Methods Flashcards
Light microscopes are considered…. since they have two lenses
Compound microscopes
Types of compound microscopes
Bright-field
dark field
Lenses that work together to magnify objects
Objective and ocular
Objectives are composed of several lens elements that magnify the object first called the …. which produces….
Primary magnification - real image
the —- is composed of several lens elements that magnify the real image, called the
Ocular … Secondary magnification
The secondary magnification produces as —– which is seen at —-
Virtual image —- eye point
Total Magnification
Product of the objective and ocular lens
Resolving power
Resolution - limits useful magnification for compound microscopes
Stage
Where the specimen is placed
Two adjustment knobs
Course - primary focusing
Fine - secondary focusing
Parfocality
Ability to move from one objective to another while using only fine adjustment knob
Working distance
Distance b/w specimen on the slide and objective lens when in focus
As magnification increases
The field of view decreases
As magnification increases,
More light is needed.
Why is light conserved in oil?
Glass and oil have same refractive indexes
Absorption
Light passes through specimen
Refracted
Light is reflected off specimen
Phasecontrast
Differentiates between specimens of different densities
Fluorescent
Koon’s fluorescent antibody technique
Resolving power
Minimum distance that can exist between two object and still allow the two objects to be seen as separate
Magnification - resolution relationship
As you increase magnification, resolution increases until about 1000x, at which time there is an inverse relationship between the two
Light is the portion of the spectrum of electromagnetic waves that is visible to the human eye and are between
400-700nm
Bright field microscopy technique normally used for
Study of live or preserved/stained material. Common multipurpose scope
Dark field microscope is equipped with ______ condenser and an objective with _______ aperture.
Dark-field
Low numerical
Result of a dark field microscope when viewing:
Only peripheral light reflected off the sides of the specimen enter the objective
Dark field microscopy is best to view
Living, unstained orgs
Examples: syphilis studies because it provides a specimen outline with reduced internal cellular details
Fluorescence microscope
Microscope is furnished with an ultraviolet radiation source and a filter that protects the eyes
Def; Fluorescence
Ability to absorb short wavelengths of light and give off light at a longer wavelength that can be seen with use of special filters
Explain prep for fluorescent scope use
Specimen is first coated with or placed in contact with fluorescent molecule. Stained with fl. Dyes or combined wit fl. Antibodies; illumination by UV radiation causes light to be given off, forming an image
Wide use of fl. Microscopy
Diagnosis of infections caused by certain bacteria, protozoa, fungi and viruses
Phase contract microscope
Equipped with a special condenser contains and annular ring shaped diaphragm.
Diaphragm function on phase contract scope
Allows direct light to pass through condenser focusing light on specimen and a refraction plate in the objective lens. Direct and reflected or diffracted light rays are brought together in phase to form the image of the specimen.
Application of phase contrast
Locating structures within live, unstained cells that are not seen under other scopes
Electron microscopes are
Use electron beams instead of life; all aspects controlled by electromagnets instead of glass. Have greater resolving power b/c of short wavelengths of the electron beams
TEM use
Beam of electrons passed through 20-100nm sections of specimen. Seen on a screen. Contrast can be increased by staining or soaking specimen in metals.
Used to eye detailed structures on preserved material (dead).
TEM reveals
Ultrastructure of cells
SEM
Produces three dimensional image of specimen, offering surface detais
How SEM works
Electron beams scan back and forth over intact, metal coated specimen from the top
Electrons leaving surface of the specimen picked up by detector
Electron patter displayed as image on television screen or photographic film
Resolving power (resolution)
The ability of lenses to distinguish between two points a specified distance apart
Types of preparations for light microscopic examinations
Wet mount/hanging drops: suspension in liquid
Dried, fixed, and stained smears
Wet mount and hanging drop techniques…
What is the quality of the resolution?
Wet mount: place liquid suspension of org on a slide and cover with a slip
Drop: suspension place on cover slip, positioned over a well on depression slide
Poorer than in dyed preparation
Staining techniques offer
Greater resolution because of color contrast
Positive stains use _____ dyes which stain _______
Basic
Cells or cell parts
Simple stains
Positive stain
One dye used to see morphology and cell arrangement
Differential stains
Positive stain
Use more than one reagent to see differentiation between cells and cell parts
Gram stain
Differential stain
Groups gram - and gram + cells
Acid Fast stain
Positive stain
Bacteria that cause tuberculosis and hansen’s disease are acid fast; most others are not
Giemsa stain
Positive stain
Used to detect parasites in blood
Endospore stain
Positive stain
Detects presence of endospores
Capsule stain and Flagella stain
Positive stains
Negative stain
Use acidic dyes that stain background around cells and leave cells colorless; can see shape and arrangement better
Stains have ______ significance
Practical diagnostic
Streak plate method
Inoculum spread over solid culture with loop
Spread plate method
Inoculum spread over surface of a solid medium with spreader
Pour plate method
Inoculum mixed in melted agar medium and poured into sterile petri dish
Enrichment culture
Culture medium used for preliminary isolation that favors the growth of a particular microorganism (blood agar, glucose, etc)
Stock cultures
Preserved for study and reference
Stock culture preservation
Short - refrigeration at 0-10 celsius
Long - liquid nitrogen at -196 celsius or lyophilization
Culture (def)
Observable growth in or on a culture medium
Inoculation
Introduction of a tiny sample of cells into medium that encourages growth
Only dead specimens can be viewed with
Electron microscope
Light microscopes have ….. lenses instead of electromagnetic beams
Glass lenses
TEM magnification
500,000X
SEM magnification
200,000-250,000x
1000-2000x
Light microscopes
Light microscope resolving power
0.2-0.3um
TEM resolving power
0.0001um
SEM resolving power
0.02um
Ultrastructure
Sub microscopic structure
Resolving power of light
Wavelength of light / 2x Numerical Aperture
