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Instrumentation
Instruments Chapter 3
| Question | Answer |
|---|---|
| compound microscope | Light microscope |
| The lens system consist of objectives and oculars | light microscope |
| provide image maginification and image resolution | Objective lens |
| Scanning lens, intermediate lens, high powered dry lens and immersion lens | types of objective lens |
| will give an image surrounded by color fringes | Uncorrected lens also know as chromatic aberration |
| are corrected for two colors red and blue and are found on most lab microscopes | achromatic objectives |
| are corrected for three colors and for other lens aberrations | Apochromatic objectives |
| are more expensive and not necessary for routine use. used for photomicrography | Apochromatic objectives |
| a defect in lens system | aberration |
| eyepiece | oculars |
| have a x10 mag and x5 oculars are frequently used on student microscope x15 oculars preferred by microscopist | oculars |
| is determined by multiplying the mag of ocular w/the objective | total mag obtained w/a microspe |
| substage found below can be moved up/down | substage |
| on modern microscopes consists of a condenser and iris diaphragm | substage |
| function primarily to concentrate light on the tissue section | condenser |
| should be centered accurately | condenser |
| regulated by iris diaphragm and should be varied w/t different objectives | amount of illumination |
| should be adjusted so that peripheral light rays are blocked and light passing through the tissue should be limited so that it fills the front lens of the objective | iris diaphragm |
| keep covered when not in use, clean lens w/lens paper, remove immersion oil immediately after use | microscope maintenance |
| use xylene on the ofectives as a last resort, dont dismantle objectives, | microscope maintenance |
| when using immersion oil becareful, make sure that the high powered dry lens inst dragged through the oil | microscope maintenance |
| reduce light t/a minimum or turn it off when not in use. remove slides from stage when not in use | microscope maintenance |
| always focus upwards, never downwards, especially w/t higher power objectives | microscope maintenance |
| do not touch lens surfaces w/fingers | microscope maintenance |
| used in identification of crystals. Talc, silica or urates | polarizing microscope |
| used in identification of amyloid stained w/congo red | polarizing microscope |
| used to examine tissue for substances exhibiting the phenomena of double refraction, anisotropism and birefringence | polarizing microscope |
| transmitting light unequally in different directions | birefringence |
| having unlike properties in different directions | anisotropism |
| is achieved by interposing a polarizing device between the light source and specimen and inserting a second polarizing filter between the specimen and the eye | polarized light |
| vibrates in many planes | natural light |
| vibrates only in one plane | light emerging from the polarizer |
| can be converted to a polarizing microscope by placing one piece of polaring film on top of light source and another on top of the microscope slide. | light microscope |
| through this microscope amyloid stained w/congo red, will show apple green birefringence | polarizing microscope |
| used for examination of unstained specimens | phase-contrast microscope |
| used to examine unstained living cells and allows transparent objects to be seen | phase-contrast microscope |
| can be converted to a phase-contrast microscope by replacing the condenser and objective w/special phase equipment | a standard binocular microscope |
| not used in routine histology | phase-contrast microscope |
| directly transmitted light is excluded | dark field microscope |
| only scattered or oblique light is utilize | dark field microscope |
| objects appear much larger than they are, bcuz of their light scattering properties and frequently fine structures | dark field microscope |
| used for the study of unstained microorganisms and is rarely used in routine histopathology | dark field microscope |
| phenomeonon in whic light of one wavelenght is absorbed by as substance and instantly remitted as light of a longer wavelength | fluorescence microscope |
| substance is bombarded w/short-wavelenght light in the ultraviolet, violet or blue range and visible light is emitted | fluorescence microscope |
| two types transmission and scanning | electron microscope |
| the specimen either transmits electrons producing electron lucent or clear areas in the image | transmission electron microscope |
| the specimen deflects electrons (producing electron-dense, or dark area in the image | transmission electron microscope |
| a two-dimensional black and white is seen on fluorescent screen | transmission electron microscope |
| cells and ultrastructure of the cells can be appreciated w/ | transmission electron microscope |
