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UND 362 instrumentat

UND 362 instrumentation

Name the 9 parts of a microscope bi/monocular eyepiece, objectives (4x etc.), stage, light source, rough focus, fine focus diaphragms, stage dial controls, dimmer switch (light on/off/intensity)
name the 9 standard steps of histolgy accessioning, grossing, processing, embedding, microtomy & drying, stainer, cover slippihng, special staining, review of slides
List 4 common things that are done during accessioning case #'s given, verification of spec types, specimen triage (split for flow/micro), labelling of cassettes and slides
name three types of specimens during accessioning fixed, unfixed (FS), cytology 1. smears (bronch wash/brush 2. cell block (concentrate spec via centrifuge 3. FNA
list 4 common things done during "Grossing" spec prep - fixing, decal, electron microscope prep, quantitative analysis
what are the steps in processing (closed system) fixation (formalin), dehydration (increasing % conc of alcohol, clearing with xylene, infiltration with molten paraffin
What takes place during embedding specs put in molten wax (in specific orientation), additional wax is added and then cooled on cold plate
what are 4 common principles of a microtome most are rotary (moved in 4-6 microns, hold block firmly, sections tissue thinly, created ribbons
What are 4 ways slides can be dried air dry (helps avoid artifacts via lower temp), dryer, incubator, microwave
H/E staining positive and drawbacks to manual and automatic processing manual- inexpensive, reusable containers, but need QC Auto-high volume, need QC but is consistent
Coverslipping pos/negs to manual and auto manual-labor intensive, inexpensive, can lead to chem exposure auto-need daily QC, high throughput
What can special stains do/types ID specific tissue components: carbohydrates, amyloid, nerve, pigments, granules, minerals
IHC (Immunohistochemistry) used two types of modes what are they 1. antibody to antigen attraction for staining (markers for the inclusion or exclusion of a diagnosis 2. In Situ hybridization- use gene probe to locate specific dna/rna and color with chromagen
list 6 types of microscopes light, polarizing, phase contrast, darkfield, flourescence, electrom (TEM -transmission, SEM-scanning)
list the 4 typical objectives of a light microscope 4x (scanning), 10x(intermediate), 40x(hidry), 100x (oil immersion) (the oculars are 10x)
total magnification is determined by oculars (10x) x the objective used =
Koehler illumination is also available on the light microscope but needs? additional optics
what is a polarizing microscope used for? ID of crystals (talc, sylica, urates)
Birefringence and double refraction is? light ray will be split into 2 that emerge from the crystal at different points
how is polarizing achieved polarizer between spec and light source, and another polarizer filter between spec and eye
what is polarized light commonly used to see? tissue stained with congo red (seen as apple green birefringence)
What is a phase contrast microscop commonly used for? for unstained specs and living specs such as protozoa.
What would a darkfield microscope be used for unstained microorganisms (spirochetes)
how is a darkfield micrscope used direct light is excluded and oblique light (light from an angle) is used
Flourescence microscopes are used for what visualization of flourescent antibodies
give 2 primary examples of spec/stain combo that is used with a fourescent microscope acid-fast bacillus w/ auramine rhodamine stain amyloid w/ thioflavin T staining method
How is flourescence achieved and what does the microscope require? light wavelength is absorbed and reemitted as a longer one (typically uv and emitted as visible light) REQUIRES mercury or halogen lamp
name 2 common flourescence dyes Fluorescein isothiocyanate, rhodamine
how is microscope set up for flourescence exciter filter between light and spec transmits desired light wave(not visible light), filter put in eyepiece to absorb UV and only allow visible light
Give 5 specifics for TEM (transmission electron micro) only in large institutions, use beams of electrons in vacuum, 1000x to 500,000x, requires special fixative, use heavy metal stains
What and how is the SEM (scanning elecron micro) used Surface or 3d imaging, back scattered electrons are picked up for image
describe and give 1 pos. and a 2 disadv. for open processors beakers with reagent(s) POS custom design, NEG evaporation and safety issues
Explain how a closed processor functions tissue in resivour and solutions are pumped in and out
give 6 adv to closed processor tissue can't dry out, QC done by instrument, many programs/schedules, heat/vacuum/agitation in one place, alarm system, safety (minimal or no vapors)
