FEU MolBio Lab 01 Basic Laboratory Practices PDF

[MOLBIO] Basic Laboratory Practices Lecturer: Prof. Adlene Atienza Transcriber/s: Chennelyn Mae R. Llanes & Celeste Ann Andales 2nd Semester...

[MOLBIO] Basic Laboratory Practices Lecturer: Prof. Adlene Atienza Transcriber/s: Chennelyn Mae R. Llanes & Celeste Ann Andales 2nd Semester (2nd Year - Section Q) MOLBIO AND DIAGNOSTICS – LAB 01 Basic Laboratory Practices 4. Safely handle sharps such as needles, scalpels, and other carefully, and place them on i. General Laboratory Practices puncture-proof containers after use. o Used needles are not removed from Introduction disposable syringes nor bent or capped. 1. Always wear appropriate personal protective equipment (PPE). ▪ When handling sharps, make sure to dispose of a. Lab coats should be worn in the lab to them in proper containers, such as puncture- protect you and your clothing from proof containers contamination. 5. No (never practice) mouth pipetting. o Lab coats serves to protect the body 6. Take care to minimize the creation of aerosols and the clothing. It should be and/or splashes. removed when leaving the o Use closed containers. laboratory. 7. Manipulation of infectious materials must be b. Lab footwear should consist of normal conducted in the biosafety cabinet. closed shoes to protect all areas of the foot 8. Decontaminate or disinfect all work surfaces from: before and after your experiments/work, and o possible puncture from sharp immediately after any spill or splash of objects and/or broken glass and, potentially infectious material with a 10% bleach o contamination from corrosive or 10% alcohol. agents and/or infectious materials. o Use 10% bleach or 70% ethanol: o Wear closed shoes for protection to disinfect all work surfaces against injury from sharp objects or before and after your laboratory broken glass, as well as from activities. corrosive reagents and infectious over any spill or splash of materials potentially infectious material. c. Gloves should be worn, and change gloves to clean laboratory equipment when contaminated. and laboratory benches. d. Protective eyewear and/or masks may 9. Decontaminate all potentially infectious need to be worn when contact with materials before disposal. hazardous aerosols, caustic chemicals o Biohazardous material should be and/or reagent is anticipated. discarded in a biohazard bag to be o used when UV lamp is involved in the autoclaved before disposal. experiment ▪ Here, laboratory coats must be prepared for 10. Report any incidents that may result in every lab class, footwear must be closed, and you exposure to infectious materials to appropriate must have your own gloves & protective eyewear personnel (e.g., laboratory supervisor, safety officer). 2. Hands are washed after removing the gloves, before and after completion of work. ii. Other Guidelines for Molecular Biology o Proper hand washing techniques Laboratory include use of germicidal soap, running water, and 10-15 seconds of → Emergency equipment friction or scrubbing action. Make sure to know the emergency exits, o Hands should be dried with an file alarms, fire extinguisher, safety automatic hand drier or with clean shower, eye wash, first aid kit. paper towels. → Ultraviolet light o Turn off the faucet using a paper Used by UV lamp for viewing of the gel towel to hold the lever. Always wear appropriate eye protection. o Throw the paper towels into a → Electricity designated trash bin. If you are operating the gel o It is preferable to have a faucet with electrophoresis apparatus, make sure a foot pedal. that the apparatus is covered – don’t dip 3. DO NOT eat, drink, smoke, handle contact your fingers into the buffer since once lenses, apply cosmetics, or store food for human you turn on the power supply, it has consumption in the laboratory. electricity (you might get an electric shock). “You are strong enough to start again.”  [MOLBIO] Basic Laboratory Practices Lecturer: Prof. Adlene Atienza Transcriber/s: Chennelyn Mae R. Llanes & Celeste Ann Andales 2nd Semester (2nd Year - Section Q) → General Housekeeping activities such as paper works and protocol Make sure there is proper labeling to that you want to do in the area avoid confusion. o Laboratories shall have adequate and Make sure your kits have pang-label. appropriate areas to safely, effectively and → Preparing for the lab efficiently provide the services to clients. One is expected to be fully-prepared ▪ Why is it necessary to have separate areas for → Following protocols each step? → Sterility To prevent contamination, laboratory Molecular Biology procedures are design must be followed. sensitive; thus, sterility must be observed If there is no contamination control, there at all times. will be an incorrect result & loss of Cover unused equipment. credibility; it will also affect the Do not touch the equipment with your performance of the laboratory bare hands – use gloves. Causes of contamination: → Hazards in the lab o Cross-contamination - if you → Equipment and reagents have a lot of specimens to Use equipment correctly. prepare If you don’t know how to use an o Unclean working areas instrument, don’t touch it – ask first how o Reagents after use were not to use it. covered immediately Before starting the experiment, identify o Laboratory personnel did not first the materials needed. follow the laboratory practices (hair not tied, skin & saliva ❖ Biological accident or an emergency contaminating the sample) 1. If the skin or face is accidentally splattered by 2. Unidirectional workflow (one-way) following corrosive or harmful chemicals, bacterial or cell the above-mentioned activities shall be culture, immediately use emergency safety maintained at all times. shower and flush skin or face with water for at o First Room → Second Room → Third Room least 15 minutes, if eyes are affected use the – you cannot go back (i.e., if you’re in the 3rd eyewash station. room and you forgot to do something, you are 2. For injury with contaminated sharp items (broken NOT allowed to go back to the 2nd room glassware, syringe, etc.), wash with water, immediately – you have to go back to the 1st consult the clinic (provide detailed information room) yourself if possible). 3. When a large volume of highly hazardous chemical occurs, immediately EVACUATE THE AREA. 4. Spillage of large volumes (>1L) of cell culture is cleaned up with paper towels (to be autoclaved prior to disposal), decontaminated with 10% bleach or 70% ethyl alcohol iii. Molecular Biology Diagnostic Laboratory ❖ Molecular Biology Laboratory Design ▪ There are three rooms in the Molecular Biology → According to DOH requirements: Laboratory: https://doh.gov.ph/sites/default/files/health- 1. Reagent preparation room – all update/ao2020-0014.pdf reagents are stored here (powdered form) 1. PHYSICAL FACILITIES OF COVID-19 2. Sample preparation room – where you TESTING will handle the specimen (e.g., bacteria) o There shall be a dedicated space for Biosafety cabinets are here specimen reception wherein the patients will 3. PCR room – where you will conduct the submit their specimens amplification, gel electrophoresis, o There must be a room for virus inactivation visualization, checking the concentration and nucleic acid extraction, reagent storage of nucleic acid and handling, and PCR, and also clerical “You are strong enough to start again.”  [MOLBIO] Basic Laboratory Practices Lecturer: Prof. Adlene Atienza Transcriber/s: Chennelyn Mae R. Llanes & Celeste Ann Andales 2nd Semester (2nd Year - Section Q) 3. The prototype floor plan and the floor plan o Standard: 121°C for 20-30 minutes at 15 checklist for constructing a COVID-19 testing psi (but it varies depending on the sample laboratory shall be used as references in load) constructing the testing laboratory. o Plastic wears have lower temperature and time compared to other heavy materials o Proof that the autoclaving was successful: Autoclave Tape - looks like a masking tape (dirty white); appearance of diagonal lines in the tape means that the materials were sterilized ▪ An autoclave is a machine that is used to sterilize materials that are resistant to heat such as biological waste, culture media, instruments, and laboratory ware. Autoclaving of laboratory and medical Actual floorplan for lab testing for COVID-19 waste that might contain bacteria, viruses, fungi and other biological ▪ All rooms are connected materials must be done before ▪ Ante Room disposal Area designed to maintain the pressure ▪ An autoclave uses a combination of steam, differential between the lab and its adjacent pressure, and time. space; not all rooms have the same pressure, For successful sterilization and it varies: decontamination, the materials are 1. Positive Pressure Room exposed to a saturated steam o Movement of air from inside to maintained at 121 ºC