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Instrumentation

Instrumentation in the histology lab

QuestionAnswer
Simple microscope (1 lens) Light microscope
2 simple microscopes used to examine tissue (Magnifying lens system) Compound microscope
Consists of an objective lens and an ocular lens Compound microscope
Most common objectives found on a histopathogy microscope x2.5-x4 (Scanning lens) x10-x20 (Intermediate lens) x40-x45 (High powered dry lens) x90-x100 (oil immersion lens)
Increasing the magnification without detail Empty magnification
Resolving power is defined as The ability to reveal fine detail or to discriminate between adjacent details
Resolving power is measured as The least distance between 2 objects at which the objects can still be discerned as 2 separate structures rather than a single blurred object
With a light microscope objects must be separated by at least _ micrometers to be seen as 2 separate structures 0.2 micrometers
Chromatic Aberration Colors are refracted at different angles, which allows them to have a different point of focus. An uncorrected lens will give an image surrounded by color fringes
Parfocal All objectives have a focal point in the same plane (Objectives can be changed without having to refocus the microscope)
Iris diaphragm Adjusts the amount of light see on the microscope
Condenser Concentrates light on the tissue section
Identifies crystals (Talc, silica or urates) Polarizing microscope
Identifies Amyloid with help of the Congo Red stain Polarizing microscope
Used to examine tissue substances that are exhibiting double refraction, an isotropism, and bifringence. Polarizing microscope
Bifringence Transmits light unequally in different directions
Anisotropism Having unlike properties in different directions
Achieved by interposing a polarizing device (polarizer) between the light source and the specimen, then inserting a second polarizing filter (analyzer) between the specimen and the eye. Polarized illumination
Polarized light vibrates in how many planes? One
This type of tissue exhibits intrinsic bifringence Collagen
Collagen will appear as this color when polarized after being stained with Congo Red Silver
Used for the examination of unstained specimens (Especially unstained living) Phase contrast microscope
Directly transmitted light is excluded and only scattered or oblique light is used in this type of microscopy Darkfield
Structures appear larger with this microscope (Good for fine structures) Darkfield microscope
To be examined by this type of microscope, sections must be thin and free of refractive material (air bubbles, oil, RBCs) Darkfield microscopy
Primarily used for unstained organisms Darkfield microscopy
Light of 1 wavelength is absorbed by a substance and almost instantly reemitted as light of a longer wavelength Fluorescence microscope
Mercury or halogen lamps are used as the light source Fluorescence microscope
Excited filter Placed between the light source and specimen to produce a specific wavelength; obscures all visible light
Barrier filter Placed in the eyepiece to absorb UV light and reduces non specific fluorescence
Collagen fibers _ naturally Fluoresce
FITC Fluorescein isothiocyanate
Can be demonstrated by use of fluorescent dyes Acid fast bacilli and amyloid
Auramine-rhodamine Acid-fast bacilli
Thioflavin T Amyloid
Thioflavin S Plaques and tangles
Subject to fading Fluorescent stains
Electron microscope magnification range 1,000-500,000 diameters
Replaces regular light source with an electron gun Electron microscopy
Transmission Electron Microscopy 2-D display of tissue
Very useful in the diagnosis of kidney and muscle diseases Electron microscopy
Scanning Electron Microscopy 3-D display of tissue
Retracting microtome is used for Plastics
Vibrating microtome is used for tissues that can't be frozen or embedded
Most frequent types of microtomes in the lab Rotary, sliding, and clinical freezing
Ultramicrotome Cuts 0.5 micrometer plastic sections for light microscopic orientation
Retracting microtome Cuts 90 nanometer sections to electron microscopy
Sliding Microtome Block is held stationary and the knife is moved along a horizontal plane past the block face
Used to section Celloidin and large paraffin blocks Sliding microtome
Provides free floating sections Clinical freezing microtome
Not good for friable tissue Clinical freezing microtome
Relatively portable Clinical freezing microtome
Glass knives are used to cut plastic embedded material
Glass (Ralph) knives are used to cut Glycol methacrylate material
Two types of disposable blades High profile and low profile
Used disposable knives or glass should be disposed of in... A puncture proof container
Blades or glass contaminated with human tissue from the cryostat or with paraffin should be disposed of how? Placed into a special biohazard "sharps" container that will later be incinerated
Section quality is determined mainly by the condition of the_ and the _ angle more than any other factor. edge and the clearance angle
Clearance angle 3-8 degrees
For H&E stains the section thickness should be how thick 3-5 micrometers
Bone marrow and kidney biopsies are cut at 2-3 micrometers
Too much or too little clearance angle can result in irregular, skipped, or excessively thick or thin section
A dull blade can result in Grooved, scored, smeared, and deformed sections
Lengthwise or vertical scratches are typically caused by A defect in the blade, calcium, or other hard materials sometimes found in tissue
Mushy sections result from Insufficient dehydration or clearing
If the tissue is mushy what can you do to correct this Back up the tissue to the dehydrating agent and then reprocessing may help. However, reprocessing the tissue will rarely be much of an improvement and will compromise the molecular or immunohistochemistry to follow.
