Ch1

• Microscopy is a scientific technique that uses microscopes to observe and study objects too small to be seen with the naked eye
• Simple microscopes have only one magnifying lens and no objectives, while compound microscopes have two magnifying lenses: oculars/eyepiece and objectives
• A compound microscope, also known as a Brightfield Microscope:

  • Light is passed through the specimen
  • Focused by glass lenses
  • Image formed on the human retina
  • Maximum magnification about 1000x
  • Resolves objects separated by 0.2 mm, 500x better than the human eye
• Magnification:

  • The number of times larger an image is than the real size of the object
  • M=I/A (I=observed size of the image, A=actual size)
  • It is like zooming in
• Resolution:

  • The ability to distinguish between two separate points
  • The higher the resolution, the greater the detail that can be seen
  • We do not improve resolution by enlarging photographs, they are just easier to see when larger
• Total magnification = eyepiece magnification x objective magnification
• Do's in using immersion oil:

  1. Use a dedicated bottle of immersion oil for microscopy

  2. Clean the objective lens before applying the oil

  3. Apply a small, precise drop of oil

  4. Use the appropriate magnification

  5. Lower the objective lens gently

  6. Focus carefully
• Don'ts in using immersion oil:

  1. Don't use immersion oil on dry objectives

  2. Don't touch the objective lens with bare fingers

  3. Don't reuse or mix different brands of immersion oil

  4. Don't use too much oil

  5. Don't use immersion oil on a wet or dirty slide
• Field of View (FOV) refers to the observable area through a microscope's objective lens at a given magnification
• Depth of Field (DOF) refers to the range of distances within the specimen that appears acceptably sharp and in focus simultaneously
• Working distance refers to the distance between the objective lens of a microscope and the specimen being observed while maintaining focus
• Parfocal refers to the ability of microscope lenses to maintain focus on a specimen when switching between different objective lenses
• Mechanical parts of a compound light microscope:

  • Base, pillar, inclination joint, arm/neck, stage, stage clips, stage opening, body tube, draw tube, revolving/rotating nosepiece, dust shield, coarse adjustment knob, fine adjustment knob, condenser adjustment knob
• Other terms in microscopy:

  • Iris diaphragm lever, rack stop
• Illuminating parts of a compound light microscope:

  • Mirror, electric lamp/light source, iris diaphragm, condenser
• Magnifying parts of a compound light microscope:

  • Ocular/eyepiece, objectives (LPO, HPO, OIO, Scanner)
• 10 Steps of Using a Microscope:

  1. Carry the microscope properly

  2. Adjust the diaphragm

  3. Adjust the light source

  4. Place the slide on the stage

  5. Center the specimen

  6. Secure the slide with stage clips

  7. Lower the body tube

  8. Focus using the coarse focus knob

  9. Switch to the high power objective lens

  10. Use the fine focus knob to focus
• Important tips in using the microscope:

  • Always carry the microscope properly
  • Start and end with the lowest powered objective
  • Keep both eyes open to reduce eyestrain
  • Adjust the diaphragm for better detail visibility
  • Use only the fine adjustment knob with the high power objective
• Safety measures in a biology laboratory:

  1. Caution, common sense, and good judgment

  2. Prohibition of eating, drinking, and smoking

  3. Proper clothing: lab coats, gloves, closed shoes

  4. Familiarize with exit locations

  5. Proper use of fire extinguishers, first aid kits, eye wash stations

  6. Note emergency hotlines

  7. Keep aisles and tables clear

  8. Report all injuries, spills, breakage, accidents to the instructor

  9. Wash hands before and after lab sessions
• Additional safety measures:

  1. Cover any abrasion, cut, or open wound before beginning work
• Safety Protocols in a Biology Laboratory:

  • Report breakage and accidents to the instructor
  • Wash hands before and after lab sessions
  • Cover any abrasion, cut, or open wound before starting work
  • Disinfect work surfaces and equipment immediately after spillage
  • Discard biohazard waste into appropriate disposal bins
  • Minimize aerosol formation during procedures
  • Sterilize inoculating needles and wire loops before and after use
  • Keep test tube cultures in test tube racks
  • Never pipette cultures by mouth
• Handling Preserved Specimens:

  • Keep the part of the specimen not being dissected in a plastic bag
  • Seal the bag after removing the specimen to confine the preservative inside
  • Notify the instructor in case of a spill of preservative
  • Do not dispose of body parts or scraps of specimens in the sink
  • Clearly label and store specimens in designated areas
• Microscope Handling:

  1. Carry the microscope with one hand supporting the arm and the other supporting the base

  2. Position the microscope safely on the table, away from the edge

  3. Drape the cord carefully onto the table and avoid letting it dangle on the floor

  4. Never use the course adjustment when the HPO or OIO lens is in place

  5. Place the microscope upright in the appropriate numbered slot in the cabinet

  6. Return all prepared microscope glass slides to their appropriate slide trays

  7. Report malfunctioning microscopes to the instructor
• Hot Plates & Water Baths:

  • Place these equipments in a safe place
  • Let the instructor regulate the temperature of the equipment
  • Use insulated gloves or tongs to move glasswares in and out of the water bath
  • Take care when working near hot plates and water baths
• Centrifuges:

  • Ensure the disconnect switch works properly and shuts off the equipment when the top is opened
  • Balance centrifuge rotors each time they are used
  • Regularly clean rotors and buckets with noncorrosive cleaning solutions
  • Clean the centrifuge lid during operation
  • Stop the centrifuge immediately if vibration occurs
• Proper Disposal of Contaminated Materials:

  1. Place contaminated syringe, lancets, blades, and glasswares in a Sharp’s container

  2. Put disposable gloves, face mask, pipets, and cotton swabs in a biohazard bag, then place in a puncture-proof biohazard bin

  3. Place bacterial cultures and agar in a biohazard bag, seal properly, then autoclave

  4. Treat biological liquid wastes with 10% Sodium Hypochlorite before pouring down the drain with running water

  5. Place uncontaminated broken glasswares in a broken glass disposal box