat/drink. This is to prevent you from accidentally digesting a dangerous chemical. 5. Be Aware of Your Surroundings: Try to stay alert and be aware of what others are doing around you to prevent accidents from occurring. Physically joking around with your lab mates is forbidden since it can lead to serious accidents. 6. Never touch your face or skin with your gloves on. The gloves are there to prevent your skin from touching the benches and chemicals. If your gloves touch to the chemicals and benches, then the gloves are now also contaminated. If you touch your face or skin with your gloves after using them, the entire purpose of using gloves is ruined! Please be mindful about contamination and try to refrain from touching your personal items as well. Handling Chemicals 1. Read Labels and Safety Data Sheets (SDS): Before using any chemical, read the label and the corresponding SDS to understand its hazards and safe handling procedures. Your instructor will also warn you depending on the experiment you’ll be working on. 2. Proper Disposal: Dispose of chemicals according to the disposal guidelines provided. Never pour chemicals down the sink unless instructed to do so. If you are not sure about how to dispose certain things you have used in during the experiment, please ask your instructor. 3. Use Fume Hoods: While conducting the experiments involving volatile or hazardous chemicals, please use the fume hood to prevent inhalation of harmful chemicals. Also please make sure there is proper ventilation in the laboratory. 4. Spills and Accidents: If you accidentally spilled a chemical or dropped something on the floor, immediately report any spills or accidents to your instructor. The spills will be cleaned as fast as possible by using the appropriate materials and procedures. Don’t be shy and try to hide the accident. Please think about everyone’s safety first. Biological Safety 1. Proper Disposal of Biological Waste: Dispose of biological materials, such as bacterial cultures and disposable gloves, should be discarded into designated biohazard containers in red color. 2. Disinfect Work Areas: Disinfect your bench by using 70% ethanol before and after experiments to maintain a clean and safe environment. This will keep your experiments and yourself cleaner as well. Equipment Safety 1. Microscopes: During our experiments you’ll mostly be working with Light Microscopes. Please handle the microscopes with care, using both hands. Clean lenses with appropriate materials and follow the instructor’s guidelines for usage and storage. If you are having problems, don’t force the equipment. Please just ask for help instead. 2. Micropipettes: One of the main laboratory equipment’s are micropipettes. Please do not use them unless you were thought how to properly use them by your instructor. After learning the basics of using a micropipette, please be mindful with how you handle the micropipettes. They are very delicate and they should not be dropped harshly on any surface. After you are done using them, please put the micropipettes back to their original position on the rack. 3. Glassware: Inspect glassware for cracks or chips before use. If there are any cracks or chips, speak to your instructor about it so the glassware can be discarded appropriately.  4. Electrical Equipment: Ensure that electrical equipment is properly grounded and in good working condition. Do not handle electrical equipment with wet hands. Emergency Procedures 1. Fire: In case of a fire, use the nearest fire extinguisher if trained to do so, or evacuate the area and activate the fire alarm. Your instructor will show you the nearest fire exit. 2. Chemical Exposure: If chemicals come into contact with your skin or eyes, rinse immediately with plenty of water and seek medical attention. You can use the nearest water source. Your instructor will show you the location of emergency eyewash and shower location 3. Injury: Report any injury, no matter how minor, to your instructor immediately. Follow first aid procedures as necessary. If you are cut with an item in the laboratory, please speak to your instructor about what exactly cut you so the appropriate steps can be taken to make sure everyone else is safe and you can receive the proper help. Conduct and Responsibilities All of our students are responsible for coming prepared to the lab. You should buy your own lab coat and make sure it’s clean throughout your studies. The single use gloves will be given to you by your instructor. 1. Preparation: Come to the lab prepared by reading the experiment protocol and safety instructions in advance. You should read your lab manuals before coming to the class. Your instructor can ask you questions and hold pop-quizzes about the experiment. 2. Attendance and Punctuality: Attend all lab sessions and arrive on time. Participation is crucial for your learning and safety. The instructor is allowed to tell the students that they cannot attend the session if they arrive later than 10 minutes. 3. Cleanliness: Keep your work area clean and organized. After the experiments, it is also your responsibility to learn how to properly clean up after yourself. 4. Respect and Collaboration: Work collaboratively with your lab partners, respect the equipment and materials, and follow the instructor’s directions at all times. 5. Ethics: Maintain academic integrity in all your work. This includes honest data collection, proper citation of sources, and respectful treatment of all living organisms. While writing your lab reports, you should check the appropriate formatting rules that are given to you by your instructor. Plagiarism and copy-pasting is absolutely not tolerated. 