for 20 - 30 outside to ensure the room is clean; minutes at 15 psi (pounds per square o Greater pressure inside the lab inch) pressure. o E.g., once a door is opened, the air o Time may be varied depending enters; but since the pressure inside on the bulk of the material a positive pressure room is high, it loaded. will push the contaminated air out o Priority: keep the germs and ▪ Biological molecules used in the molecular contaminants OUT laboratory can be degraded by substances such 2. Negative Pressure Room as nucleases that are found in non-sterile o air movement is not allowed to be surface. exhausted outside Therefore, most consumable materials o the air in the room is contaminated; (tips, tubes, etc.) are sterilized by the room is dirty autoclaving. o air is filtered Usually, an autoclave tape is attached to o E.g., Isolation Rooms of TB patients the packages to be sterilized, which will form dark stripes if the materials have ❖ Instruments and Equipment Used in Mol Bio Lab been autoclaved. → Facilities and Other Resources: (Reference Plan: o Types of Autoclaves Free-Standing COVID-19 Testing Laboratory using RT-PCR) a. Pressure Cooker Type (usually used in 1. Autoclave small labs or dental clinics; bench type o Provides a physical method of autoclaves) sterilization by killing bacteria, viruses, and even spores present in the material put inside of the vessel using steam under pressure (heat). o Has large capacity to sterilize materials (e.g., buffer, containers) all at once. “You are strong enough to start again.”  [MOLBIO] Basic Laboratory Practices Lecturer: Prof. Adlene Atienza Transcriber/s: Chennelyn Mae R. Llanes & Celeste Ann Andales 2nd Semester (2nd Year - Section Q) b. Common Laboratory Autoclave - Aerosol particles are around 5 micrometers & some droplets are around 5 to 100 micrometers in diameter; but they are invisible to the naked eye o If used properly, it can reduce laboratory- c. Vertical Autoclave acquired infection and cross contamination of specimen. o How are aerosols created? (You can’t see it). - Rapid hand movement - When you open a reagent stocked for a long time d. Horizontal Autoclave - When you use a vortex or centrifuge o Types of BSCs e. Large Automatic Hospital Autoclave Class I Class II Class III For disposal of waste such as bacteriology wastes (after bacteria is cultured), and sterilization of the instrument You can’t dispose the wastes directly to trash bins since it can harm others – Class III you must first autoclave it to kill the microorganisms a. Class I BSC is the first designed and simple Biological Safety Cabinet ▪ Basic principle of autoclave: Open front with directional airflow (hindi All the items within the autoclave will pabalik-balik), thus, protect personnel come in direct contact with the steam or and environment heat for a particular period of time. Operates under negative pressure ▪ If the temperature exceeds the standard, release Has HEPA filter exhaust the pressure. ❖ Any contaminated air that is released is microbe free 2. Biological Safety Cabinet (BSC) BSL 1, 2, 3 containments o Is an engineering control intended to No product protection since air flows protect laboratory workers, laboratory inside the work surfaces/cabinet; the environment and work materials from unsterilized room air will go into your exposure to infectious or biohazardous work surfaces – not reliable for product aerosols and splashes. protection o Such aerosols and splashes may be Cell & tissue culture, propagation of generated while manipulating materials viruses cannot be done in BSC 1 containing infectious agents, such as Media culture cannot be performed in a primary cultures, stocks and diagnostic BSC 1 specimens. “You are strong enough to start again.”  [MOLBIO] Basic Laboratory Practices Lecturer: Prof. Adlene Atienza Transcriber/s: Chennelyn Mae R. Llanes & Celeste Ann Andales 2nd Semester (2nd Year - Section Q) ▪ How does Class I BSC works? o In addition, Class II Type B biological 1. The room air (A - contaminated) goes inside safety cabinets require very specific the cabinet. installation and operating conditions to 2. It will flow in the product/work surfaces. function properly. 3. When it goes outside, it is already clean air/filtered air. b. Class II Biological Safety Cabinet (BSC) is a ventilated cabinet, which provides personnel, ▪ How does air flow in class II? product and environmental protection. 