| useful in dx of kidney disease and in tumor identification | transmission electron microsocpe |
| has replaced the electron microscope, in tumor diagnosis | immunoperoxidase |
| a dramatic three-dimensional image | scanning electron microscope |
| the electron beam sweeps the surface of the specime and releases secondary electrons | scanning electron microscope |
| has a great depth of focus | scanning electron microscope |
| very expensive, requires skilled operator | scanning electron microscope |
| found in large instistutions | scanning electron microscope |
| Rotary, sliding and freezing microtomes | most commonly used microtomes in histology |
| used for sectioning glycol methacrylaten and paraffin embedded material | rotary microtome |
| operates w/a screw feed | rotary microtome |
| block moves up/dowm and either the knife or block advances a preset number of micrometers w/each revolution of the wheel | rotary microtome |
| used for sectioning celloidin and large paraffin blocks | sliding microtome |
| block held stationary and the knife is moved along a horizontal plane past the block face | sliding microtome |
| not used in routine histology | sliding microtome |
| has been replaced by the cryostat | clinical freezing microtome |
| free-floating sections for some special stains are easier to obtain with this than with the cryostat | clinical freezing microtome |
| a sharp knife must have and edge free of defects, is essential to obtain a good section | microtome knifes |
| successful sectioning of poorly processed tissue is possible w/a | good knife |
| nondiagnostic sections m/b obtained w/a | poor knife |
| used for cutting plastics | glass knifes |
| used to section glycol methacrylate embedded materials | ralph knifes |
| used for sectioning, paraffin, celloidin, carbowax and frozen tissue | microtome knives |
| made from high-grade tempered steel and are wedge-shaped, biconcave or planoconcave | microtome knives |
| used for paraffin, carbowax, and frozen sections | wedge-shaped knife |
| preferred for celloidin | planoconcave knife |
| requires both block and knife to be kept wet | the wet celloidin method |
| planoconcave knife can be used for this method | the wet celloidin method |
| used for early hand sectioning | bioconcave knife |
| has been replaced by the plane wedge knife | bioconcave knife |
| rarely used | bioconcave knife |
| wedge angle 15 to 18 | microtome knife |
| 27 to 32 | bevel angle |
| condition of knife is critical, c/b determined w microscopic examination w/x100 mag | mictrome knife |
| are stainless steel razor blades that are held secure in a holder on the microtome | disposable blades |
| used to cut ultrathin sections for EM | diamond knifes |
| should be discarded in punture-proof containers | disposables blades and glass knifes |
| if used in cryostat, they need to be autoclaved before disposal | disposable blades |
| knife tilt | clearance angle |
| when cutting sections, angle should be 3 to 8 | clearance angle (knife tilt) |
| should be determined with each knife | clearance angle (knife tilt) |
| c/b corrected by adjusting the knife so that the correct clearance angle between knife and specimen is obtained | irregular, skipped or thick and thin sections |
| horizontal edges of block are not parallel | ribbons are crooked |
| mictoromy artifact | holes in sections |
| should be performed less aggresively | block facing |
| when holes appear in ribbons, an there is sufficient tissue in block, | ribbons should be cut until holes disappear |
| dull knife | ribbon will not form |
| depends on enough heat being generated by friction occurring as each section is cut to cause the sections to adhere to each other | ribbon formation |
| caused by dull knife or too little knife tilt | section lifts from the knife as the block is raised |
| warm room or paraffin to soft | section lifts from the knife as the block is raised |
| occurs in very hard tissue like uterus | washboarding or undulations |
| occurs when a block or knife has not been tightly clamped | washboarding or undulations |
| too much knife tilt or worn microtome parts | washboarding or undulations |
| overdehydration or lack of moisture | chatter, microscopic vibration appear in sections |
| treating faced block for a few sections with wet cotton, or dip thumb in flotation bath and rugh the block | to remove chatter, or microscopic vibration |
| dull knife, too much tilt, or cutting to rapidly | microscopic chatter |
| too little knife tilt | sections are skipped or vary in thickness |
| dull knife or gummed w/paraffin | sections are compressed, wrinkled or jammed |
| too little knife tilt, too rapid cutting or too warm room | sections are compressed, wrinkled or jammed |
| keep free of paraffin by wipping up w/a gause damped w/xylene (nerver wipe down | knife edge |
| defect in knife | lenghtwise scratches or splits in ribbons |
| static electricity, add moisture to air by breating on block and knife or boil water in open pan near microtome. | section flies or sticks to nearby objects |