6. Organization: For every lab session, you are responsible for bringing a lab notebook. This notebook should only be used in the lab for you to take notes and record your experiment results. In the cases where you don’t bring your notebook and your data  is recorded wrong, the lab instructor is allowed to deduct points from your upcoming lab reports. Part II: Using a Light Microscope The light microscope is one of the main essential tools in the field of biology, enabling us to visually examine structures and organisms that are too little to be observed without using a tool. This manual offers details on the correct usage, maintenance, and upkeep of a light microscope that makes sure students can accurately and successfully view small objects. Components of a Light Microscope The microscopes we are using in the student laboratories are SOIF Smart-2 Binocular Laboratory Light Microscopes (Fig 1). Figure 1. SOIF Smart-2 Binocular Laboratory Light Microscope (AN-KA, 1989). Before doing anything at all with the light microscope, it is important to familiarize yourself with its parts. If you have no idea where to start first, it could be a great idea could be to start by researching the brand and the website to see if they have any informative videos or documents you should be aware of. During this laboratory class, we will provide you with detailed instructions and key points. Here are some main important components of a light microscope: 1. Eyepiece (Ocular Lens): This is the lens you look through to see the specimen. Most of the times, you can adjust the space between the lenses to fit your eye width. 2. Objective Lenses: These are the lenses above the black stage where you can adjust the magnification. Typically, three or four lenses with varying magnifications (e.g., 4x, 10x, 40x, and 100x) are found of the microscope. 3. Stage: The flat black platform where you place the sample slide. It usually has clips to hold the slide in place so that is does not move while you image your sample.  4. Stage Controls: These are knobs and buttons that can move the stage left/right and forward/backward. This way, you don’t have to adjust your view by touching the sample slide multiple times. 5. Light Source: This is the light source/lamp that provides illumination for us to see the sample in clear image. You can adjust the intensity of the light depending on the visual you are trying to see. Guide to Using a Light Microscope Setting Up the Microscope The microscope is situated on a flat dry surface. To protect the equipment, microscopes will have a plastic cover. You can start by carefully lifting the cover. Connect the microscope to a power source if it has an electric light source by plugging it in. Then, turn on the light source. Preparing the Slide Your sample can be different depending on what you are trying to observe. During this class, we will check the already prepared slides under the microscope. Your instructor will give you random sample specimens. You should make sure the slide is clean and then place it on the stage of the microscope. You should make sure the secure the slide with the stage clips. If you are having any troubles placing your sample, please ask your instructor for help. Initial Observation Start your visualization with 4x objective lens. You can move towards the higher magnification lenses as you and your lab groups are all done with the visualization. You can adjust the sample location using the knobs and make sure the visual you get is not hazy. You should make adjustments to center the image and get a clear visual. Depending on your eyesight, the adjustments can be different. During this adjustment, the instructor will show you which knobs to use for fine focus. You will get to observe many different specimens. When you are observing a slide, please take notes in your lab notebook about what you are seeing, which magnification you had to use and is there any certain details that catches your eye. Changing Magnification When you change your magnification, you will have to readjust the focus and location of the slides. As you repeat these steps multiple times, you’ll be able to adjust the microscope settings much quicker and better in no time. Recording Observations While you are recording the visuals in the specimens, you can draw sketches of what you see and write important points that seem to catch your eye. Also, the microscope can come with a camera attachment so you’ll be able to take a picture and the lab instructor can send the visuals to you so that you can include them in your lab report. Turning the Microscope off When you are done with using the microscope, you should lower the stage away from the lenses to its lowest position and turn off the light source. You should also remove all of the slides from the stage and reorganize them as your instructor suggests. After cleaning up the bench you worked on, you can place the dust cover back on the microscope and start to take off your protective equipment. Conclusion The biology laboratory is a place of discovery and learning at the same time. By following the guidelines and procedures outlined in this manual, you can ensure a safe, productive, and enjoyable lab experience. Your adherence to safety protocols not only protects you but also your peers and the integrity of the scientific work you will be conducting. In order for us to maintain a nice educational environment, it is at utmost importance for you to follow laboratory rules. Welcome to the world of biological exploration—let's make it a safe, educational and fun journey together! Notes For When You Are Writing Your Lab Report While writing your lab report, you should follow the general guidelines given below: Introduction: This is where you explain the importance of lab safety and write a paragraph about the things we discussed in the lab. Here are some examples, why do we wear gloves all the time when we are in the laboratory? Why do we wear lab coats, etc. Also, you should give an introduction about what is light microscope and give a brief background about its history (No more than a paragraph. We’d