Research I Quarter 3 - Week 1: Standard Methods and Techniques Used in Performing Experiments PDF

# Research I Quarter 3 - Week 1: Standard Methods and Techniques Used in Performing Experiments ## What is It Experiments are tests done to learn something or discover if something works or is true. A chemical laboratory is a place for doing tests and research procedures and preparing chemicals an...

# Research I Quarter 3 - Week 1: Standard Methods and Techniques Used in Performing Experiments ## What is It Experiments are tests done to learn something or discover if something works or is true. A chemical laboratory is a place for doing tests and research procedures and preparing chemicals and some medications. It is designed to support and illustrate chemical concepts studied in the lecture portion of the course and introduce essential laboratory techniques and encourage analytical thinking. In addition to a productive experiment, safety in the laboratory can be assured by mastering these techniques and operations. That is why, before conducting such, it is imperative to know the following: safety precautions, the laboratory tools and equipment, and the different methods and techniques used in performing experiments. Attention should also be given on Potentially Hazardous Biological Agents Risk Assessment and Rules for Studies with Potentially Hazardous Biological Agents (PHBA) for safety purposes and ethical considerations. ## General Safety Laboratory Rules It is vital to observe precautionary measures when doing some experiments to avoid accidents. Below are some of these laboratory rules: 1. Know locations of laboratory safety showers, eyewash stations, and fire extinguishers. The safety equipment may be located in the hallway near the laboratory entrance. 2. Know emergency exit routes. 3. Avoid skin and eye contact with all chemicals. 4. Minimize all chemical exposures. 5. Do not play inside the laboratory. 6. Assume that all chemicals of unknown toxicity are highly toxic. 7. Post warning signs when unusual hazards, hazardous materials, hazardous equipment, or other special conditions are present. 8. Avoid distracting or startling persons working in the laboratory. 9. Use equipment only for its designated purpose. 10. Combine reagents in their appropriate order, such as adding acid to water. 11. Avoid adding solids to hot liquids. 12. Never leave containers of chemicals open. 13. All containers must have appropriate labels. Unlabeled chemicals should never be used. 14. Do not taste or intentionally sniff chemicals. 15. Never consume and/or store food or beverages or apply cosmetics in areas where hazardous chemicals are used or stored. 16. Do not use mouth suction for pipetting or starting a siphon. 17. Wash exposed areas of the skin before leaving the laboratory. 18. Long hair and loose clothing must be pulled back and secured from entanglement or potential capture.. 19. No contact lenses should be worn around hazardous chemicals - even when wearing safety glasses. 20. Laboratory safety glasses or goggles should be worn in any area where chemicals are used or stored. They should also be worn anytime there is a chance of splashes or particulates to enter the eye. Closed-toe shoes will be worn at all times in the laboratory. Perforated shoes or sandals are not appropriate ## Safety Precautions for Handling Hazardous Materials When you work with an experiment that requires hazardous materials, the safety of the students and laboratory personnel is the most important consideration. The following are the general safety precautions you must keep in mind when working on hazardous materials: 1. Follow all existing and established procedures. 2. Be cautious and prepare accordingly. Think of what could go wrong as you work and pay careful attention to what you're doing. 3. Always use the appropriate PPE - and carefully check it before of use to ensure that it is safe to use. 4. Ensure that all containers are correctly labelled and that the content is in a suitable jar. Don't use any substance which is not properly contained or labelled. Report immediately to your laboratory custodian or teacher any broken containers or illegible marks. 5. Know the material safety data sheet (MSDS) before using any material to make sure you understand hazards and precautions. 6. Use all materials solely for their intended purpose. Don't, for example, use solvents to clean your hands, or gasoline to wipe down equipment. 7. Never eat or drink while handling any materials, and if your hands are contaminated, don't use cosmetics or handle contact lenses. 8. Read the labels and refer to MSDSs to identify properties and hazards of chemical products and materials. 9. Store all materials properly, separate incompatibles, and store in ventilated, dry, cool areas. 10. Keep you and your work area clean. After handling any material, wash thoroughly with soap and water. Clean work surfaces at least once a shift so that contamination risks are minimized. 11. Learn about protocols and supplies for emergencies. Awareness of emergency procedures means understanding of evacuation procedures, emergency reporting procedures, and fire and spill handling procedures. It also involves understanding what to do when a colleague is injured or overcome by chemicals in a medical emergency. ## Proper Disposal of Hazardous Chemical Waste Appropriate disposal of hazardous chemical waste is as important as the proper storage and handling of it. Improper dumping of chemical waste may cause more serious problems not only in the laboratory but also in the nearby community. Read the following guidelines: 1. Store chemical wastes in appropriate containers; plastic bottles are preferred over glass for storing hazardous waste when compatibility is not an issue. Segregate chemical waste by compatibility, and not alphabetically. 