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