Pre-ANAPhY LAB (MOD1).pdf

LABORATORY RULES AND REGULATION University of the Assumption Basic Requirements Individual: Lab gown Gloves Face Mask Activity sheet/manual Alcohol 70% Drawing materials ( colored pencils and pencil) Group: Disinfectant (zonrox, lysol :liqui...

LABORATORY RULES AND REGULATION University of the Assumption Basic Requirements Individual: Lab gown Gloves Face Mask Activity sheet/manual Alcohol 70% Drawing materials ( colored pencils and pencil) Group: Disinfectant (zonrox, lysol :liquid and spray) Antibacterial soap ( e.g. safeguard) Tissue and paper towels Yellow Sando plastic garbage bags (waste) Sponge Alcohol Big Scissors Lighter Glass slide and cover slips University of the Assumption Rules of Laboratory Safety Before you start and experiment Never enter the laboratory room unless you are permitted to do so. Always come on time. Read carefully the experiment procedure before coming to class. know the theories and principals involved. You are expected to know this when performing and experiment Make a flow chart of the procedure so it will be easier for your to performed the proper sequencing of the procedures. Place your things on the appropriate location so they will not clutter your working area. Do not follow directions mechanically, but know the reason(s) for every step. Know why as well as how. This will make you more aware of what you are expecting from the outcome of a proper done experiment. Clean your assigned working area before beginning your experiment use disinfectant always. Good work, good results can only be attained when you observe neatness, cleanliness and exactness. University of the Assumption Rules of Laboratory Safety Always keep your working are free of clutter. Work on the experiment only when the instructor is present or when you have given permission to start. Be sure that your apparatus is clean before doing your experiment. Clean them with detergent, wash thoroughly with water and dry them up. Check glassware for any crack before using for it might weakened and break during the use and may cause injury. Always know the precautions (chemicals used and the procedures) to be observe for each experiment Know the location of the fire extinguisher, eyewash, fire safety blanket and the first aid kit (this may be requested from the lab technician if needed) Mobile phones are not allowed during the class unless instructed for documentation purposes only. Notify the instructor or lab tech for any spills and drips on the working table or floor for proper cleaning instructions. University of the Assumption  When performing an experiment Wear your PPEs Read the labels on every chemical reagent bottle before taking out sample. Put label on your specific test tube or container on where you are going to transfer the reagent. Never touch chemicals with your bare hands nor taste any chemicals even the label indicated like SALT. If live specimens are to be use, execute the procedure with all precautions. University of the Assumption  Avoid contamination or reagents. use spatula separately for solid (powder or crystal reagents). Use pipette or dropper (base on the amount to be extracted) separately for liquid reagents Never return excess clean chemical back to the reagent bottle, instead place them in a clean vessel (test tube or beaker with label) and you may give it to you other classmate who will be using the same reagent. If not you may place them in a collecting bottles or jars right after the experiment. When transferring large amount, you may pour from the reagent bottle to the experiment container, this needs practice to minimize of prevent drips and spill. Make sure your hand is over the label, not on the neck of the bottle nor at the base, this will help you control the pouring better. University of the Assumption  Avoid wasteful use of reagent, get only the specified amount stated from the procedure. Lost or damage apparatus borrowed and used by any group must be the responsibility of the entire group. Secure a breakage form from the laboratory technician. Never smell reagent or chemicals being tested directly, instead practice “wafting technique” to prevent any effect of the strong scent or smoke from the test tube. Inhale cautiously. Never look down directly from the container (test tube or beakers) being heated or use in mixing chemical, view the reaction from the outside. Do not perform unauthorized experiment. Never leave the experiment without someone to look after the reaction that taking place. University of the Assumption  Do not leave the burner during heating. Always use non- luminous flame unless instructed to use yellow flame. Extinguish the flame when not in use. Do not pour the used chemical directly on the sink, ask the lab instructor or technician for the waste chemical container. Do not point the test tube being to anybody angle the test tube at least 45-90 degrees when heating reagent in the test tube For the chemicals that are allowed to be poured directly into sink, dilute them first with water before draining down the sink. University of the Assumption  When diluting acids, ALWAYS add acid to water, NEVER pour water to acid. Strictly eating and drinking is not allowed inside the laboratory. Stay on your designated area to do loiter around. Our school is a non-smoking campus, more so smoking is strictly prohibited in the laboratory. Carry the microscope properly with both hands University of the Assumption After the experiment Before returning the microscopes, make sure to disinfect them, position or align the LPO on the stage, place the mirror in upright position, adjust the coursed adjustment knob to lower the body tube close to the stage. Return the microscope back to the stockroom. Dispose all used chemical to their proper collecting bottles. Dilute with water those chemicals that are allowed to be drained into the sewage. Clean all the apparatus with water and detergent before returning them back to the stock room and or lockers. Clean your working area, never leave the room with a dirty working table. Close all gas cock/valve. Make sure the faucet is proper close to prevent leaking of water Wash your hand with soap and water before touching your things and or your face and hair. Grooming is not allowed inside the lab University of the Assumption Strictly No Eating and Drinking inside the Lab University of the Assumption  Chapter 1: Basic Microscopy University of the Assumption  THE MICROSCOPE University of the Assumption  Kinds of Microscope 1. Optical / Light microscope It makes use of convex lenses to focus the light and produce an image that can be viewed either directly by the naked eye or through the use of a monitor Resolution limit of about 1000 nanometer A. Simple microscope A type of microscope that uses a single lens for the magnification of the sample. A simple microscope is a