I. Microscopy:
A. Units of measure:
1. convert between the following units of measure using proper scientific notation
a. meter (m), centimeter (cm), millimeter (mm), micrometer (um), nanometer (nm)
b. example #1) 10,000 = 104 and 0.0001 = 10-4, etc.
c. example #2) 234,000 = 2.34x105 and 0.00357 = 3.57x10-3, etc.
B. Major terms & concepts in microscopy:
1. magnification
a. increase in specimen image size (ocular X objective lenses = Total Magnification)
2. resolution
a. smallest distance between two objects that allows each to be seen as separate objects
i. for light microscope, about 0.2um is maximum resolution possible
ii. affected by: refractive index of specimen and wavelength of light used for illumination
3. refraction
a. ability to "bend" light passing through a medium
b. immersion oil reduces refraction of light passing between glass and air, thus increasing resolution
i. "refractive index" is a measure of refractive ability of an object.
C. Identify major parts of light microscope and their function:
1. ocular lens (eyepiece) -
2. objective lens -
3. condenser -
4. course & fine focus knobs -
5. iris diaphragm -
6. stage -
C. Various forms and types of microscopy:
1. brightfield microscopy
a. "typical" light microscopy; background is illuminated
2. darkfield microscopy
a. uses special filter in condensor to illuminate specimen, leaves background dark
3. phase-contrast microscopy
a. uses annular diaphragm to shift light coming through specimen and background "out of phase"
4. fluorescence microscopy
a. uses UV light and special fluorescent dyes/stains to illuminate specimen
5. electron microscopy
a. scanning EM: electron beam bounces of surface of specimen and produces 3-D surface images
b. transmission EM: electron beam penetrates specimen to show internal structures
c. EM has extremely high resolution, good for viruses and small cellular components.
6. scanning-tunneling and atomic force microscopy
a. uses probes to "scan" surface of specimen, shows resolution down to individual atoms.
II. Specimen Preparation:
A. Smear preparation:
1. small sample of specimen is placed on slide (smaller & more spread out is best)
B. Fixing of specimens:
1. usually heat-fix will adhere specimens to slide for most general staining procedures
2. other methods call for chemical fixing and using precoated "subbed" slides to help adherence
C. Staining of specimens:
1. basic dyes use cationic chromophores, binds to negatively charged cell components
a. more common type of bacterial stains - crystal violet, methylene blue, safranin
2. acidic dyes use anionic chromophores, binds to positively charged cell components
a. acidic dyes usually are repelled by negative bacterial cell charges, stains background instead
3. simple stain - one stain used and one color results
4. differential stain - more than one color results (usually more than one stain used)
a. most common and most useful of staining procedures (Gram stain, acid-fast stain etc.)
b. Gram stain - most important stain in bacteriology
i. four step staining procedure using: Primary stain, Mordant, Alcohol rinse, Counterstain
ii. we will cover details in lab... you're still responsible for knowing technique in lecture though
c. acid-fast stain - important in diagnosing/identifying tuberculosis (Mycobacterium tuberculosis)
5. negative stain - background is stained, specimen is not
a. capsule stain is example, capsule is unstained but cell and background are stained
6. endospore and flagella stain
a. specialized stain to reveal particular structures on specimen.
This is only a general outline.
There may be material that has been discussed in lecture that is not included in this outline
and there may be material on this outline that has not been discussed in lecture.
Any material discussed in lecture or listed in this outline is "fair game" for the test.