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Instruments Chapter 3

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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