Observing microorganisms through a microscope
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Observing Microorganisms Through a Microscope. Chapter 3. Units of Measurement. Sizes and Magnification. Check Your Understanding. Differentiate between the different metric units used to measure the sizes of microbes. Microscopy: The Instruments. Light Microscopy.

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Check your understanding
Check Your Understanding

  • Differentiate between the different metric units used to measure the sizes of microbes.

Light microscopy
Light Microscopy

  • Uses light to illuminate a specimen to be viewed

  • Useful for objects larger than 0.2μm in size

    • Bacteria, eukaryotic cells

  • Allows scientists to view living specimens

Compound light microscopy
Compound Light Microscopy

  • Visible light provides the source of illumination

  • Image passes from Objective to Ocular Lens

    • TotalMagnification

  • Resolution (Resolving Power) is ability of a lens to distinguish fine detail

    • 0.2 μm

Immersion oil
Immersion Oil

  • Only used with 100X objective lens

  • Reduces refraction of light rays passing from one medium to another

    • Has same refractive index as a glass slide or lens

Brightfield microscopy
Brightfield Microscopy

  • Background is brightly illuminated

  • Condenser focuses light directly on specimen

  • Typical compound light microscope

Darkfield microscopy
Darkfield Microscopy

  • Useful for living specimens or that cannot be stained while maintaining integrity

    • Treponemapallidum

  • Condenser is opaque

    • Light is reflected off the speciment

    • Background is darkened

Phase contrast microscopy
Phase-Contrast Microscopy

  • Allows visualization of large internal structures in living specimens

    • Structures are well-defined

  • Uses “in-phase” and “out-of-phase” light rays to produce areas of contrast

Differential interference contrast dic microscopy
Differential Interference Contrast (DIC) Microscopy

  • Uses two light sources to produce differences in phase contrast

  • High resolution is possible

  • Image is bright and has a three-dimensional quality

Fluorescence microscopy
Fluorescence Microscopy

  • Fluorescence is ability to absorb one wavelength of light and give off at another

  • Antibodies are immune particles produced against a specific molecule(Antigen)

  • Allows scientists to “paint” specific molecules to make them highly visible

Confocal microscopy
Confocal Microscopy

  • Uses fluorescent antibodies in conjunction with light microscopy

    • Specimen is scanned in series of focal planes and then reassembled via computer

    • Produces a detailed, 3-dimensional image

Electron microscopy
Electron Microscopy

  • Uses a beam of electrons to resolve the object being “viewed”

  • Provides a highly detailed image for objects smaller than 0.2μm

    • Organelles and viruses

Transmission electron microscopy
Transmission Electron Microscopy

  • Uses a focused beam of electrons that penetrate the specimen

    • Provides detailed images of internal structures

  • “Stains” produced by various metals can enhance the contrast of the image

Scanning electron microscopy
Scanning Electron Microscopy

  • Electron beam is focused across the surface of the specimen

    • Gives a three-dimensional view of the specimen

    • Provides detailed images of external structures

Check your understanding1
Check Your Understanding

  • Describe the path of light through a compound microscope.

  • Explain the role of immersion oil in light microscopy. When is it used?

  • Define total magnification and resolution. What is the significance of a 0.2μm resolution for light microscopes.

  • Differentiate between electron and light microscopy. Why do electron microscopes have a greater resolution than light microscopes?

  • Differentiate between the different types of light and electron microscopy techniques available. When would each be used?

Preparing smears for staining
Preparing Smears for Staining

  • Specimen are added to the slide using water and then spread to a thin film called a smear

  • Heat-fixing kills the microbes and adheres them to the slide BEFORE stains are applied

    • Performed only after the smear has completely air-dried

  • Staining requires adding dyes to a specimen to add contrast so it is more visible under the microscope

    • Stains (dyes) are liquids that can remove specimens

Types of dyes
Types of Dyes

Basic Dyes

Acidic Dyes

Chromophores are negatively charged

Repelled by the the negatively charged cell walls of bacteria

Cells appear colorless against a dark background

Negative Staining


  • Chromophoresare positively charged

  • Attracted to the negatively charged cell walls of bacteria

  • Cells appear colored against a clear background

  • Crystal Violoet, Methylene Blue, Malachite Green, Saffranin

Simple stain
Simple Stain

  • A single dye is added to a specimen providing contrast

  • A mordant may be added to help the specimen retain the dye

Bacillus subtilis

Gram stain
Gram Stain

  • Differential stain

  • Developed by Han Christian Gram (1884)

  • Helps to begin classifying bacteria

    • Differences are based on differences in the cell wall of bacteria

    • Gram (+) bacteria have a thick peptidoglycan (PG) cell wall

    • Gram (-) bacteria have a thin PG cell wall and outer layer of lipopolysaccharide (LPS)

Acid fast stain
Acid-Fast Stain

  • Differential stain

  • Dyes adhere strongly to the thick layer of mycolic wax found in some bacteria

    • Wax resists decolorization even with acid-alcohol

  • Useful in identifying bacteria in the genus Mycobacterium

Capsule stain
Capsule Stain

  • Capsules enhance the virulence of an organism

    • Prevent its phagocytosis by leukocytes

  • A type of negative stain preventing removal of the capsule by excessive washes


Endospore stain
Endospore Stain

  • Endospores are resistant structures containing the DNA of a bacteria

    • Formed under adverse environmental conditions

  • A type of differential stain

  • Uses heat to allow primary dye to penetrate the spore coat

Bacillus megaterium

Flagella stain
Flagella Stain

  • Flagella are normally too small to be seen with the light microscope

  • A type of simple stain

  • Uses a mordant and dye to build up the layers of dye on the flagella

Proteus vulgaris

Check your understanding2
Check Your Understanding

  • What steps are needed to make a smear prep? Why is it necessary to heat-fix a specimen prior to staining?

  • Differentiate between an acidic and a basic dye. Why does a negative stain color the background and not the specimen?

  • What is a simple stain and what is its role in the microbiology lab? Why does a simple stain color the specimen and not the background?

  • Describe the steps in a Gram stain. Differentiate between a Gram-positive and a Gram-negative organism based on color.

  • Compare and contrast the Gram stain and the acid-fast stain. Provide two examples of organisms that can be identified with an acid-fast stain.

  • What is the purpose of a capsular stain, endospore stain, and flagellar stain? Give examples of organisms that could be identified with each type of staining technique.