just like you to research about the first microscope and who invented it. What was its importance?) Materials and Methods: You should make a table where you write all of the equipment and materials that you used while conducting your introduction experiment in the laboratory. All of the materials and equipment’s that are found in our student lab can be found in the lab manual (Appendix A). You should also write how did you use the light microscope in your lab report in detail. Results: While writing your results, you should include the picture of the specimens you observed under the microscope and explain what you see and point out some distinctive structures that are unique for the specimen. Your instructor will also point out these things for you to notice. Don’t forget to learn about the formatting of these images and figure legends. Organization is very important while writing your lab report. Discussion and Conclusion: Finally, you should finish of by summarizing everything you learnt in the laboratory about lab safety and how to use the light microscope. References: Any information you have gained from a website, paper or book has to be referenced. All of these references need to be included in at the end of your lab report as the bibliography. Your instructor will show you an example lab report prior to your submission. There are certain rules that you need to consider while citing a source. Citations allow the readers to locate your source and confirm your statements. Moreover, readers can learn more about the topic your article is based on by checking your sources. There are different formats that you can use for article citation (Such as APA -American Psychology Association and AMA - American Medical Association). We will use APA format in this course. Keep in mind that each type of citation (article citation, book citation and web page citation) has certain set of rules you need to follow. Journal article citation: Name of author or authors (last name, first initial, middle initial) Year of publication Article title Journal title Volume followed by the issue number (issue number is in parenthesis) Page numbers Example: Ahmed, A. A., Z. Lu, et al. (2010). "SIK2 is a centrosome kinase required for bipolar mitotic spindle formation that provides a potential target for therapy in ovarian cancer." Cancer Cell 18(2): 109-121. Book citations: Name of author or authors (last name, first initial, middle initial) Year of publication Title of book Edition (if more than one) Place were published (city) followed by a colon Publisher name. Page number(s) Example: Karp, G. (2013). Cell and Molecular Biology: Concepts and Experiments, 7th edition. John Wiley and Sons Inc., NY. Pp 435-476. Web page: Author (year). Title [Type of medium]. The available Web address, Retrieval Date Jones, J. (1991, May 10). Networks (2nd ed.) [Online]. Available: http://www.atm.com, Retrieval Date: 09.02.2017 Things you mustn’t do while preparing your reports: Cheating : Copying from another student, using unauthorized materials during exams/quizzes, and providing answers to other students. Plagiarism : Using other people’s ideas, writing as one’s own without giving proper credit to the source. You are expected to cite all printed & online sources you use in your assignments. Fabrication & Falsification : Generating data or information that never existed Changing or omitting data Multiple Submission : Submitting the same work, as a whole or partially, in more than one course without the explicitly informing the instructors. You will be instructed in the courses on the ways of referencing sources to avoid inadvertent plagiarism cases. When in doubt, please ask the instructor. IMPORTANT: All your written assignments will be evaluated by multiple AI detection tools. Any assignment that is generated by AI algorithms will be graded as 0 (“zero”). 2- LB-AGAR PLATE POURING AND BACTERIUM COLONY OBSERVATION Introduction During this experiment, you will learn what is agar, what is Escherichia coli (E. coli) bacteria and why do these substances are important for Molecular Biology laboratory experiments. To start our background information, we should first discuss about what is agar and why is it important for us. Agar is a gelatinous substance derived from the cell walls of certain species of red algae. It is composed mainly polysaccharides, agarose and agaropectin. Agar has unique properties that make it invaluable in various scientific and industrial applications, especially in molecular biology. Firstly, it has slightly transparent, gelatin like structure. Below 40°C temperatures, it can solidify and create a smooth solid surface on top. It can melt temperatures above 85°C. It creates a perfect base line solid structure that can be supplied with additional nutrients for microorganisms and can be sterilized using autoclaving technique that utilizes high temperature and pressure. This can ensure of a sterile, highly efficient surface that a diverse range of microorganism can thrive on for us to study. A great example for this is called Luria broth (LB), which is a highly nutritious medium frequently employed for controlled bacterial growth in laboratory settings. Agar is added to LB medium, which forms a gel matrix that provides a solid surface for bacterial growth. Bacteria are unable to break down the solid structure made possible by agar but can obtain nutrients from the LB medium (Figure 2.1). Figure 2.1 LB-agar plates that are prepared on a 100mm petri dish. These plates can be used for seeding bacteria to grow colonies on a solid surface. One additional benefit of use LB-agar plates is the ability to modify the selectivity of your experiment. Antibiotics can be employed to selectively cultivate specific types of bacteria by eliminating any undesired microorganisms that are not intended to thrive (AddGene, 2016). During our experiment, we