U1 Characterization of Cytogenetics & Molecular Biology Lab PDF

# U1 Characterization of the cytogenetics and molecular biology laboratory ## Introduction - **Cytogenetics:** A branch of genetics that studies metaphase chromosomes contained within the dividing cells, and their potential alterations. - **Molecular Biology:** Studies the properties (composition,...

# U1 Characterization of the cytogenetics and molecular biology laboratory ## Introduction - **Cytogenetics:** A branch of genetics that studies metaphase chromosomes contained within the dividing cells, and their potential alterations. - **Molecular Biology:** Studies the properties (composition, structure and function) of the molecules within the cell, as well as the processes involved. ## Functions of the cytogenetics laboratory - Two areas are needed: - **Culture Room:** Where cell cultures are performed. Dividing cells are needed in order to lyse them and obtain the nuclei and its chromosomes. - **Cytogenetics Area:** Where the techniques are performed. ## Culture Room - Cell cultures are performed and must maintain the same conditions as the cells in vivo (where they were extracted from). - The room must have optimal conditions of pH, temperature, etc. as well as optimal sterility conditions. This means they must be free from contamination from other organisms. ### Equipment: - **Laminar Flow Cabinet:** Has a continuous flow of air that removes particles larger than 0.2 micrometers, protecting the samples from contamination from other microorganisms. It consists of: - Transparent front panel - Front grid - Mechanical system - Large-surface HEPA filter - Second HEPA filter - **CO₂ Incubator:** Maintains the optimal conditions required for the growth of cells in each culture. It consists of: - Temperature control - CO₂ control - Humidity control - Air recirculation - **Inverted Microscope:** Needed to observe the evolution of the cultures. - **Conventional Optical Microscope:** Used to view genetic material of cells using staining techniques. It starts at the lowest magnification and then zooms in. - **Fluorescence Microscope:** Allows visualizing certain structures stained with fluorescent probes. - **Camera and image analysis software:** Images are recorded and viewed through the software. - **Cryogenic equipment:** Tank of liquid nitrogen for the freezing and storage of samples. - **Autoclaves:** Used for sterilization. It is like a pressure cooker that maintains high temperatures for sterilizing samples and materials. - **Water purification system:** Provides distilled water. ## Functions of the molecular biology laboratory - Molecular biology focuses on studying the molecules contained within cells, specifically nucleic acids (DNA and RNA), and the processes involved, like replication. - The most widely used technique in this laboratory is **PCR (Polymerase Chain Reaction).** This technique amplifies a DNA chain exponentially. ### Different working areas: - **Pre-PCR Area:** Samples and nucleic acids are prepared. - **PCR Area:** Temperatures are increased and decreased to perform the synthesis of DNA chains. - **Post-PCR Area:** Results are analyzed and prepared. ### Equipment: - **Laminar flow cabinet** - **Water purifier** - **Autoclaves** - **Centrifuge** - **Incubators and heating blocks** - **Refrigerators and freezers** - **Pipets, tips, tubes and Eppendorf tubes** - **Spectrophotometer** is an essential tool used to measure the concentration of the samples. It emits a beam of light and measures the light absorbed by the substance under analysis. - **Thermocycler** is the main instrument used during PCR. It alters the temperature to separate and reunite double DNA chains, ultimately doubling the DNA. It includes: - A heating block that heats the tubes uniformly. - A thermal lid that prevents convection and condensation in the samples. ## Post-PCR Area - **Gel Electrophoresis** is used to visualize the PCR results. This process separates molecules by applying an electric current. - **"-foresis"** comes from the Greek word "phoresis" which means "transport". - DNA molecules have a negative charge and therefore move towards the positive pole. Larger molecules take longer to move than smaller molecules. - The following components are needed for gel electrophoresis: - Electrophoresis chamber - Adjustable power supply - Agarose or polyacrylamide gel - Fluorescent dye - The results are visualized in a dark room using: - **UV transilluminator** - **Image capture system** ## Manipulation of sterile materials - The large variety of processes and materials used in the laboratory increases the probability of contamination. - **Cross contamination** may occur due to microorganisms present in the environment, by genetic