Lab Discussion 1

• Microscopy is a technical field using microscopes to view samples and objects that cannot be seen with the unaided eye
• COMPOUND LIGHT MICROSCOPE also known as Brightfield Microscope
• CLM Light is passed through the specimen and focused by glass lenses
• CLM The image is formed on the human retina
• CLM Maximum magnification is about 1000x
• CLM Resolves objects separated by 0.2 mm, which is 500x better than the human eye
• Mechanical parts:

  • Base: Bottommost portion supporting the entire microscope
  • Pillar: Part above the base supporting other parts
  • Inclination Joint: Allows tilting of the microscope for user convenience
  • Arm/Neck: Curved part held while carrying the microscope
  • Stage: Platform for the object
• Magnifying parts:

  • Objectives: Metal cylinders containing ground and polished lenses
  • LPO/Low Power Objective: Gives the lowest magnification, usually 10x
  • HPO/High Power Objective: Gives higher magnification, usually 40x or 43x
  • OIO/Oil Immersion Objective: Gives the highest magnification, usually 97x or 100x, used wet with cedar wood oil or synthetic oil
• Illuminating parts:

  • Substage: Iris Diaphragm regulates light amount
  • Condenser: Set of lenses between the mirror and stage concentrating light on the specimen
  • Ocular/Eyepiece: Another lens set on top of the body tube further magnifying the image produced by the objective lenses
• step 1 To carry the microscope, grasp the arm with one hand and hold the base with the other hand
• step 2 Adjust the diaphragm while looking through the eyepiece
• step 3 Adjust the light source (mirror) until you see the circle of light
• 4 Place the slide on the stage and 5 center the specimen over the small opening
• 6 Secure the slide with the stage clips
• 7 While looking at the STAGE from the side, turn the COURSE FOCUS KNOB to lower the BODY TUBE until the LOW POWER OBJECTIVE almost touches the slide
• 8 Carefully move the coarse focus knob until the specimen comes into view
• 9 Switch to the high power objective lens by moving the nosepiece until it "clicks" into place
• 10 Turn the fine focus knob until the specimen comes into focus
• Make sure to follow important tips such as carrying the microscope properly, starting and ending with the lowest powered objective, and adjusting the diaphragm for better visibility
• High Power Objective (40x) is very close to the slide
• Use only the fine focus knob on High Power to avoid scratching the lens and cracking the slide
• A compound light microscope consists of two lenses separated by a tube
• Lenses magnify objects by bending the light passing through them
• Magnification is the ability to make things appear larger than they are
• Resolution refers to the fineness of detail that can be seen in an image
• To mount a specimen:

  • Place the slide on a flat surface
  • Add a drop of water to the slide
  • Add the specimen to the drop of water
  • Hold the coverslip by its sides and lay its bottom edge close to the specimen
  • Slowly lower the coverslip to spread the water out, removing air bubbles if present
• Staining a slide:

  • Place a drop of stain on one edge of the coverslip and a piece of paper towel on the other edge
  • Allow the stain to cover the area containing the specimen
  • Wipe off excess stain with a paper towel to avoid staining the objective lenses
• • Acetocarmine: stains chromosomes and connective tissue red/pink
  • Sudan III or IV: used as a lipid indicator, stains red
• Common stains for slide preparations include:

  • Iodine: stains carbohydrates brown or blue-black, stains glycogen red
  • Methylene blue: stains acidic cell parts blue, used on animal, bacteria, and blood specimens
  • Eosin: stains alkaline cell parts pink, used on plants, animals, and blood
• To measure the size of a specimen:

  • Fit an ocular micrometer into the eyepiece or replace the ordinary eyepiece with a micrometer eyepiece
  • Mount the stage/slide micrometer and focus under LPO
  • Adjust the scales to superimpose on each other
  • Count the number of ocular units and stage micrometer units between two marks
  • Divide the number of stage micrometer units by the number of ocular units to get the calibration constant
• Drawing observations:

  • Examine the specimen carefully and draw only what you see
  • Use pencil for drawings and make them large and clear
  • Use stippling or dots for darker areas, do not shade
  • Include a title, magnification, labels of important features, and a scale bar in all drawings
  • Underline scientific names and write them as Genus species
• Parts of a clm:

  Mechanical, Magnifying and Illuminating parts
• Simple microscope has only one magnifying lens no objs
• compound microscope has two magnifying lenses oculars/eyepiece and Objs
• MP

  • Stage Clips: Secures the specimen to the stage
  • Body Tube: Attached to the arm and bears the lenses
  • Draw Tube: Cylindrical structure holding the ocular lenses
  • Revolving/Rotating Nosepiece –Rotating disc where the objectives are attached
  • Dust Shield –Lies atop the nosepiece and keeps dust from settling on the objective
• • Condenser Adjustment Knob –Elevates and lowers the condenser to regulate the intensity of light
  • Iris Diaphragm Lever –Lever in front of the condenser and which is moved horizontally to open/close the diaphragm •
  • Rackstop was created to protect the microscope objectives from hitting the stage. • Before it was invented, objectives were sometimes damaged when they hit the stage. • The rack stop is set by the factory when the microscope is manufactured.
• stains

  • Safranin: stains red, mainly used for plant tissues
  • Toluidene blue: stains acidic cell parts dark blue, good for showing mitosis in plant cells
  • Wright's stain: stains red blood cells pink/red
  • Leishman's stain: stains nucleus of WBC blue and blood cells pink
  • Crystal Violet: stains bacteria purple