Ch. 2 Microscopy

Bio 3340

Microorganism

organism not visible to naked eye

Electron microscope

It was possible to view viruses only after the invention of the electron microscope because they are too small to be seen with a light microscope

Light

refracted (bent) when passing from one medium to another

Refractive index

bends the path of light and slows the velocity of light. Air < Glass

Focal point (F)

Focus light rays at a specific place

Focal length (f)

Distance between center of lens and focal point is the f.

Confocal Microscope

creates sharp composite 3D image of specimens by using laser beam

Staining of specimens

Staining allows for better visibility of specimen

Increases visibility of specimen

Accentuates specific morphological features

Preserves specimens

Fixation

Preserves internal and external structures and fixes them in position, Organisms usually killed and firmly attached to microscope slide

Heat fixation

routinely used with bacteria and archaea

Preserves overall morphology but not internal structures

Chemical fixation

often used for eukaryotes, but also bacteria and archaea, particularly with the use of electron microscopy

techniques

Protects fine cellular substructure and morphology

Dyes

Make internal and external structures of cell more visible by

increasing contrast with background

Basic dyes

ex: Methylene blue, basic fuchsin, crystal violet, safranin, malachite green

Have positively charged groups;

bind to negatively charged

molecules such as nucleic acids,

many proteins, and the surfaces

of bacterial and archaeal cells

Simple staining

•Ionizable dyes have charged

groups

•Basic dyes have positive charges

•Acid dyes have negative charges

•Simple stains

•A single stain is used

•Use can determine size, shape, and

arrangement of bacteria

Differential staining

• Divides microorganisms

into groups based staining

properties

• Gram stain

• Acid-fast stain

• Detect presence or absence

of structures

• Capsules

• Flagella

Gram staining

•Most widely used •Divides bacteria into two groups

• Gram-positive (purple)

• Gram-negative (pink)

• Based on differences in cell wall structure (peptidoglycan)

Electron microscopy

Electrons replace light as ‘illuminating’ beam

• Wavelength of electron beam much shorter than light resulting in higher resolution

• Allows for study of microbial morphology and intracellular structures in great detail

• Analogous to procedures used for light microscopy

• For transmission electron microscopy, specimens must be cut very thin

• Specimens are chemically fixed and stained with electron dense materials, such as heavy metals, that differentially scatter electron

Transmission Electron Microscope

Electrons scatter when they pass through thin sections of a specimen

Scanning Electron Microscope

•Scans a narrow electron beam back and forth over the specimen

•Produces a realistic 3-D image from electrons released from a specimen’s surface, visualizing the features in great detail