material from laboratory personnel (hair, skin cells), or by contaminants present on the work surfaces. - **Contamination of reagents and single-use materials**: Even if materials are sterile, genetic material may be present. ### Measures to reduce the risk of contamination: - Separate working areas physically. - Establish unidirectional work flow. - Control the dates of opening and expiration of reagents. - Perform negative controls in all techniques: A control tube with no genetic material is used. If contamination is present in the control tube, it is a sign of contamination. - Follow aseptic techniques. - Sterilize all materials. ### Aseptic technique - Ignaz Semmelweis (1818-1865) is commonly referred to as the father of aseptic techniques. He was a surgeon and obstetrician who discovered that mortality in the delivery rooms decreased when the surgeons disinfected their hands before attending to the delivery. - His proposals for achieving asepsis in the medical environment were ridiculed by his colleagues. After his death, however, his discoveries were confirmed by Louis Pasteur, who discovered the microbial source of infections. ## Sterilization of materials - **Disinfection:** Removes most of the active microorganisms from a surface or material. It can be achieved by: - **Chemical Methods:** Cleaning with ethanol, sodium hypochlorite, etc. - **Physical Methods:** Boiling. - **Sterilization:** The complete elimination of all life forms (active and latent) from a surface. It can be achieved by: - **Chemical Methods:** Immersing in ethanol or cleaning them with a disinfectant. - **Physical Methods:** UV radiation, autoclave, incineration. ## Laboratory safety - Laboratory safety in cytogenetics refers to preventing exposure of personnel to hazardous substances, as well as preventing their release into the environment. ### Types of safety: - **Biosafety:** Prevention of exposure to pathogenic agents and toxins. This is considered a biological risk. - **Chemical safety:** Prevention of exposure to chemical agents. This is considered a chemical risk. - **Physical safety:** This refers to the design and structure of the building and the equipment used in the laboratory. It includes the following: - **Ergonomics:** Incorrect postures - **Psychosocial:** Poor working environment. - **Biosafety:** This entails following rules, techniques, practices and work habits to prevent accidental exposure of personnel to potentially hazardous biological agents, or their release into the environment. This is particularly important in the case of genetically modified organisms (GMOs). - The main biological risk in cytogenetics and molecular biology laboratories are microorganisms. All biological samples should be treated as potentially hazardous to lab personnel as well as the environment. ### Actions to prevent exposure to biological risks: - Use appropriate PPE (personal protective equipment): lab coats, gloves, and goggles. - Do not pipette by mouth. - Handle microorganisms in class II biological safety cabinets. - Avoid spills and leaks. - Sterilize waste. ## Chemical safety - Laboratories that handle molecular biology and cytogenetics work extensively with chemical products. These products are categorized according to the regulation on classification, packaging and labeling of hazardous substances. - These include: - **Extremely flammable** - **Explosive** - **Flammable** - **Oxidant** - **Corrosive** - **Irritant** - **Sensitizer** - **Toxic** - **Harmful** - **Carcinogen** - **Mutagen** - **Teratogen** - **Hazardous to the environment** - **Reproductive toxicant** ### Actions to prevent exposure to chemical risks: - Read the safety data sheets of products and reagents. - Store products appropriately based on their hazard class. - Label products correctly. - Use appropriate PPE (personal protective equipment) for each product. - Use a fume hood for volatile substances. - Do not pipette by mouth. - Manage waste properly. - Use flammable products away from heat or flames. ## Physical safety - Physical risks in laboratories are related to the building design and equipment. This includes: - **Temperature:** It should be comfortable. The equipment used in the laboratory can be a source of heat, so it's necessary to consider this factor. - **Lighting:** Adequate lighting is necessary. Natural light should be maximized. - **Noise:** The equipment and apparatus in the laboratory shouldn't cause unpleasant noise. It shouldn't affect conversations or the concentration of the workers. - **Humidity:** The humidity level shouldn't be too high or too low. It should be conducive to a comfortable environment. - **Radiation:** Keep radiation levels to a minimum. Be very careful when using UV lamps and