Crooked ribbons Horizontal edges of the block are not parallel to the blade edge when sectioning(Top and bottom)
Ways to correct crooked ribbons -Ensure the block is evenly chilled -Check for imperfections in the blade -Ensure horizontal edges are parallel
Block face unevenly sectioned Can cut through one side of the tissue quicker than the other
Ways to prevent unevenly sectioning the block face Ensure block holder is parallel to the blade
Holes in section Occur when the block is faced too aggressively or if all of the air is not displaced from the tissue during infiltration
Liver, brain, lymph nodes are especially prone to this Holes in section
Ways to prevent holes in section -Soak the block in ice -Cut the tissue till the holes disappear -Face the block less aggressively -Face large brain sections at 5 micrometers -Know the type of tissue
Failure of ribbon to form Most commonly caused by a dull blade. But can also occur from paraffin that is too sticky (not enough plastic) or too hard (too high a melting point), too much blade tilt, or room temp that is too high or low
Ways to prevent a ribbon from not forming -Choose paraffin with a low melting point -Decrease tilt of the blade (smaller clearance angle) -Change room temp
Lifting of the section from the blade as the block is raised Frequently cause by dull blade or too little blade tilt
To prevent the section lifting from the blade -Increase the tilt of the knife -Change to a harder paraffin
Washboarding or undulations Commonly occurs in hard tissue such as uterus or in over fixed tissue
Chatter seen macroscopically Washboarding
Washboarding can occur from Worn microtome parts and loose clamping of the blade or block
Washboarding can be prevented by -Ensuring the blade and block are tightly clamped -Ensuring the paraffin is filled on the top of the cassette to provide adequate support -Checking that the block shaft is not over extended -Ensure microtome is working properly -Decrease blade tilt
Chatter or microscopic vibration Primarily from over dehydration. Can also be from dull blade, too much blade tilt, and cutting too rapidly
Chatter can be prevented by -Re evaluating the processing cycle -Restoring moisture to tissue -Decrease tilt of blade -Decrease cutting speed
Skipped or varied thickness of section Can be caused from too little blade tilt
Alternating thickness of sections can be prevented by -Increasing blade tilt -Ensure microtome is working properly
Compressed wrinkled of jammed sections Commonly cause from dull blade or blade gummed with paraffin. Also, too little knife tilt, too rapid cutting, or too warm of a room
Compressed sections can be prevented by -Keeping the paraffin from building up on block holder -Ensuring that a sharp blade is in use; change if necessary -Increase knife tilt -Decrease cutting speed
Lengthwise scratches or splits in the ribbon Defect in the blade or hard particle in the block
Ways to prevent lengthwise scratches -Avoid metal objects that may damage blade -Ensure paraffin is not collecting on the front or back of blade -Move to new area of blade -Surface decal if needed
Fragmented or torn section Incomplete fixation or poor processing, over stretching on the water bath, mechanical tearing, mechanical tearing with instruments, or water bath temp too high
Sections flying or sticking to nearby objects or other parts of the microtome Static electricity
Ways to prevent static electricity -Add moisture to the air -Ionize the air
Cryostat Refrigerated chamber containing a microtome
Tilt of the blade edge for the Cryostat should be around 30 degrees
Cryostats operate at around -20 degrees Celsius
Brain, liver, spleen lymph nodes, and endometrial scrapings section better on a cryostat at what temp? -10 degrees Celsius
Tissue containing fat such as breast requires much colder temp -25 degrees Celsius or lower
Slow freezing of tissue cause _ to form. When the tissue is defrosted later the artifact leaves behind holes in the tissue. This can be horrible in the diagnosis of muscle diseases especially. ice crystals
Isopentane and liquid nitrogen Preferred reagents to freeze specimens
CAP requires the cryostat to be cleaned how often Once a week
Poorly adjusted anti roll plate If plate is too warm, sections will stick; if it is too far above the blade edge, sections will rub the plate; if it is damaged, sections may catch on damaged area if it is too far below blade edge sections will not slide between the plate and blade
Incomplete sections on a cryostat Dull knife or incorrect temp to cut specimen at
Flotation baths temp 5-10 degrees below melting point of paraffin
Methods to get tissue to stick to slide Chromium potassium sulfate covered slides Poly-L-lysine coated slides Aminoalkylsilane treated slides Postively charged slides Glue added to the water bath Agar
Sources of contamination on the slide (Typically fungi or acid fast organisms) Water and adhesives
If flotation bath is contaminated with the debris of an unfixed tissue exploding, it is recommended that you do what? Dump the water, clean the water bath, and re fill with clean water
Chemicals that lower the surface tension TritonX-100 or Brij-35
Chemicals that reduce the surface tension do what to the tissue sections? Flatten the sections on the water bath
Other ways to flatten the tissue sections better -Use two water baths with different temps -Lay the ribbons in alcohol before placing on the water bath
High temps may cause Nuclear bubbling
Incomplete removal of paraffin is indicated by White spots on tissue as the slides are removed from xylene
If white spots occur on tissue from residual xylene what should be done to correct this? Slides should be treated with absolute alcohol and then placed in xylene once again. Then the slides can be run down starting with 100% to 95% and then water to be stained.
Dryers and ovens are typically maintained at what temp? 60 degrees Celsius
Incubator ovens are kept at what temp? 37 degrees Celsius
For patient safety, a cassette should have how many types of identification present? At least two (Name and surgical number for example)
Fridges are typically maintained at this temp 4 degrees Celsius
Fridges should never reach a temp of <10 degrees Celsius
Typical lab freezer operates at this temp -20 degrees Celsius
Freezers can store concentrated antibodies when at this temp -70 degrees Celsius
pH scale represents A 10 fold change in hydronium or hydroxyl ion concentration
What do the p and H stand for in pH p=negative power (the number 10 must be raised to express moles per liter concentration of the solutions hydrogen ions) H=Moles per liter concentration of solutions hydrogen ions
When preparing the acidulated water for Warthin-Starry stain pH should be calibrated to what? 4.0
Paraffin in the embedding center should be kept how many degrees above melting point? 2-4 degrees Celsius
If paraffin gets too hot when embedding what happens? The nature of the paraffin changes and the resultant sectioning qualities are affected
High temps can destroy what? Tissue and nuclear morphology
Created by: Ziek98