1. Once room air enters, instead of directly It is commonly found in clinical and research proceeding to the work surface, it will first go laboratories working with infectious agents in to the open/front exhaust grill, Risk groups 2,3, and 4 (if positive-pressure 2. Then it will go down, then up, then some air suits are used) or with tissue culture. will be exhausted (clean air) and some air will Most common be recirculated (clean air) Open front with directional flow (walang Clean air because of HEPA filter pabalik so may personnel protection) Air that will go to the work surfaces is HEPA filter laminar downflow and exhaust clean HEPA Filter (has two HEPA filter, 1 pang 3. And then the air in the work surfaces will go exhaust, 1 pang re-circulate thus has product to the front or rear exhaust grill and so on… protection) ▪ It is important that the rear and front exhaust grill BSL 1, 2, and 3 containments mustn’t be blocked by your materials (so the air There are four types (A1, A2, B1, and B2) flow won’t be disturbed) of Class II BSCS. ▪ According to DOH, pwede sa Class II BSC yung o The main differences between the types COVID-19. are the ratio of air exhausted from the BSC to the air that is recirculated within c. Class III Biological Safety Cabinet provides the the BSC, and the type of exhaust highest level of personnel protection and is system present (some ay naka-dikit sa used for Risk Group 4 agents. roof: hard-duct OR canopy: wherein may It is suitable for work in Biosafety Level 3 gap before yung roof) and 4 laboratories. 1. A1 and A2 Totally enclosed ❖ 70% of air circulate and There are already rubber gloves attached ❖ 30 are exhausted to the BSC 2. B1 Negative pressure (all BSCs are) ❖ 30% air circulation, Has double HEPA filter exhaust; ❖ 70% exhausted autoclave and dump tank for disposal 3. B2 Handles Risk Group 4 agents such as ❖ 0% circulated and Ebola Virus ❖ 100% exhausted There is a presence of double door o About 90% of all biosafety cabinets are autoclave to ensure that any exiting Type A2 cabinets. materials that you used inside are o There is a limited need for Class II Type sterilized once you remove it. B biological safety cabinets. “You are strong enough to start again.”  [MOLBIO] Basic Laboratory Practices Lecturer: Prof. Adlene Atienza Transcriber/s: Chennelyn Mae R. Llanes & Celeste Ann Andales 2nd Semester (2nd Year - Section Q) Personnel, product and environment - Thermocycler is a device used to protection amplify a specific region of any DNA sample; the target is amplified - Thermocycler is like a photocopier or xerox machine ❖ Has a screen where you can set the temp, time, & how many cycles ❖ Has 3 steps: denaturation, annealing, elongation ❖ Once set, you will play/start it then wait for 2-3 hours for it to be amplified a. In conventional PCR → HEPA (High Efficiency Particulate Air) filters: o they are constructed of pleated borosilicate glass and arranged into random fibers; the HEPA filter traps 99.97% of particles of 0.3 µm in diameter - anything larger to 0.3 can be efficiently Also uses gel electrophoresis filtered by HEPA filter Endpoint; you will only see the result ❖ that’s why it can now effectively trap after the allotted time all known infectious agents Purpose is migration in the electrical ❖ but, ensure that only micro-free field exhaust air is discharged from the E.g., sample is nucleic acid/DNA cabinet since it is already filtered by (negatively charged molecule) the HEPA filter o Since it is negatively charged, - however, since gases and vapors are it will migrate to positive relatively smaller, they cannot be electrode (if the sample is removed by HEPA filtration positively charged, then it will migrate to the negative ▪ How can BSLs be effective? electrode). This is because of the HEPA filter o When using gel Already installed in BSLs electrophoresis, make sure that the wells are aligned to its corresponding electrode. - If sample is DNA (negatively charged), then you align the well to the negative electrode so the sample will migrate downward - If the well is aligned to the positive, then it will migrate to the buffer ▪ After electrophoresis, you need to view the gel. 3. Real Time Polymerase Chain Reaction (RT- Use UV transilluminator. Then compare. PCR) Machine (Eyewear must be used since UV light is used) o Both conventional and real-time uses/needs thermocycler “You are strong enough to start again.”  [MOLBIO] Basic Laboratory Practices Lecturer: Prof. Adlene Atienza Transcriber/s: Chennelyn Mae R. Llanes & Celeste Ann Andales 2nd Semester (2nd Year - Section Q) b. Real Time PCR Machine o The wavelength of maximum Used in COVID absorption for both DNA and RNA is Only needs thermocycler and 260 nm. computer, then after a few hours, you can see the result You can see the movement of the threshold value in the graph; can interpret immediately Faster than the conventional 5. Vortex Mixer o Simple device used commonly in laboratories to mix small vials of liquid. o Purpose: mix all of the liquids o For the mixture to be uniform, you need to c. Conventional vs RT-PCR have a vortex mixer 6. Centrifuge o Centrifugation is a procedure that separates components of liquids that have 4. Spectrophotometer different weights. o Is an instrument used to measure how - if denser, tendency is that it will settle much a chemical substance absorbs on the bottom light by measuring the intensity of light as a - if less dense, it will float or settle at beam of light passes through sample the top solution. o measures light absorbed and light transmitted (you can see it in the screen) o know the concentration of the sample ▪ The basic principle is that each compound absorbs or transmits light over a certain range of wavelength. The amount of absorbed light (absorbance) is in direct proportion to the amount of substance in a solution ▪ In molecular biology, the spectrophotometer is o The device used for this purpose is called a used to measure the concentration and purity centrifuge, in which a centrifugal force is of DNA, RNA or protein. applied that moves liquid components Nucleic acids absorb ultraviolet (UV) light away from the center. because of the presence of heterocyclic - there will be a separation after rings in the bases. centrifugation “You are strong enough to start again.”  [MOLBIO] Basic Laboratory Practices Lecturer: Prof. Adlene Atienza Transcriber/s: Chennelyn Mae R. Llanes & Celeste Ann Andales 2nd Semester (2nd Year - Section Q) For large capacity refrigerated centrifuges, rotor chambers may have varying sizes. ▪ For complete and rapid sedimentation of the precipitate to the bottom of the vessel, the revolution per minute (rpm) or g force (gravitational force) may be increased. ▪ The remaining solution is called supernatant. ▪ The speed by which particles settle to the bottom ▪ In Molecular Biology, PCR/Eppendorf tubes are is determined by the size and shape of the used particles, the centrifugal speed, the difference Has two parts: the liquid part in density between the particles and the liquid, (supernatant) and the pellet (precipitate) and the viscosity. o Precipitate (pellet) ▪ When using a centrifuge, it is important to always - stands for the balance the tubes before being run. concentrated particles in a The tubes placed directly opposite tube after successful each other must have equal weight. centrifugation If the tubes do not have identical - solid form at the bottom of components or do not have the same the container volume, either weigh the tubes and o Supernatant make them equal or you may use another - is the remaining solution tube of the same capacity and fill it with a above the pellet solution with equal volume. ▪ when precipitate is needed, you need to decant ▪ Nevertheless, with low-speed microcentrifuge the supernatant (quickly pouring off the liquid) (maximum rpm ≦ 15000), weighing the tubes is then store it in the refrigerator not necessary but be sure that the tubes have identical contents and volumes. ▪ Types of centrifuges: ▪ Once the centrifuge is loaded, the rotor lid is 1. Microcentrifuge - used in Molecular Biology placed on the rotor, the centrifuge lid is since only small tubes are used closed, the required speed is set, and the timer ▪ designed for small tubes of between is also set. 0.2 mL and 2.0 mL Always place the tubes with the hinge ▪ In some centrifuges, the rotor can be facing the outer rim of the rotor. This switched or replaced to be able to fit will make it easier to find the pellet. tubes of different sizes. 