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Lab

Equipment

QuestionAnswer
Simple vs compound microscope? One lens vs two (light scope)
Scanning lens magnification? x2.5 to x4
Intermediate lens magnification? x10 to x20
High-powered dry lens magnification? x40 to x45
Oil immersion lens magnification? x90 to x100
4 common light microscope objectives? Scanning, intermediate, high-powered dry, oil immersion
What is resolving power? Closest two objects can be & still appear separate
What is chromatic aberration? Distortion of color in image due to lenses being incorrectly angled
Total magnification = Objective magnification X oculars magnification
Polarizing microscope used to? ID crystals (talc, silica, urates); amyloid stained w/ Congo red
What is used to view substances exhibiting double refraction, anisotropism, & birefringence? Polarizing microscope
What is birefringence? Property of refractive index being dependent on polarization & propagation of light
Convert light scope to polarizing by? Analyzer between specimen & eye, polarizer between light source & specimen
Anisotropic or birefringent objects appear? Bright against dark background
Phase-contrast microscope used to? View unstained, esp. living, specimens, transparent objects
Dark-field microscope used to? Study unstained microorganisms
Dark-field microscope works by? Excluding direct light, only scattered/oblique light, small objects appear larger & luminous
Fluorescence microscope works by? Emitting light λ that's absorbed by substance & reemitted as longer λ
Fluorescence scope light source? Mercury or halogen lamp, exciter filter between light & specimen, barrier filter in eyepiece to absorb UV
Primary/autofluorescence means? Substance naturally fluoresces, e.g. collagen
What is a fluorochrome? Dye that fluoresces if excited by UV light
What is auramine-rhodamine? Fluorescent dye for acid-fast bacilli
What is thioflavin T? Fluorescent dye for amyloid
Two types of electron microscopy? Transmission & scanning
Transmission microscopy works by? Specimen transmitting/deflecting electrons; 2D black & white image on fluorescent screen
Scanning microscopy works by? electron beam causes specimen to release electrons, 3D image, great depth of focus
Transmission microscopy great for? Diagnosis of kidney dieases, tumor ID
4 types of microtomes? Rotary, sliding, ultra, clinical freezing
Ultramicrotome section thicknesses? 0.5 μm plastic for light, 90 nm for EM
Rotary microtome mechanism & block type Block moves past knife, frozens, paraffin, GMA
Sliding microtome mechanism & block type Knife moves past block, large paraffin blocks, celloidin
Clinical freezing microtome Mostly replaced by cryostat, bad for friable tissue, easier free--floating sections, use CO2 to freeze blocks, infectious agent inhalation hazard
Wedge-shaped knife used to section? Paraffin, Carbowax, frozens
Planoconcave knife used to section? Celloidin, blade must be kept wet
Average paraffin knife clearance angle? 3° to 8°
What is a cryostat? Refrigerated chamber containing (usually rotary) microtome
Optimum cryostat knife tilt? 30°
Average cryostat operating temp? -20°C, colder for fat, warmer for brain, liver, spleen, lymph node, endometrial scrapings
Slides must be completely dry before deparaffinization because? Water contaminates xylene, incomplete deparaffinization, white spots in tissue
Incubator temperatures? 37°C, body temp; for enzyme rxns, special stains
Fridge/freezer temperatures? 4°C to 10°C / -20°C
Linear stainers Move slides from one container to next, same time each container
Revolving stainers Move slides from one solution to next, can set times
Robotic stainers Allow total programming, slides can go in containers in any order
Diamond knives Used for v. hard resin
Glass knives Used for resin, e.g. Spurr's, make just before use
Disposable knives Used for paraffin, GMA, frozens, high profile - used w/ firm, difficult tissues, or range of tissues, low profile - Cryostat & soft tissues
Steel knives No longer used in histology, sharpened by hand
Bevel angle aka facet angle, angle on tip of microtome blade
Common section thicknesses Routine 3-5 µm (~1 cell thick), some biopsies 2 µm, brain & nervous 8 µm
Cryostat should be cleaned with? 70% ETOH
Cryostat blades should be changed: Between cases to avoid risk of exposure & cross-contamination
Created by: CCF