MAIN CONCEPTS OF MICROSCOPY

Total Magnification = power of objective x power of ocular.

Determines how much the image is enlarged.

Resolution Power = ability to show detail or to separate two adjacent objects or points from one another.

R = l/2 NA

Numerical Aperture (NA) = a mathematical way of describing the light-gathering ability of a lens system.

At 100x: the lens can distinguish any cell or cell apart as it is at least 0.22 µm in diameter.

As far as they are not closer to than 0.22 µm.

Focal length = tube length/magnification

--------------------------------------------------------------------------------
Constraints to a good image

Quality of lens

Light source

Lack of contrast in the specimen

Spherical aberration

Distortion in the image caused by irregularities in the lens (curved image)

Chromatic aberration

Blurry, rainbow-like image

Lens acts as a prism breaking light apart into its colored bands

Brightness

Direction of illumination

-------------------------------------------------------------------------------
Types of microscopes

Bright Field

Forms image when light is transmitted through the specimen.

Oil immersion: to maximize resolution power at the 100x objective.

1,000X maximum practical magnification.

Dark Field

A disk 'stop' in the condenser blocks all light that is reflected off the sides of the specimen.

Black background.

1,000X maximum practical magnification.

Phase Contrast

Greater internal detail of the specimen.

For internal structures.

1,000X maximum practical magnification.

Fluorescence

UV/fluorescent dyes (acridine, fluorescin).

Dyes emit visible light when bombarded with UV light.

1,000X maximum practical magnification.

Confocal

Fluorescence light scanned with laser beam.

Provides detailed view in 3-D

1,000X maximum practical magnification.

----------------------------------------------------------------------------------
Electron Microscopy

Electron Microscope

Uses electrons and magnets in vacuum instead of light.

Transmission (TEM) magnification >10⁶X

Scanning (SEM) 3-D magnification 10⁶X

Scanning Probe Microscopes (SPM)

Atoms, patterns of molecules.

Scanning Tunneling Microscope (STM) >100⁶X

Atomic Force Microscope (AFM) >100⁶X

-----------------------------------------------------------------------------
Techniques for Mounting a Sample on a Slide

Smear: spread of a thin film on a slide

Fixation: exposure to rapid heat, kills specimen and adhere it to the glass

Staining: dyes affix to the specimen by chemical reaction

-------------------------------------------------------------------------------
Stains

Are diagnostic chemical tools in microbiology that make it possible to observe microorganisms and their structures.

Can be basic (cationic, positively charged) or acidic (anionic, negatively charged).

Bacteria have negatively-charged molecules on their surfaces.

Bacterial cells stain with BASIC dyes (crystal violet, methylene blue, safranin, malachite green, etc.).

Positive Stain

The dye sticks to specimen and gives its color. (e.g. Gram stain)

Negative Stain

The reverse of positive.
Nigrosin, India ink (e.g. in spore staining).
Acidic dyes are repelled by cells.

------------------------------------------------------------------------------
Simple Stain

One single dye.

Reveal size, shape and internal granules in bacteria
(malachite green, crystal violet, fuchsin, safranin, methylene blue)

Differential stain

Use two different colored dyes, the primary dye and the counterstain to distinguish between cell parts.

Examples: Gram stain (crystal violet, safranin)

Acid-fast stain (carbol fuchsin, methylene blue)

Endospore stain (malachite green, safranin)
A structural stain.

Capsule stain (India ink, Congo red)

Flagellar stain (Gray's mordant, carbol fuchsin)

End of chapter 3.
Make sure that you have prepared well the previous two chapters.

-----------------------------------------------------------------------------

Remember to read your textbook, study tables, graphs and illustrations.
Develop a strategy to administer your time so that when exams come you do not have to cram.
Attend lectures and ask questions.

Lecture notes are posted BEFORE lecture is given thereafter they will be removed.