| refrigerated chamber containing a microtome (rotary) | cryostat |
| cooled by mechanical regrigeration | cryostat |
| sections are picked up directly onto slide | cryostat |
| knife m/b sharp and edge free of defects | cryostat |
| wedge-shaped knife is commonly used. | cryostat |
| antiroll devices are designed for this knife | wedge-shaped knife |
| operated at -20c | cryostats |
| slow freezing allows water present in the tissue to form | ice crystals |
| are seen as artifacts | ice crystal formation |
| open and closed | two types of processors |
| tissue is transported from one solution to the next | open processors |
| tissue is stationary and fluids are pumped in and out of the chamber | closed processors |
| are computerized w/a digital readout to indicate the exact instrument status | closed processors |
| major advantage is that specimen cannot dry out w/i the chamber | closed processors |
| disadvantage is that not all reagents c/b used on these processors | closed processors |
| mercury, dichromate containing fixatives and chloroform, cant b/used on this processor | closed processors |
| heat and vacumm | closed processors |
| speed up processing | heat and vacumm |
| use carefully on bx specimens | heat and vacumm |
| m/b kept clean and a routine reagent rotation | processors |
| fluid levels m/b higher than tissue | processors |
| temp of paraffin m/b carefully adjusted to no more than 2C to 4C above melting point | processors |
| temp m/b monitored and recorded daily | processors |
| used to float out ribbons | flotation baths |
| maintained at 5C to 10C below the melting point of paraffin used for embedding | flotation baths |
| ribbons should be stretched gently, small wrinkles and folds should be teased out | flotation baths |
| a mixture of egg white and glycerin w/elmers glue | albumin |
| gellatin, agar and elmers glue | water bath additives |
| poly-L lysine-coated slides | used for frozen sections, some to be stained in microwave and paraffin sections to be stained w/immunoenzyme technique |
| postive charged attracts frozen and paraffin tissue sections elctrostatically | positive charged slides |
| no adhesive needed | positive charged slides |
| background staining is eliminated and section loss w/staining procedures is reduced | positive charged slides |
| fairly expensive and not recommended for routine work | positive charged slides |
| dissolve 1g of gelatin in 1L of distilled water w/heat. cool and add.1g of cromium potassium sulfate, store in fridge. | subbed slides |
| after section is mounted on slide | it should be dried in the oven, hot air dryer or warming plate |
| slides are dried to | remove water |
| white spots that can be seen in tissue when slides are removed from xylene | incomplete deparaffination |
| maintain a temp just above the melting point of paraffin | dryers and ovens |
| can be used for overnight drying of immunoperoxidase slides but temp must be kept below 60C | ovens |
| are maintained at 37C | incubators |
| used for enzyme reactions and for some special stains | incubators |
| used for storing may of the reagents used in histology, enzyme histochem reagents, ihc reagents, and buffer solutions | freezers and refrigeratos |
| maintained at about 4C and should not be allowed to reach a temp above 10C | The refrigerator |
| -20C | Freezer temp |
| for IHC surface markers of lymphoid tissue or estrogen/progesterone receptor the freezer should be at what temp | -70C |
| are used for performing special stains, for fixation, processing, drying of sides and other procedures | microwave ovens |
| is dependent on exposure of time, solution volume and oven wattage | heat generated by microwave |
| linear,revolving and robotic | automatic stainers found in histo |
| transfer slides from one container to the next w/t same time allowed in each container | linear stainer |
| also dries slides | linear stainer |
| operate on the same prinicple as open tissue processor | revolving stainer |
| are the most flexible stainer | robotic stainers |
| allow computerized programming w/ability to return slides to same container used in a previous step | robotic stainers |
| vary in slide capacity but slides may be continously loaded | linear stainer |
| are batch stainers, one batch of slides m/b finisher before the next one is started | robotic and revolving stainers |
| used to measure acidity. | pH meters |
| scale goes from 0 to 14 | pH scale |
| defined as netgative power to which the number 10 m/b raised to express the moles per liter concentration of solutions hydrogen ions | PH |
| has melted paraffin w/warm storage for embedding molds, small warming and chilling plates, and larger chiller plate for rapid chilling of embedded tissue blocks | embedding center |
| paraffin is kept 2C to 4C above melting point of parffing used | embedding center |
Created by:
nperez
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