transilluminators. - **Heat:** Some equipment can produce burns. Always wear appropriate protective gear, such as heat-resistant gloves. - **Sharp Objects:** Always handle sharp objects carefully. Use specific containers for disposal. ## Laboratory safety manual - The safety manual is a mandatory document that all laboratory personnel must be familiar with. It must include the following information: - Assessment of the risks inherent to each workstation in the laboratory. - Description and use of laboratory safety equipment (biological safety cabinets, fume hoods, PPE, etc.). - Appropriate work habits to minimize risks. - Rules for the storage of chemicals. - Rules for waste disposal and management, including spills. - Rules for acting in emergency situations, biological accidents and chemical accidents. - All risks and safety precautions must be legally enforced. If any laboratory does not comply with regulations, they can be reported. ## Waste management - Laboratories have a waste management plan for toxic and hazardous waste. The plan must include measures for selective collection, classification and temporary storage until collected by the specialized company. There are specific measures for each type of waste: ### Types of Waste: 1. **Special biosanitary waste:** - Sharps and cutting tools that have been in contact with biological agents, including: - Needles - Scalpels - Slides - Cell cultures - Large volumes of blood and other biological fluids. 2. **Chemical waste:** - Containers that have contained chemical products. - Expired or unnecessary reagents. - Contaminated PPE. 3. **Cytotoxic waste:** Includes leftover products and containers that contained: - Carcinogens - Mutagens - Teratogens 4. **Radioactive waste:** Includes any contaminated materials. - Radioactive isotopes - Waste is collected in separate containers for solid and liquid waste. These containers must be approved, labeled and only filled to two-thirds of their capacity. They must always be closed tightly. - The waste management plan must also include rules for handling spills. ### Spills - Spills are one the most common accidents in laboratories, and they can be hazardous. Therefore, action must be taken immediately. The steps to take are: - **Neutralization:** The appropriate neutralization method depends on the chemical characteristics of the spilled product. - **Absorption:** The type of absorbent material used for the spill depends on the nature of the spill. Here are some options: - Sawdust: For non-flammable, non-toxic, and non-corrosive liquids. - Charcoal: For flammable, toxic, or corrosive liquids. - Absorbent-neutralizing commercial products: Choose the right product for the spill as indicated by the manufacturer. - Cellulose: For small spills. - **Disposal:** Put the waste in an appropriate container and eliminate it according to regulations. - Spills of powdered products are collected directly. Make sure to avoid generating dust clouds and suspension. - Spills of volatile, toxic products (inhalation hazard) are collected with cellulose. They must be eliminated quickly in appropriate containers with a tight seal. The cellulose can also be placed in a fume hood with an activated carbon filter, allowing the product to evaporate. The remaining cellulose is then treated as chemical waste. - In all spill situations, ensure the following precautions are taken: - Restrict access to the lab. - The personnel responsible cleaning the spill must wear appropriate PPE for that specific product. - Exercise caution. ## Efficient Use of Resources - Efficient management of resources is crucial in cytogenetics and molecular biology laboratories, as there are significant costs involved. ### Key aspects of efficient resource management: - **Facilities:** Minimize and optimize the use of resources. For example, consider installing low-consumption light bulbs and low-flow faucets. Proper thermal insulation is also crucial. - **Equipment:** Periodically check the equipment to ensure proper function and maintain it in good working order. - **Reagents and Products:** Implement a robust system for storing reagents and products to ensure they are in good condition. Maintain traceability of materials from the time of ordering to the time of use. - **Waste Management:** Reduce waste, as much as possible, by reusing or recycling materials. This is particularly important for common items like paper and glass containers. - **Procedures and Techniques:** Utilize standardized procedures to ensure consistency and high quality. This ensures everyone follows the same steps for each technique. - **Human Resources:** Provide staff with comprehensive training on safe and efficient techniques, protocols, and procedures for managing waste.