2. Refrigerated centrifuge - if samples are sensitive & the temperature is maintained ▪ Refrigerated centrifuges are used for samples that require a constant range of temperature. Thus, these centrifuges must run at maximum speed at a constant temperature. Such centrifuges usually have a temperature range between -20 and -40c, which are ideal for molecular biology work, such as working with DNA, RNA, or PCR. ▪ These centrifuges can also achieve 7. Automatic Pipettors / Pipettors/Micropipettes speeds of more than 30,000 rpm with o Pipettes are equipment used to deliver an relative centrifugal force of greater accurately measured volume of solution. than 65,000 x g. o In a molecular biology laboratory, They may have rotors with a micropipettes are most frequently used. swing bucket, fixed-angle ▪ Micropipettes measure volumes type or both. that range from 1000 µl (1 ml) to less than 1 µl. “You are strong enough to start again.”  [MOLBIO] Basic Laboratory Practices Lecturer: Prof. Adlene Atienza Transcriber/s: Chennelyn Mae R. Llanes & Celeste Ann Andales 2nd Semester (2nd Year - Section Q) o Always check the capacity of pipette before using. o Different tips are used depending on the capacity. 10. Laminar Airflow (LAF) Hood 11. Pipette Filtered Tips 12. Freezer 13. Personal Protective Equipment (PPE) Cabinet 14. First Aid Kit 15. Cold Rack 16. Laboratory Deep Sink with Eye Wash 8. Refrigerator 17. Computer o Storage of sample and reagent 18. Pass Box o When reagents are ordered from the 19. Waste Bin manufacturer, it is in Lyophilized form 20. Laboratory Stool (powdered), so once it must be used, they 21. Computer Chair will need to be diluted with water or saline 22. Hand Washing Skink with Eye Wash solution and then be stored within the 23. Laboratory Counter needed temperature to maintain the 24. Minifuge/Mini Centrifuge function (and para hindi masira) 25. Conventional PCR Machine o Extracted materials are still viable to be 26. Stainless Steel Utility Sink used if refrigerated in correct temperature 27. Spill Kit (i.e., 5 years storage) 28. PCR Hood 29. Thermocycler o A thermocycler is a device is used to amplify (like a photocopier) a specific region of any DNA sample with polymerase chain reaction (PCR) in a test tube. Once amplified, the PCR product (also called amplicon) can be used in many analytical procedures including detection of bacteria or viruses (particularly AIDS), and diagnosis of genetic disorders. o The temperature of a thermocycler is programmed to change at a given time 9. pH Meter to allow amplification. o very sensitive to changes in pH and hence, The sample is first denatured buffers are used to stabilized the pH by heated forming two single- o test the acidity and alkalinity stranded DNA. These single-stranded DNAs ▪ A pH meter is a special device used to determine serve as templates for the the acidity or alkalinity of a substance. synthesis of new DNA strands The unit of measure expressing the level of with the use of a heat-resistant acidity or alkalinity of a substance is pH enzyme such as Taq ▪ The pH scale typically ranges from 0 to 14. polymerase. A pH of 7 is a neutral pH and a pH less than This copies the original DNA 7 is acidic, while a pH greater than 7 is forming a new molecule alkaline. consisting of one old and one new DNA strand. “You are strong enough to start again.”  [MOLBIO] Basic Laboratory Practices Lecturer: Prof. Adlene Atienza Transcriber/s: Chennelyn Mae R. Llanes & Celeste Ann Andales 2nd Semester (2nd Year - Section Q) Each of these strands now is used as a template for synthesis of two new copies. This process is repeated in about 30 to 40 cycles multiplying the original DNA copies by a factor of one billion. 30. UV Transilluminator o An ultra-violet (UV) transilluminator is an apparatus used for viewing DNA or protein molecules separated by agarose or polyacrylamide gel electrophoresis. o The UV radiation emitted by the transilluminator causes the dye used to stain the DNA or protein molecules to fluoresce making them visible as bands. 31. Electrophoresis Apparatus o is used in molecular biology to separate charged molecules such as DNA, RNA, or proteins using an electric field. o The process, called electrophoresis, enable movement of molecules from one end of the electrode to another based charge, in which negatively charged molecules move toward the anode (positive electrode) and positively charged molecules move toward the cathode (negative electrode). o The rate of migration depends upon its net charge, size, shape, and the applied electric current. – Nothing follows – NOTE: Black text – lecturer’s ppt content Blue text – reference book Red text – lecturer’s discussion Purple text – internet “You are strong enough to start again.”