will grow the Escherichia coli (E. coli) bacterium on the LB-agar plates we prepared. Here are some of the main reasons for us to be working with E. coli. E. coli is a rod-shaped, Gram-negative bacterium that is frequently observed in the intestines of both humans and animals. Some strains of E. coli can cause food poisoning and other problems, while the majority are harmless and part of the normal gut flora. Theodor Escherich, the bacteriologist who discovered the species, was the one who gave it its name (American Society for Microbiology, 2011). Figure 2.2. Visual representation of E. coli bacterium which has a rod like shape (CDC, 2024). E. coli is also considered a model organism which allows us to manipulate it into studying important biological processes and understand these natural phenomena in better detail. These bacteria are mainly used in gene expression and genetics recombination experiments due to their ability for being able to grow very quickly and adapt fairly easy. They can also be genetically manipulated; they are very cheap to maintain and therefore great for producing desired proteins in bulk or for cloning experiments. In conclusion, E. coli is a fundamental organism in molecular biology, serving as a versatile and powerful tool for genetic manipulation, protein production, pathogenicity studies, and synthetic biology. It is very important to learn how to cultivate them and grow them as it can be considered as a basic fundamental molecular biology and genetics laboratory skill. Materials and Equipment Materials  Agar powder or pre-prepared agar medium  Distilled water (autoclaved)  LB powder  Sterile Petri dishes  Sterile pipettes or inoculation loops  Ethanol (70% or higher)  Bunsen burner or alcohol lamp  Permanent marker or labeling tape  Glass slides  Cover slips  Gram staining kit Equipment  Autoclave  Microwave  Beaker or flask (for preparing agar medium)  Balance (for weighing agar powder)  Graduated cylinder (for measuring water)  Incubator (set to appropriate temperature for bacterial growth)  Gloves  Lab coat  Safety goggles Safety Precautions  Lab coats and gloves are mandatory. If you touch any surface in the laboratory with your gloves on, you cannot touch your face, skin or personal items while wearing those gloves otherwise you’d be causing cross contamination! Procedure Preparing Agar Plates 1. Prepare the Agar Medium:   Measure the required amount of agar powder and add it to a flask.  Add the appropriate volume of distilled water carefully  Mix well until the agar powder is fully dissolved. 2. Sterilize the Agar Medium:  Cover the beaker or flask with aluminum foil or use a cap that allows steam to escape.  Place the beaker or flask in an autoclave and sterilize at 121°C for 15-20 minutes. The autoclave can only be used with the instructor present.  Allow the sterilized agar medium to cool to approximately 50-55°C. This can be done by placing the container in a water bath or letting it sit at room temperature. 3. Pour the Agar Plates:  Once the sterilized agar cooled down to a temperature where you can hold the flask without being burnt, you can arrange Petri dishes on a clean, flat surface.  Working near a flame or in a sterile environment, carefully pour the cooled agar medium into each Petri dish to a depth of about 3-4 mm (approximately 15-20 mL per petri dish). This is where we will prepare 2 mixtures, 1 falcon will have LB-agar, another falcon will be prepared with antibiotics supplied to it. Make sure to label the falcons and petri dishes “no antibiotics (-), with antibiotics (+) accordingly.  Avoid creating bubbles or overfilling the plates. The surfaces of the plates should be flat. 4. Solidify the Agar:  Allow the poured agar plates to solidify at room temperature with their lids partially ajar to prevent condensation on the agar surface.  Once solidified, cover the plates with their lids. They can be used immediately after they solidify or you can carefully store them in the fridge to use later.  Add 50 microliter of antibiotic solution onto one solidified agar plate (one plate per group) and spread the solution across the agar surface. MARK THE PLATE WITH ANTIBIOTICS! Seeding E. coli into Agar Plates  1. Label the Plates:  Use a permanent marker or labeling tape to label the bottom of each agar plate with relevant information, such as the date, type of medium, and antibiotic type. 2. Prepare the Bacterial Culture:  Take the determined (1 microliter) bacteria amount from the E. coli supply your instructor provides to you using micropipettes. Mix the bacterial stock with 1 ml water. Then take 1 microliter of bacterial solution from the dilution you prepared and repeat the step with 1 ml water.  Take 50 microliter of diluted bacterial solution by using micropipettes.  Spread the diluted bacteria solution evenly across the plates surface without damaging the solid flat surface of the agar plate.  Take 50 microliter of stock bacterial solution and spread the volume on the antibiotic-containing agar plate you prepared previously. Spread the bacteria as before. 3. Incubate the Plates:  Place the seeded agar plates upside down (lid down, agar up) in the incubator set to the appropriate temperature for the bacterial strain being studied (commonly 37°C for many bacteria).  Incubate for 24-48 hours or until colonies are visible.  You can come the next day at the same time to see your bacterial colonies. Your instructor will also send you the pictures of your results. Disposal and Clean-up  Right After the Experiment: It is really important to discard your trash into biohazard trash. Anything and everything that touches the E. coli experiment and the benches, will either have to be discarded into biohazard trash bag or has to be cleaned by the instructor accordingly. Do not put your gloves and other plastic waste into regular trash can. Observing Bacterium Colonies (1 day later)  After incubation, you can observe the colonies without opening the plates to prevent contamination. The bacterial colonies can be visible to the eye.  