lab 1 micro

SAFETY RULES

  • USE OF THE MICROSCOPE

SAFETY RULES

  • Location of Safety Equipment in Lab

    • Shower

    • First Aid Kits

    • Fire Extinguisher

    • Fire Blanket

    • Eye Wash Stations

  • Protective Wear Requirements

    • Lab coats must be worn.

    • No open-toe shoes must be worn in the lab.

    • Gloves and goggles are mandated in the lab.

CONTAMINATED MATERIAL DISPOSAL

  • Proper Disposal Locations

    • Contaminated gloves and paper towels must be disposed of in red bags on the lab bench.

    • Sharps (e.g., pipette tips, broken glass, and slides) should go into the red box on the lab bench.

    • Contaminated liquids containing bacteria should be placed in a large red container near the window/back of the room.

    • Waste from dyes used for staining must be disposed of in satellite disposal containers located near the fume hood.

HANDWASHING TECHNIQUE

  • Importance of Proper Handwashing

    • A layer of oil and the skin structure inhibit microorganism removal with simple handwashing.

    • Soap aids in the removal of oil, while the scrubbing action facilitates the removal of microorganisms.

    • Reference for Proper Handwashing

    • Source: http://sct.poumon.ca/protect-protegez/germs- microbes_e.php

LIGHT MICROSCOPES

  • Types of Light Microscopes

    • Bright Field Microscope

    • Dark Field Microscope

    • Phase Contrast Microscope

    • Fluorescence Microscope

BRIGHT FIELD LIGHT MICROSCOPE

  • Functionality

    • Produces a dark image against a bright background.

    • Resolves objects down to approximately 10 μm.

COMPONENTS OF THE LIGHT MICROSCOPE

  • Eyepiece (Ocular Lens)

    • Provides final magnification, typically 10X.

  • Objective Lenses

    • Scanning Objective Lens

    • Identified by a red band, magnifies objects 4X.

    • High Dry Objective Lens

    • Identified by a blue band, magnifies objects 40X.

    • Low Power Objective Lens

    • Identified by a yellow band, magnifies objects 10X.

    • Oil Immersion Objective Lens

    • Identified by black and white bands, magnifies objects 100X.

HIGH POWER OBJECTIVE LENSES

  • Utilization of Immersion Oil

    • Oil has the same refractive index as glass, minimizing light refraction and improving resolution at high power.

TOTAL MAGNIFICATION

  • Calculation Formula

    • Total Magnification = (Magnification power of the objective) × (Magnification power of the eyepiece)

    • Example Calculation:
      rac40.10=40extXrac{4}{0.10} = 40 ext{X}

MICRON MEASUREMENTS

  • Micrometers (μm) Scale

    • 0 to 500 μm visual representation

    • Oil immersion field of view approximately 180 μm, high power field of view approximately 450 μm.

MICROSCOPE STAGE COMPONENTS

  • Stage

    • The platform that supports the specimen below the objective lens.

    • Substage Condenser

    • A lens that concentrates light on the specimen.

    • Iris Diaphragm

    • Regulates amount of light passing through, analogous to the iris of the eye.

FOCUSING MECHANISMS

  • Coarse Focus

    • Utilized for focusing with larger lens adjustments.

  • Fine Focus

    • Utilized for focusing high-power images through minor lens adjustments.

MICROSCOPE COMPONENTS

  • Eyepiece/Ocular

  • Trinocular Head

  • Revolving Nosepiece

  • Condenser

  • Stage

  • Lamp

  • Base

  • Objective Lenses

  • Power Switch

  • Light Intensity Regulator

  • Coarse Focus

  • Fine Focus

  • Mechanical Stage Knobs

DARK FIELD MICROSCOPE

  • Principle of Operation

    • Forms a bright object against a dark background.

    • Especially useful for viewing extremely small living organisms that are invisible in light microscopes.

PHASE CONTRAST MICROSCOPE

  • Functionality

    • Enhances contrasts of transparent and colorless objects by influencing the optical path of light.

FLUORESCENCE MICROSCOPY

  • Applications

    • Used for imaging specific features of small specimens (like microbes).

    • Visually enhances 3-D features at small scales, can stain specific cellular structures.

    • Facilitates identification of cellular locations and interactions of specific molecules within the cell.

    • Reference image: http://www.sciencecodex.com/aggregated-images/body/oV94aKJ2s2jn60dq.jpg

OCULAR AND STAGE MICROMETERS

  • Ocular Micrometer

    • Small glass disk with uniformly spaced lines (0 to 100), inserted into the ocular and calibrated against a stage micrometer.

  • Stage Micrometer

    • A micrometer with uniformly spaced lines of known distances, typically divided into 0.01 millimeter and 0.1 millimeter.

CALIBRATING OCULAR MICROMETER

  • Process:

    • Adjust ocular in the body tube until lines of both micrometers are parallel.

    • Align left edges of both by moving the stage micrometer.

    • Count ocular lines that overlap stage micrometer lines to calculate distance.

    • Distance should be expressed in micrometers (µM).

    • Conversion for lines: Each small line = 0.01 mm or 10 µM, and each large line = 0.1 mm or 100 µM.

OCCULAR MEASUREMENTS EXAMPLES

  • Sample Problem

    • If 20 ocular spaces align with the 5th small stage line, what is the value of 1 ocular space?

    • Calculation:
      1extocularspace=racextspacesonstageimes10extµmextspacesonocular1 ext{ ocular space} = rac{{ ext{spaces on stage}} imes 10 ext{ µm}}{{ ext{spaces on ocular}}}

    • 1extocularspace=rac5extspacesonstageimes10extµm20extocularspaces=2.5extµm1 ext{ ocular space} = rac{{5 ext{ spaces on stage} imes 10 ext{ µm}}}{20 ext{ ocular spaces}} = 2.5 ext{ µm}