convex lens with a small focal length. B. Compound Microscope A compound microscope offers a better magnification than a simple microscope. It has combination of lenses and optical parts; objectives and ocular that function to magnify an object. The lens closest to the eye is called the ocular. The lens closest to the object is called the objective. It has four objectives lenses; scanning lens (4X) it is the shortest of the four, low-power lens (10X), high-power lens (40 X), oil-immersion lens (100 X) it is the longest of the four. University of the Assumption  A compound light microscope often contains four objective lenses: a.scanning lens (4X) it is the shortest of the four b.low-power lens (10X) c.high-power lens (40 X) d.oil-immersion lens (100 X) it is the longest of the four University of the Assumption 3. Electron microscope Electron beams are focused using various magnets There are two kinds namely; transmission electron microscope (TEM) and the scanning electron microscope (SEM) 4. Stereo microscope It is also called as a dissecting microscope. It provides a three- dimensional view of a specimen. It has separate objective lenses and eyepiece such that there are two separate optical paths for each eye. University of the Assumption  Magnification 2phase: 1st lens closest to the specimen = objective lens ( forms the initial image) 2nd lens closest to the eye = ocular lens / eye piece (forms the virtual image ) Magnification power: derived by multiplying the magnifying power of the ocular / eye pc by the magnifying power of the objective used. Achieving total magnification is possible with the magnification achieved by the objective multiplied by the magnification achieved by the ocular lens. Parfocal microscopes means that the microscope remains in focus when one switches from one objective to the next objective Computing the magnification power University of the Assumption  Resolution - is the ability to distinguished two very small and closely- spaced objects as separate entity Resolving power- The resolving power of an objective lens is measured by its ability to differentiate two lines or points in an object. The greater the resolving power, the smaller the minimum distance between two lines or points that can still be distinguished. The larger the N.A., the higher the resolving power. The numerical aperture (NA) refers to the widest cone of light that can enter the lens; the NA is engraved on the side of the objective lens. University of the Assumption  Factors affecting the formation of clear image 1. Quality of lens Typical problem: spherical aberration – a distortion in the image caused by the irregularities in the lens which create a curved rather than a flat image 2. Light source Brightness and direction of illumination Too much light can reduce contrast and burn out the image (iris diaphragm) 3. Lack of contrast Using special lenses (phase contrast) and by adding dyes University of the Assumption  PARTS OF THE MICROSCOPE Eye piece / ocular Draw tube Coarse adjustment knob Body tube Fine adjustment knob Revolving nose piece Objectives Arm Stage Stage Clip Diaphragm/ condenser Inclination joint Mirror (concave/ convex) Base University of the Assumption University of the Assumption  Mechanical parts Magnifying parts Illuminating parts- - use to support and - use to enlarge the - use to provide light and adjust the parts: specimen have a good vision of the specimen: 1. eyepiece or 1. draw tube ocular 1. mirror- collect 2. body tube 2. objectives- natural light 3. revolving nose piece LPO 2. light bulb 4. stage HPO 5. stage clip OIO 6. arm Scanning 7. base inclination joint 8. iris/diaphragm 9. condenser 10. coarse adjustment knob 11. fine adjustment knob University of the Assumption  1. Eyepiece / ocular lens- the first magnifying lens where the image is being observe (10x) 2. Draw tube – the upper tube that supports the eyepiece 3. Body tube- attaches the upper part of the microscope to the arm and support the objectives. 4. Revolving nose piece- located at the lower part of the body tube and bears the objectives. Low power objective- shorter lens serve to form image within the body tube (10x) High power objective – longer lens, serves to form a more close up and bigger image of the specimen (40x) Oil immersion objective – the longest objective. Should only be used with oil that helps in increasing the resolving power of a microscope. (100x) University of the Assumption  5. Arm- short curve handle used in carrying the microscope and at the same time connect the upper portion of the microscope to the lower part 6. Pillar-short metals supporting and connecting the microscope’s upper portion to the base. 7. inclination joint- help in tilting / inclining the microscope and connect the arm to the base 8. Base- serve as the foot on which the microscope stands 9. Course adjustment knob- used to bring the object into focus (bigger knob) 10.Fine adjustment knob- use to have a better and closer view of the part being observe. (smaller knob) University of the Assumption  11. Dust shield- round metal that protects the lenses attached to the revolving nose piece. 12. Stage – a platform with central aperture and stage clips that hold the slide in place. 13. Mechanical stage - mechanism attach to the stage for ease in moving the slide. 14. Mirror – collects natural light that serve as source of illumination. 15. Iris diaphragm – located on the condenser and controls the amount of light coming through it. It consisting of blades that form circular opening that may be enlarged or reduced to control the amount of light reaching the object. 16. Condenser – found immediately below the stage, it serves to concentrate the light rays on the specimen. The condenser and diaphragm makes up the sub stage. University of the Assumption  Using the Microscope 1.Always carry the microscope by the base and the arm. 2.Clean the microscope before and after using. 3.Position the slide on the center of the stage. Secure with the stage clip. 4.Use the lowest objective power upon initial observation and focusing the image. Slowly lower it down near the slide using the coarse adjustment knob 5.Adjust the angle of the mirror in collecting light for illumination. 6.Use the higher power objective for a more clear image. 7.Always place the LPO over the stage and be sure the stage is at its lowest position before putting the microscope away. 8.Always turn off the light before putting the microscope away (for electric microscope). 9.If the source of light is a mirror, position the mirror vertically. 10.Always wrap the cord correctly before putting the microscope away. 11.Always return the microscope to the correct cabinet. 12.After cleaning and position the lowest objective in line with the aperture and bring it down or bring the stage up near the lowest objective University of the Assumption