give 2 disadv to closed processors and 4 QC properties can't/may not be able to use all reagents, heat and vacuum must be used carefully. QC frequent reagent rotation, fluid above cassettes, paraffin 2-4○ above melt pt., document qc
describe microwave tissue processors and give 3 adv closed system, only large instiutions, expensive ADV- shortens processing time, avoids xlyene use, used only 4 reagents after preprocessing solution
what is the melting point of paraffin in the embedding station 2-4○ above melting point
name the 5 types of microtomes rotary, cryostat, sliding, ultra, clinical freezing
explain rotary microtome most common, hand wheel moves block forward typicall 4-5 microns, routine cleaning needed
what is the typical configuration of the cryostat and its normal temp chill chamber with rotary microtome, 10-25○ below 0○C
what is the typical knife point for the cryostat 30○ (w/ wedge shaped knife)
what is a artifact to avoid with cryostats ice crystals can form if not frozen quickly enough (can use liquid nitrogen to snap freeze which will avoid crystals and therefore holes in the sample)
what is the QC for the cryostat universal precautions, keep clean, decontaminate
explain how/what a clinical freezing microtome is portable cryostat of sorts (uses constant CO2 to freeze)- block is moved up and the knife moves horizontally above it
what are 2 drawbacks to the clinical freezing microtome? poor for friable tissue, airborne debris may result from the C02
how is the sliding microtome used and for what used for research (block and knife are horizontal) - used on celloidin and large paraffin blocks
what type of knives are used in the ultra microtome? diamond or glass knives
what it he ultra microtome used for and how cuts .5 micrometer sections for light microscope and 90nm (ultra thin) for eletron microscope (requires training)
what are the four types of microtome knives steel, disposable, glass, diamond
what are the three shapes of microtome knives wedge, planoconcave and biconcave
What are the wedge/planoconconcave/biconcave knife shapes used for wedge - paraffin, carbowax, frozen, celloidin plano- celloidin biconcave - rarely used
what is the clearance angle on a microtome? wedge angle? cutting facet angle? 3-8, 15-18, 27-32
give three facts about water baths allow paraffin to decompress, high heat causes parched earth, kept at 5-10○ below melting point of paraffin (stretch ribbon gently)
what are 6 types of slide adhesives gelatin, albumin, glue, poly L lysine, charged slides, silane
how is a slide dryer typically used dries paraffin off sections of slides (typically set just above paraffin melting point around 60○)
name the 4 types of stainers manual, linear, revolving, robotic (L/R/R are expensive)
name two types of special stainers Immunohistochemistry (IHC and In Situ), auto "routine" special stainer
describe the method for manual staining use coplin jars in sequential order (drying, deparaffin, rehydration, dye uptake, counterstain, clearing, coverslip)
describe linear stainers transfers slices from one container to the next, can be continually loaded, staining times can ONLY be varied by the # of containers (of each chem), dries the slides
what is the main difference between linear and revolving stainers they are the same but revolving can have its times varied
what are 3 descriptions of a robotic stainer computerized, some do the drying, no particular order for slides since the stainer knows where they need to go
what are 2 properties and 2 adv. of auto coverslippers must be cleaned and QC/glass and plastic covers ADV. hight output and reduces xylene exposure
regarding ph if acid is added what will occur what if a base is added acid = hydroniom ion conc. increase = lower ph base = hydroxy ion conc. increase = higher ph
how do you calibrate a solution use a standard (known spec) that is close to the solution to be calibrated
what temp are incubators typically set at and what are they used for 37○, enzyme stains (not recommended for slide drying)
what are the typical temps for lab fridges and freezers fridge (2-10○C), Freezer (-20○C)
what can fridge and freezers be used for? storing aliquots (acetone and ether must be in explosion proof fridge/freezer)
what is a hydrometer used for measures specific gravity -or- % alcohol
define koehler illumination extremely even illumination of a sample (needs additional optics)
what is auto flourescence naturally occuring fourescence (ie collagen fibers)
define achromatic, apochromatic, parfocal achromatic (corrected for 2 colors) apochromatic (corrected for 3 colors and other lens aberrations) parfocal- all objectives have focal point in same plane and magnification
Created by: mustangvxd



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