2. Chemical waste containers must be labeled with the following information: Full chemical name and quantity of the waste. For mixtures, each chemical must be listed. Abbreviations, acronyms and ditto marks ('') to replace words are not allowed; date of waste generation; place of origin (department, room number); bottle number assigned on corresponding waste sheet; and the tag or label must have the words: "Hazardous Waste." ## Potentially Hazardous Biological Agents Risk Assessment Before conducting experiments, it is also necessary to know the risk of the agent to be studied. Risk assessment defines the potential level of harm, injury or disease to plants, animals and humans that may occur when working with biological agents. The end result of a risk assessment is the assignment of a biosafety level which then determines the laboratory facilities, equipment, training, and supervision required. Biological agents, plant or animal, are classified according to biosafety level risk groups. These classifications presume ordinary circumstances in the research laboratory, or growth of agents in small volumes for diagnostic and experimental purposes. 1. BSL-1 risk group contains biological agents that pose low risk to personnel and the environment. These agents are highly unlikely to cause disease in healthy laboratory workers, animals or plants. The agents require Biosafety Level 1 containment. Examples of BSL-1 organisms are: Agrobacterium tumifaciens, Micrococcus leuteus, Neurospora crassa, Bacillus subtilis. 2. BSL-2 risk group contains biological agents that pose moderate risk to personnel and the environment. If exposure occurs in a laboratory situation, the risk of spread is limited and it rarely would cause infection that would lead to serious disease. Effective treatment and preventive measures are available in the event that an infection occurs. The agents require Biosafety Level 2 containment. Examples of BSL-2 organisms are: Mycobacterium, Streptococcus pneumonia, Salmonella choleraesuis. 3. BSL-3 risk group contains biological agents that usually cause serious disease (human, animal or plant) or that can result in serious economic consequences. Projects in the BSL-3 group are prohibited. 4. BSL-4 risk group contains biological agents that usually produce very serious disease (human, animal or plant) that is often untreatable. Projects in the BSL-4 group are prohibited. ## Levels of Biological Containment There are four levels of biological containment (Biosafety Level 1–4). Each level has guidelines for laboratory facilities, safety equipment and laboratory practices and techniques. 1. BSL-1 containment is normally found in water-testing laboratories, in high schools, and in colleges teaching introductory microbiology classes. Work is done on an open bench or in an appropriate biosafety hood. Standard microbiological practices are used when working in the laboratory. Decontamination can be achieved by treating with chemical disinfectants or by steam autoclaving. Lab coats and gloves are required. The laboratory work is supervised by an individual with general training in microbiology or a related science. 2. BSL-2 containment is designed to maximize safety when working with agents of moderate risk to humans and the environment. Access to the laboratory is restricted. Biological safety cabinets (Class 2, type A, BSC) must be available. An autoclave should be readily available for decontaminating waste materials. Lab coats and gloves are required; eye protection and face shields must also be worn as needed. The laboratory work must be supervised by a scientist who understands the risk associated with working with the agents involved. 3. BSL-3 containment is required for infectious agents that may cause serious or potentially lethal diseases as a result of exposure by inhalation. Projects in the BSL-3 group are prohibited. 4. BSL-4 containment is required for dangerous/exotic agents that pose high risk of life-threatening disease. Projects in the BSL-4 group are prohibited. ## Rules for Studies with Potentially Hazardous Biological Agents (PHBA) To safeguard the welfare of the researcher and all other adults involved in a research project and to ensure strict compliance with the international guidelines and regulations on biosafety, rules adapted from ISEF must be considered and reviewed prior to the conduct of the experiment. 1. Experimentation involving the culturing of potentially hazardous biological agents, even BSL-1 organisms, is prohibited in a home environment. However, specimens may be collected at home as long as they are immediately transported to a laboratory with the BSL containment determined by the affiliated fair SRC. 2. Research determined to be at Biosafety Level 1 (BSL-1) must be conducted in a BSL-1 or higher laboratory. The research must be supervised by a trained Designated Supervisor or a Qualified Scientist. The student must be properly trained in standard microbiological practices. 3. Research determined to be a Biosafety Level 2 (BSL-2) must be conducted in a laboratory rated BSL-2 or above (commonly limited to a Regulated Research Institution). The research must be reviewed and approved by the Institutional Biosafety Committee (IBC) if the Regulated Research Institution requires the review. The research must be supervised by a Qualified Scientist. For a high school BSL-2 laboratory, the SRC must review and approve. The research must be supervised by a Qualified Scientist. 4. Students are prohibited from designing or participating in BSL- 3 or BSL-4 Research. 5. The culturing of human or animal waste, including sewage sludge, is considered a BSL-2 study. 6. Naturally-occurring plant pathogens may be studied (not cultured) at home, but may not be introduced into a home/garden environment. 7. All potentially hazardous biological agents must be properly disposed at the end of experimentation in accordance with their biosafety level. For BSL 1 or BSL 2 organisms: Autoclave at 121 degrees Celsius for 20 minutes, use of a 10% bleach solution (1:10 dilution of domestic bleach), incineration, alkaline hydrolysis, biosafety pick-up and other manufacturer recommendations are acceptable. 