Note the size, shape, color, and texture of the colonies. Use a magnifying glass or dissecting microscope if necessary. Compare the plates with antibiotic and without  antibiotic. What did you expect to see? What did you see? Please record these findings to your lab notebook. You can also take photographs to include in your lab reports. Classification of Bacteria with Gram Staining The largest group of organisms that share our environment with us are microorganisms. Among various microorganisms, bacteria constitute a a large domain of prokaryotic life. There are a fairly high number of bacterial species with different features and traits; however, it is possible to examine bacteria in two basic classes depending on the features of their cell walls. The chemical staining method developed by Danish scientist Hans Christian Gram allows us to identify bacteria with a thick peptidoglycan wall as "Gram-positive" and bacteria with a very thin wall as "Gram-negative". Figure 2.3 Essential steps of Gram staining. Gram staining is a frequently used technique in many areas, such as food industry and medicine. It is commonly performed when a bacterial infection is suspected. To check if an infection has bacterial origin, biopsy samples taken from patients can be examined with Gram staining providing valuable medical information in short time. In today's lab session, we will stain the E. coli bacteria we used previously with a Gram staining kit and observe them under light microscope, and thus we will obtain information about the cell walls of E. coli bacteria. Figure 2.4. A Gram stain of mixed bacteria. Gram-negative bacilli are in red and Gram-positive cocci are in purple. Procedure: Fixation, staining and visualization:  "Heat-fix" the slide with the specimen by passing it over a heat source, such as a flame, several times using a clothes pin or forceps. The slide should be passed very quickly through the flame and not be heated excessively. Place slide on the staining tray.  Flood the fixed smear with crystal violet solution and allow to remain for 1 minute.  Rinse off the crystal violet with distilled or tap water.  Flood the slide with iodine solution. Allow to remain for one minute.  Rinse off the iodine solution with distilled or tap water.  Holding slide on a tilt with a clothes pin, flood the slide with decolorizer for one to five seconds.  Rinse off the decolorizer with distilled or tap water.  Flood the slide with safranin. Allow to remain for 30 seconds.  Rinse off the safranin with distilled or tap water.  Dry the slide on bibulous paper or absorbent paper and place in an upright position. *All ingredients mentioned in the protocol are found in the kit and will be provided by the instructor. ** This protocol is reprinted from Cornell University College of Veterinary Medicine website: Available: https://www.vet.cornell.edu/animal-health-diagnostic-center/testing/testing- protocols-interpretations/gram-stain-protocol Retrieval date: 10.09.24 3- IMAGING WITH LIGHT MICROSCOPY: WHAT’S UNDERNEATH OUR FINGERNAILS? Observing Material Underneath Fingernails with a Microscope Introduction Everybody says it is bad to bite your nails and not good for your health but what’s the actual reason for this? Well, even if you wash your hands frequently, underneath the nails can be the perfect narrow moist environment for bacteria, fungi and yeast to grow. By biting your nails, you are taking a very serious risk of E. coli infection or salmonella infection. We touch many dirty areas without even realizing and these microorganisms can grow in a very short amount of time. During this experiment, we are visualizing what’s underneath our finger nails and we are learning to prepare our sample specimen ourselves this time rather than using previously prepared slides. At the end of this experiment, you’ll be able to prepare your own microscopy samples and also, you’ll be visually aware of what else might be inhabiting on your skin. Being mindful about these microorganisms will definitely change your perspective on hygiene and certain bad habits. Figure 3.1. Underneath your finger nails might be filled with a diverse range of microorganisms. Our purpose for this experiment is to visualizing them under the microscope and trying to identify several of them. As an example, nail biting is not merely a gross habit; it can have serious effects on one's health and well-being. Taking measures to break the habit and comprehending these risks can result in a more positive self-image and improved overall health. It may also be helpful to consult with a doctor or therapist if nail biting is persistent and linked to symptoms of anxiety or compulsive behavior (UCLA Health, 2022). Materials and Equipment  Microscope: A light microscope with various objective lenses (4x, 10x, 40x, and 100x).  Slides and Cover Slips: For mounting samples.  Sterile Tweezers or Nail Scraper: For collecting material from under the fingernails.  Sterile Swabs: For collecting samples.  Distilled Water or Saline Solution: For preparing wet mounts.  Staining Reagents (Optional): Such as Gram stain or methylene blue for better visualization of microorganisms.  Pipette or Dropper: For applying liquid to the slide.  Gloves and Lab Coat: For safety and to prevent contamination.  Waste Disposal: Biohazard bag for disposing of samples and used materials. Procedure 1. Sample Collection 1. Control group and test group: In one of the sessions, half of the students can collect samples before they wash their hands and the other half can collect samples after washing their hands. Let’s try to determine if a quick wash can help with what’s underneath our nails. While washing your hands, try use a lot of soap and water to remove surface contaminants. 