Brightfield Microscopy Study Notes

Brightfield Microscopy

Lab Exercise Overview

  • Lab Exercise Title: Brightfield Microscopy
  • Course: SSC Laboratory Exercises for Microbiology (MCB2010C)

Laboratory Safety Procedures

  • Personal Protective Equipment (PPE) Requirements:
    • Face mask
    • Lab coat
    • Gloves
    • Safety glasses
    • Long pants
    • Close-toed shoes
  • Procedure Compliance:
    • Follow the lab manual step-by-step.
    • Quizzes and exams will be based solely on the procedures detailed in the lab manual.
  • Supplemental Videos:
    • Videos provided are for visual demonstration only and should not replace the lab manual as they may contain differing content.

Goals of Microscopy

  • Primary Goals:
    1. Generate a magnified image.
    2. Allow details to be seen clearly (resolution).

Lab Exercise #1 Goals

  • I. Microscope Nomenclature:
    • Identify the basic components and functions of a brightfield microscope.
  • II. Proper Focusing Technique:
    • View a specimen using a compound microscope.
  • III. Microscopic Measurements:
    • Identify length and width of a specimen.

I. Microscope Nomenclature

Microscope Parts and Functions
  • Rotating Head:
    • Contains mirrors, allowing the body tube to rotate 360°.
  • Objective Lens:
    • Produces the majority of magnification:
    • High-Power Lens (blue stripe): magnifies 40x.
    • Low-Power Lens (yellow stripe): magnifies 10x.
  • Stage:
    • Holds the slide with an opening that allows light to pass through the specimen.
  • Diaphragm:
    • Rotating dial controlling light passage through the stage;
    • Numbers indicate relative light amounts (5 = most, 1 = least).
  • Body Tube:
    • Passes light from the head to the eyepiece.
  • Eyepiece (Ocular):
    • Where the enlarged object is viewed; contains a lens that magnifies 10x.
  • Arm:
    • Supports lenses, mirrors, and body tube; carry microscope with one hand on the arm and another under the base.
  • Coarse Focus Knob:
    • Moves the stage up/down quickly; used to find a specimen at low power.
  • Fine Focus Knob:
    • Makes small focus adjustments, especially at high power.
  • Base:
    • Supports the microscope and houses electronics; carry with one hand under the base.
  • Power Switch:
    • Turns the light source on and off.
  • Light Source:
    • Illuminates the specimen by shining bright light through it.

Figure 5: The Compound Light Microscope with Descriptions of its Parts

II. Proper Focusing Technique

Viewing a Specimen with a Compound Microscope

How Light Microscopy Works

  • Description:
    • Utilizes compound light microscopes where light passes through the specimen generating a magnified image.
    • Key Concept:
    • The image is not identical to the specimen.

Specimen vs Image

  • Characteristics of the Image:
    • Inverted
    • Reversed
    • Enlarged
  • Focal Point and Convex Lenses:
    • The role of convex lenses is significant in magnification and clarity of the image.

Magnification

  • Definition:
    • Apparent increase in the size of an object, indicated by a number followed by “x” (e.g., 400X indicates 400 times magnification).
  • Total Magnification Formula:
    • TotalextMagnification=(magnificationextofocular)imes(magnificationextofobjective)Total ext{ Magnification} = (magnification ext{ of ocular}) imes (magnification ext{ of objective})
  • Examples of Objective Magnifications:
    • Low Power: 10x
    • High Dry Power: 40x
    • Oil Immersion: 100x

Magnification and Working Distance

  • Definition of Working Distance:
    • The space between the tip of the objective lens and the specimen.
  • Relationship Between Magnification and Working Distance:
    • As magnification increases, working distance decreases.
  • Coarse Adjustment Limitations:
    • Should only be used with 4x, 10x, and 20x objectives.

Magnification and Field of View

  • Field of View Definition:
    • The area visible when looking through the microscope.
  • Relationship Between Magnification and Field of View:
    • As magnification increases, the field of view decreases.
    • Significance:
    • Always center the specimen before increasing magnification.

Microscope Resolution

  • Also Known As:
    • Resolving power.
  • Definition:
    • The ability of a lens to separate or distinguish two small objects that are close together.
  • Enhancement of Resolution:
    • Oil immersion improves resolution.