8. Research involving human and/or non-human primate established cell lines and tissue culture collections (e.g., obtained from the American Type Culture Collection) must be considered a BSL-1 or BSL-2 level organism as indicated by source information and treated accordingly 9. The collection and examination of fresh/frozen tissues or body fluids or meat, meat byproducts, pasteurized milk or eggs NOT obtained from food stores, restaurants, or packing houses may contain microorganisms. Because of the increased risk from unknown potentially hazardous agents, these studies must be considered biosafety level 2 studies conducted in a BSL-2 laboratory under the supervision of a Qualified Scientist 10. Human breast milk of unknown origin, unless certified free of HIV and Hepatitis C, and domestic unpasteurized animal milk are considered BSL-2. 11.All studies involving human or wild animal blood or blood products should be considered at a minimum a Biosafety level 2 study and must be conducted in a BSL-2 laboratory under the supervision of a Qualified Scientist. Known BSL-3 or BSL-4 blood is prohibited. Studies involving domestic animal blood may be considered a BSL-1 level study. Any tissue or instruments with the potential of containing blood-borne pathogens (e.g. blood, blood products, tissues that release blood when compressed, blood contaminated instruments) must be properly disposed after experimentation. 12. Studies of human body fluids, where the sample can be identified with a specific person, must have IRB review and approval, and informed consent. 13. Any study involving the collection and examination of body fluids which may contain biological agents belonging to BSL-3 or -4 is prohibited. However, there are studies that do not require pre-approval from the Scientific Research Committee but need risks assessment such as studies involving protists, research using manure for composting, fuel production or other nonculturing experiments. On the other hand, studies using/involving baker's and brewer's yeasts, lactobacillus, water or soil microbes, mold growth on food items, slime molds and edible mushrooms and E. coli do not require both SRC preapproval and no additional forms are needed. ## Methods and Techniques Used in Performing Experiments It is essential to have proper knowledge of using standard laboratory equipment and the basic techniques and operations to execute correct and precise experimentation and research. By being knowledgeable of these techniques and operations, students will be able to garner good results from their conduct experiments. These techniques range from simple measuring liquids to complex laboratory works. Among the commonly used are as follows together with their basic set-up. ### Life Science **Autoclaving** is a sterilization method that uses highpressure steam. The autoclaving process works by the concept that the boiling point of water (or steam) increases when it is under pressure. **Culture medium or growth medium** is a liquid or gel designed to support the growth of microorganisms. **Rotary evaporation** is the process of reducing the volume of a solvent by distributing it as a thin film across the interior of a vessel at elevated temperature and reduced pressure. It promotes the rapid removal of excess solvent from less volatile samples. Most rotary evaporators have four major components: heat bath, rotor, condenser, and solvent trap. An aspirator or vacuum pump, bump trap, and a round bottom flask containing the concentrated sample should be attached to the body. **Streak plate technique** is used to isolate the pure culture of the organisms (mostly bacteria) from a mixed population. The inoculum is streaked over the agar surface so that it "thins out" the bacteria. Some individual bacterial cells are separated and well-spaced from each other. ### Physical Science **Acid-base extraction** is a type of liquid-liquid extraction. It typically involves different solubility levels in water and an organic solvent. The organic solvent may be any carbon-based liquid that does not dissolve very well in water; the common ones are ether, ethyl acetate, or dichloromethane. Acid-base extraction is typically used to separate organic compounds from each other based on their acid-base properties. **Crystallization** is used to purify a solid that requires a suitable solvent. **Distillation of compounds** is a separation method that exploits the differences in the boiling point of a crude mixture. **Drying Agents** -when an organic solvent has been exposed to aqueous solutions, it will contain a small amount of water, depending on the solubility of water in the solvent. **Extraction** is a method for moving a compound from one medium to another. For example, if you make coffee from coffee beans, you extract some flavorful components of the bean and some caffeine into the water. **Melting Point Determination** is the standard physical property of a solid is its melting point.. The melting point is a melting point range. It is used to help determine the purity of a solid. It helps to verify the identity of the compound. **Titration** is the slow addition of one solution of a known concentration (called a titrant) to a known volume of another solution of unknown concentration until the reaction reaches neutralization, which is often indicated by a color change. The solution called the titrant must satisfy the requirements to be a primary or secondary standard. In a broad sense, titration is a technique to determine the concentration of an unknown solution. Most of the laboratories available in school do not have such kind of resources. However, what is right, the researcher can always collaborate with other laboratories to perform such a method/technique.