2. Collect Material: Use sterile tweezers, a nail scraper, or a swab to gently collect material from underneath your fingernails. Be careful not to cause injury. If a student is giving out samples from underneath their nails, they will have to take of their gloves so, one of your lab mates who is wearing gloves can gather the material under your nails as you stand by. 3. Prepare Sample: If using a swab, you can directly smear the collected material onto a slide. If using tweezers or a nail scraper, transfer the material onto a glass slide carefully. Ask your instructors help if you are struggling. 2. Preparing the Slide o Place a small drop of distilled water or saline solution on the slide. o Using the collected material, mix it with the drop of liquid on the slide. o Carefully place a cover slip over the drop to spread the sample evenly and to avoid air bubbles. 3. Examination 1. Turn on the microscope and adjust the light source. o Place the slide on the stage and secure it with stage clips. o Start with the lowest magnification (4x objective lens) to locate the sample. o Use the coarse focus knob to bring the sample into focus. o Switch to higher magnifications (10x and 40x) to observe more details. o Use the fine focus knob to adjust the clarity of the image.  o Observe the material under different magnifications and note any microorganisms, dirt, or other particles. o Sketch or take photographs of your observations for documentation. o Try to identify couple of microorganisms if possible. You can ask the instructors help for this stage. What You Might See  Bacteria: Various shapes and sizes can be observed from different students.  Fungi: Yeasts or mold spores.  Skin Cells: Dead cells shed from the skin that accumulated overtime.  Dirt and Debris: Inorganic and organic particles.  Other Microorganisms: Protozoa or small arthropods which still might be moving around! Safety and Disposal  Wear Protective Gear: Always wear gloves and a lab coat to protect yourself from potential pathogens.  Dispose of Samples Properly: Place all used slides, cover slips, swabs, and any other disposable materials in a biohazard bag.  Clean the Work Area: Disinfect your work area and wash your hands thoroughly after completing the examination. Conclusion Underneath your fingernails, microscopic study of material can expose a wide spectrum of germs and particles. This straightforward but educational approach shows the need of excellent cleanliness habits and helps grasp the microscopic world living on our bodies. PART II: Observation of mitotic stages on onion root tips through acetocarmine staining Cell division plays a crucial role for living organisms; it is an essential step for events like development, growth, and maintenance of tissue integrity. Our somatic cells increase their numbers by giving rise to daughter cells with the same number and kind of chromosomes as their parental cells. This type of cell division is called mitosis, and it is essential for tissue growth. Mitosis is divided into four sequential stages: prophase, metaphase, anaphase, and telophase. The cells that are not in any of these phases are in another metabolic stage of the cell cycle called interphase. Each stage has its own characteristic features, which give us hints for labelling the cells properly. Prophase: Chromatin fibers become visible. Nuclei start degrading, but nuclear boundaries are still visible. Metaphase: Nuclear boundaries are not visible. Chromosomes are fully condensed and arranged at the midline. Anaphase: Chromosomes become V-shaped; one set keeps getting distant from the other set. Telophase: Chromosomes are completely pulled to opposite poles. New nuclear membranes are partially formed. Interphase: Nuclear boundaries and nuclei can be clearly seen. Figure 3.2: Representative image of acetocarmine staining under light microscope. Highly prolific tissues display a large number of cells undergoing mitosis. It is easy to capture and visualize mitotic stages accurately by using cationic dyes, such as acetocarmine solution (carmine in acetic acid). Carmine is a basic dye extracted from the insect Coccus cacti and has high binding affinity to negatively charged nucleic acid molecules. In this part of our lab session, we will take advantage of carmine’s unique DNA-binding ability to observe mitotic stages of cells in the onion root tips, a highly prolific plant tissue frequently used for mitotic analysis. Procedure   Fixated onion root tips will be provided by the instructor.  Rinse the fixated onion tips with water 3 times.  Take 1-2 tips and put them on a glass slide.  Add one drop of 1/10 N HCl.  Add two drops of acetocarmine solution.  Wait for 12-15 minutes.  Rinse the tips with water and cut 2-3 mm portions from the tips.  Carefully put the portions on glass slide again, add one drop of water and carefully mount them with a cover slip.  Examine the samples under light microscope as you did for the fingernail samples during the first part of the session.  Count the total number of cells you see under microscope. Then count cells from each stage and calculate the mitotic index. Mitotic index: ratio between the number of cells undergoing mitosis to the total cell number.  