How Oil Immersion Increases Resolution

  • Mechanism:
    • Immersion oil prevents refraction and loss of light.
    • Reason:
    • Oil has the same refractive index as the glass slide, directing more light through the objective for a clearer image.
    • Outcome:
    • Increased light results in clearer and crisper images.

Image comparison without and with immersion oil

Applying and Using Oil Immersion

Procedure for Oil Immersion:
  1. Follow steps 1 through 19 as outlined in the lab manual.
  2. With the 100x objective, leave an opening between the 40x and 100x objectives for oil application.
  3. Place a drop of immersion oil on the specimen before rotating the 100x objective into place.
  4. Always use the fine adjustment knob exclusively for focusing at the 100x objective.
  5. When removing the slide, rotate back to the 10x objective, using the coarse adjustment to increase the working distance.
  6. Clean oil immersion lens only with lens cleaner and lens paper.
    • Important Note:
      • Only a small piece of lens paper is needed for cleaning; DO NOT clean other lenses unless they are dirty.

Contrast in Microscopy

  • Definition of Contrast:
    • Differences in intensity between two objects or an object and its background; critical in resolution determination.
  • Enhancement of Contrast:
    • Staining increases contrast observed between unstained and stained cells.
    • Example:
    • Stained vs unstained cheek cells.

Brightfield Microscopy Settings

  • Iris Diaphragm Functionality:
    • Increases contrast and resolution; most useful at higher powers.
    • Located under the stage adjusting light quantity passing through the stage opening.
    • Each opening varies in diameter controlling the light passing.
    • Typically set at a value matching the objective lens used.

Brightfield Microscopy Characteristics

  • Appearance:
    • Background (field) appears bright while the specimen appears dark.
  • Ideal Usage:
    • Best suited for stained or dead specimens.
    • Example:
    • Works excellently with prepared slides, ineffective for living specimens of bacteria.

Exercise 1: Identification of Microorganisms

  • Expected Task:
    • Identify microorganisms observed under 400X and 1000X magnification.
    • Determine if the organisms are prokaryotic or eukaryotic cells.

III. Microscopic Measurements

Identifying Length and Width
  • Measurement of specimens includes both length and width assessment.

Size in the Microbial World

  • Microbial Size Scale:
    • 1 micron (µ) = 10−610^{-6} meters
    • Common size references include:
    • Nucleus: Small
    • Proteins: 1 nm, 10 nm, 100 nm (e.g., ribosomes)
    • Most bacteria: 1 µm to 10 µm
    • Most eukaryotic cells: 10 µm to 100 µm
    • Human height: ~1.8 meters

Measuring Size of a Specimen

  • Methodology:
    • Ocular Micrometer Values:
    • At 40X: 2.5 µm per ocular division.
    • At 100X: 1 µm per ocular division.
  • Examples for Size Calculation:
    • At 40X:
    • Length = (7 ocular lines) x (2.5 µm) = 17.5 µm
    • Width = (3 ocular lines) x (2.5 µm) = 7.5 µm
    • At 100X:
    • Length = (15 ocular lines) x (1 µm) = 15 µm
    • Width = (3 ocular lines) x (1 µm) = 3 µm

Questions for Consideration

  • Regarding Measurements:
    • Are the length and width measurements the same at 40x and 100x?
    • The measurements should be consistent as they refer to the same specimen.
    • Which objective lens offers the most accurate measurements?
    • The 100x lens is advisable for accuracy due to its proximity to the specimen.

Post-Exercise Procedures

  • Cleaning the Microscope Before Storage:
    • Ensure the following standards:
    1. Stage and lenses are clean and free of any contaminants.
    2. The 10x objective is positioned correctly.
    3. Stage height is adjusted to midpoint.
    4. Turn off the microscope.
    5. Wrap the cord properly around the cord keeper.
    6. Position the oculars facing the arm and power cord.

Final Knowledge Check: Exercise Completion

  • Key Areas to Review:
    • Understand the parts and functions of a light microscope.
    • Definition of resolution.
    • Calculation of total magnification.
    • Relationship of diameter of the field and working distance to magnification.
    • Proper focusing technique.
    • Mechanism on how oil increases resolution via refraction properties.
    • Distinction between prokaryotic and eukaryotic cells under microscopic observation.
    • Brightfield microscopy use cases, specimen appearance, and background illumination.