4- IMAGING WITH LIGHT MICROSCOPY: WATER SAMPLE FROM HALİÇ Introduction During this lab experiment, we’ll be comparing between sterile water and water sample that is taken from Haliç. The purpose of this experiment is to understand the importance of sterilization. Although rivers and lakes might seem to be easy sources of water, ingesting unclean water from these sources carries major health hazards. Before consumption, water should be correctly treated to guarantee safety by removing bacteria, chemicals, and other pollutants. The main reason why we don’t use water from lakes and rivers before sterilizing it is that it might be containing many different microorganisms. Depending on the environmental factors, bodies of water usually house bacteria, viruses, parasites and protozoans of all kinds. There are also many different types of sterilization that is done to prevent infections from water prior to drinking. These can be filtration, chemical sterilization or reverse osmosis. In our laboratory, we will use autoclaving to see what happens to these microorganisms after boiling in high temperature and pressure. Figure 4.1. Unsterilized water under light microscope. A lot of accumulated debris accompanies bacteria, protozoans and chemical hazards can be seen. There are several different microorganisms presents in the image above. Autoclave can be described as a controlled pressure cooker. Most of the microorganism when boiled, are not able to survive thus the heat and the pressure kills them (Black, 1993). In summary, examining unclean water using a light microscope can reveal a diverse range of microbes, particles, and other impurities. This experiment will help you understand the importance of sterilization while collecting samples from your environment and visualizing them under the microscope can both be a wonderful way to start asking research questions and also trying to find answers to these questions as well. You will also get more experience in preparing and examining a specimen of contaminated water using light microscopy. The objective of this activity is to recognize and write down the diverse constituents found in the sample, encompassing bacteria, protozoa, algae, and inanimate particles then visualizing it once again after autoclaving it to see if high temperature allows us to sterilize the dirty water. Do you think, if the contaminated water also has other type of impurities like chemical components and harmful ingredients, autoclaving will be enough to make it drinkable? Please discuss this in the class with your instructor. Materials and Equipment  Light microscope with objective lenses (4x, 10x, 40x)  Glass slides and cover slips  Droppers or pipettes  Sterile falcons for collecting water samples  Staining reagents (methylene blue)  Distilled water  Gloves and lab coat  Waste disposal container Procedure 1. Collecting the Samples  In order to collect the samples, wear gloves in the lab and follow your instructor to the side of Haliç and use a disposable falcon to collect some samples from the water. Make sure to be mindful to your steps and follow your instructor’s guidance throughout this process.  Bring the collected sample in to the lab. Wear your lab coat before starting to work with the water sample.  Before preparing the sample for visualization, in order to make most of our time, immediately put some of the contaminated water sample into the autoclave and start the sterilization cycle. 2. Preparing the Slide  Ensure the glass slide is clean and free from dust or debris.  Using a dropper or pipette, place a drop of the dirty water sample onto the center of the glass slide. Take another clean glass slide and this time, use sterilized autoclaved water sample onto the center. Don’t forget to label the glass slides accordingly.  Gently lower a cover slip over the drop of water. Avoid trapping air bubbles under the cover slip as they can obstruct the view.  If necessary, use a piece of lens cleaning paper to blot any excess water around the edges of the cover slip. 2. Examination  Let the water dry for a few minutes, then add one drop of 1% methylene blue.  Mount the slide carefully with the cover slip.  Turn on the microscope and adjust the light source.  Place the slide on the microscope stage and secure it with stage clips. Start with the 4x objective lens to locate the sample and bring it into focus using the coarse focus knob.  Switch to higher magnifications (10x, 40x) to observe more details. Use the fine focus knob to sharpen the image.  Systematically scan different areas of the slide to get an overview of the sample.  Look for and identify microorganisms such as bacteria, protozoa, and algae. Also note any non-living particulate matter. Is there any movement? Try to observe and describe the type of environment you are seeing under the microscope.  Write detailed descriptions of the observed components, including shape, size, color (if stained), and movement (if applicable).  To the exact steps once again for sterilized water sample and compare the differences between each sample.  5- ASSESMENT OF AMYLASE ACTIVITY WITH BENEDICTS’S SOLUTION Introduction In today's lab session, we will learn to observe a biological reaction and evaluate the results. For this, we will use the chemical reagent called Benedict's solution. Benedict's solution is an effective tool for determining simple carbohydrates classified as reducing sugar. If Benedict’s solution is mixed with another liquid that contains reducing sugar, the sugar oxidizes at high temperatures and causes accumulation of red colored copper oxide. Depending on the concentration of copper oxide, the solution’s color can shift from blue to yellow or red. In this way, we have an idea about the amount of sugar. While monosaccharides and disaccharides react very quickly in Benedict's solution, polysaccharides such as starch do not cause color change. However, starch can be easily broken down to glucose that is capable of changing the solution’s color. One way to quickly and effectively convert starch into reducing sugar is enzymatic digestion. Amylase enzyme, which we produce in the salivary gland in our body, is widely used in the industry to process starch for this purpose. In this experiment, we will examine the conversion process of starch digested with amylase enzyme for different periods of time into glucose, depending on the color change of the Benedict solution. Figure 5.1: Application of Benedict’s solution is very fast and easy. The solution is mixed with a few drops of sample and heated briefly. The color change in the liquid provides preliminary information about the amount of sugar in the sample. Materials and Equipment  Amylase (Commercially available)  Benedict’s Solution  Starch (Commercially available)  Glass tubes  Droppers or pipettes  Heater  Distilled water  Gloves and lab coat  Waste disposal container Procedure 1) Preparing the Solution:  1% starch solution will be provided by the instructor. The solution will be distributed as 4 separate tubes per group.  Add a determined amount of amylase powder into starch tubes.  Label the tubes and start incubating them at 37 degrees.  Take one tube out of the incubation chamber each 5 minutes.  Prepare a 1% glucose solution separately.  Add 1-2 drops of Benedict’s solution to all tubes (4 amylase-treated starch tube + 1 glucose-containing tube)  Incubate all tubes at boiling temperature for 10 minutes.  Observe the color changes in each tube. 6- MODEL ORGANISMS AND THEIR USE IN BIOSCIENCE Model organisms are non-human species that are used in labs to examine specific biological processes. They are widely preferred to study diseases when human experimentation is not feasible. Animal models, which constitute a large portion of model organism studies, have been central to medical research for decades and contributed to our understanding of various physiological phenomena. In this lab session, you will explore about the necessity of model organism use in contemporary research. Your instructor will introduce several of commonly used model organisms, explain their characteristic features, and give some examples from scientific literature to deepen your understanding. To give a brief idea, we share a few examples with you below: Drosophila melanogaster (fruit fly) The fruit fly is one of the oldest model organisms in modern biology, and its use dates back to 1901. The low cost of maintenance and wide availability of genetic tools make the fruit fly one of the most ideal model organisms in bioscience. Flies have certain characteristic advantages, such as a short lifespan (3 weeks), rapid reproduction and minimal culturing requirements. A female fruit fly can lay up to 1500 eggs in her lifetime, and development of an adult fly from egg takes only 10 days. These characteristics make the fruit fly an excellent model for studying early developmental stages. Dario rerio (zebrafish) As a vertebrate model, zebrafish have become increasingly valuable for research. They are considered a suitable model for investigating a large variety of biological topics, including cancer, development, physiology and behavior. The zebrafish genome is fully sequenced, allowing researchers to investigate evolutionarily conserved traits and compare them with more complex organisms, including humans. Other advantages include short developmental time (3 months) and high fecundity. An adult female zebrafish can lay more than 100 eggs per breeding cycle. Moreover, embryos have certain characteristics (such as external development and transparency), which allow for live observation of developmental processes with advanced microscopy techniques. Due to the genomic similarities with humans, zebrafish are widely used to model various diseases, including muscular dystrophy, melanoma, polycystic kidney disease, nephronophthisis, Parkinson’s disease, Huntington’s disease, and Alzheimer’s disease. Mus musculus (mouse) Mice have always remained important as model organisms throughout the history of modern research. The mouse genome was sequenced successfully in 2002, making it a crucial tool for comparative genomics. Due to the genetic and physiological similarities to humans, mouse models have been frequently used to study human biology and diseases. The mouse genome is 99% similar to the human genome, and there are a wide range of gene editing tools available for research. It is a cost-effective choice for the studies of mammalian vertebrates, and mice are capable of giving birth to a large number of offspring in a short time. There have been debates about the use of animal models in science and industry. In academic settings, researchers are obliged to follow 3 principles (also known as “3-R rules”) while experimenting with animals. Replacement: Use of animals must be avoided and replaced, if there are alternative approaches (e.g., computer simulation, cell culture studies, etc.) Reduction: If replacement is not possible to answer a particular scientific question, the researchers should use the minimum number of test subjects to conduct their experiments. Refinement: The animals must be kept in optimal conditions, and stress and pain should be minimized during the experiments. Appendix A Table 1: Materials and equipments used in Biology laboratory. Material Producer / Product code Refrigerator Beko B1 9459 NMN Refrigerator (4 C and -20 C) Oven Beko Microwave oven Light Microscope Small Autoclave Nüve OT102 Sterilization Chamber Thermocycler Electrophoresis Centrifuge Nüve NF800R Cooling centrifuge Incubator N-Biotek NB205 Tabletop shaking incubator Micropipettes VWR pipettes, 613892, 613893, 613895, 613896 Glassware VWR glass bottles, 1 L, 500 ml, 250 ml Petri dish LP-121518, 100 mm Plastic tubes, round Corning, 734-0445 (5 ml) bottom Plastic tubes, pointed Sarstedt, 62.547254 (50 ml), 62.554502 (10 ml) bottom Agarose Prona Biomax Agarose TBE buffer ChemBio TBE buffer CB6680, 10X Loading dye Thermo Scientific R0611, 6X SYBR® Safe DNA Gel Stain ThermoFisher S33102 DNA ladder (100 bp) SibEnzyme M15 DNA ladder (1 kb) Fermentas SM0311 Ampicillin Sigma A9393-5 Sodium chloride Merck M106404.1000 Tris base VWR - 33621.260, powder Ethylenediaminetetraacetic VWR - 280214S, powder acid (EDTA) Isopropanol VWR - 437423R Acetic acid Millipore - 1.00063.2511 Ethanol Pharmaceutical grade Bacteria Medium, liquid Sigma LB L3022 Bacteria Medium, solid VWR LB agar - 84684.0500 Distilled water Onur Kimya