Instrumentation Flashcards
Describe Polarizing Microscopy
Used to examine tissue for substances exhibiting the phenomena of double refraction, anisotropism, and birefringence
Describe Phase-contrast microscopy
Used for the examination of unstained specimens, especially unstained living cells, and allows almost transparent objects to be seen clearly. Can be made by replacing the condenser and objectives with special phase equipment
Describe Darkfield Microscopy
Directly transmitted light is excluded, and only scattered or oblique light is used. Objects will appear self-luminous against a dark background as well as appearing much larger - allowing fine structures to be seen much more easily. Primarily used for the study of unstained microorganisms and for silver grains in radioactive staining procedures
Describe Fluorescence microscopy
A substance is bombarded with short-wavelength light in the UV, violet, or blue range and visible light is emitted
State the most common objectives used in light microscopy and the approximate magnification of each.
○ Scanning lens: x2.5 to x4
○ Intermediate lens: x10 to x20
○ High-powered dry lens: x40 to x45
○ Oil immersion lens: x90 to x100
Describe the method of determining total magnification.
Multiply the magnification of the ocular and the objective
x10 oculars and x45 objective = x450 total magnification
Define: clearing agent
Removes alcohol from tissues and is replaced by fluid which is miscible with wax with which tissue must be impregnated
Define: Bevel angle
Angle made when the 2 cutting facets of a microtome knife intersect. Standard 28-32 degrees
Define: Resolution
The optical ability to distinguish 2 objects a minimal distance apart as 2 objects. The light microscope has a resolving power of 0.2um (objects closer than 0.2um can no longer be distinguished as 2 separate objects and will be seen as 1 object)
Define: Ocular
The eye piece of the microscope, or the lens found at the upper end of the barrel or body tube
Define: Objective
The lens at the lower end of the barrel, or body tube, of the microscope. Several objectives, yielding different magnifications, are found on most microscopes
Define: Micrometry
The measurement of minute distances with the microscope
Define: Microtomy
The cutting of thin sections of tissue for microscopic examination
Define: Microscopy
Study of the microanatomy of cells, tissues, and organs as seen through a microscope
Define: Achromatic
Without color, or not easily stained. Microscopy - achromatic lenses are corrected for 2 color, red and blue = helps produce images free from chromatic aberrations.
Define: Apochromatic
Corrected for both spherical and chromatic aberration.
Define: Binocular
Having 2 oculars (2 eye pieces on a microscope)
Define: Parfocal
In the same plane of focus. Microscope objectives are said to be parfocal when they can be changed without the need to refocus.
List 3 types of microtomes.
Rotary, sliding, and clinical freezing
Identify the use of the rotary microtome.
Operates with a screw feed or a computerized motor; the block moves up and down, and either the blade holder or the block advances a preset number of micrometers with each revolution of the wheel. Most commonly used for glycolmethacrylate and paraffin embedded material
Identify the use of the sliding microtome.
Used for sectioning celloidin and large paraffin blocks
Not used for routine histopathology
Identify the use of the clinical freezing microtome.
(has been replaced by the cryostat)
It is easier to obtain free-floating sections required for some special stains than with the cryostat
Describe routine microtome maintenance.
- Clean the microtome thoroughly at the end of each day or shift by carefully removing all accumulated paraffin with a soft brush or soft cloth moistened with xylene and then drying the microtome thoroughly
- If the model requires, apply microtome oil or grease to all sliding parts as indicated by the manufacturer
- Document the service, repair, or routine preventative maintenance performed.
- Cover the microtome when it is not in use.
Describe glass knives (microtome blades).
Used for cutting sections of plastic embedded material (Ralph knives are used to section glycol methacrylate embedded material, not discussed further)
Describe disposable microtome blades.
Standard in labs, made of austenitic stainless or carbon steel with or without various coatings and found in high and low profile form.
Define and describe clearance angle on the microtome.
Blade tilt - routinely 3-8 degrees
○ Clearance angle is too slight: results in thick/thin sections, wrinkled
○ Clearance angle is too great: cause chatter, or washboarding
What is the cause & corrective action of crooked ribbons?
Cause: the horizontal edges of the block are not parallel
Corrective action:
○ Ensure the upper and lower block edges are parallel
○ Ensure the lower block edge is parallel to the cutting edge of the blade
○ Ensure there are no problems with the blade edge
○ Ensure the block is evenly chilled
What is the cause & corrective action of block face unevenly sectioned?
○ Cause: 1 side of the block is exhausted while attempting to get a complete section of the block face because the block holder was not parallel to the blade prior to sectioning
○ Corrective Action: Ensure the block holder is adjusted so that the block face and the blade are perfectly parallel (use of calibration tools to align the chuck with the blade provides a standardized method to avoid unevenly faced sections
What is the cause & corrective action of holes in the section?
Cause: block is faced too aggressively
Corrective action: face the block to expose the tissue then soak, face less aggressively, cut until the holes disappear
*Liver, brain, and lymph nodes are especially prone to this artifact
What is the cause & corrective action of failure to form ribbon?
Cause: a dull blade, paraffin that is too sticky (not enough plastic) or too hard (too high a melting point), too much blade tilt, improper room temperature
Corrective Action: choose a paraffin with lower melting point, decrease blade tilt, change room temp
What is the cause & corrective action of lifting of the section from the blade as the block is raised?
Cause: dull blade, too little blade tilt
Corrective Action: increase blade tilt (greater clearance angle), change to harder paraffin (higher melting point)
What is the cause & corrective action of water trapped underneath tissue during section pick-up onto slide?
Cause: Incorrect technique when mounting tissue from the floatation bath (scooping)
Corrective action: mount sections using a pulling method by positioning the slide at an angle of about 30 degrees and then pulls the slide forward to pick up the section
What is the cause & corrective action of washboarding or undulations in the section?
Cause: very hard tissue (uterus), over-fixed tissue, worn out microtome parts, loose clamping of the blade or block
Corrective Action: ensure paraffin is filled on the top of the cassette to provide support, ensure block and blade are tightly clamped, ensure block holder shaft is not overextended, decrease blade tilt (smaller clearance angle)
*Can be easily seen when sections are the water bath
What is the cause & corrective action of chatter in the section?
Cause:
Corrective Action:
What is the cause & corrective action of thick and thin sections?
Cause:
Corrective Action:
What is the cause & corrective action of compressed, wrinkled, or jammed section?
Cause:
Corrective Action:
What is the cause & corrective action of lengthwise scratches or splits in the ribbon?
Cause:
Corrective Action:
What is the cause & corrective action of fragmented or torn sections?
Cause:
Corrective Action:
Identify 4 artifacts that may occur during section flotation.
○ Parched Earth (exploding)
○ Wrinkles/folds
○ Bubbles
○ Contamination with a tissue from another source
○ Squamous cell contamination from an ungloved operator
Describe the mechanisms of closed tissue-processing systems
○ The tissue is stationary and fluids are pumped in and out of the pressurized chamber holding the tissue
○ Can assist with keeping exposure to toxic vapor to a minimum
○ Specimens cannot dry out in the tissue chamber (retort) in the event of a malfunction
Identify critical factors in processing tissue on a short cycle
Only very thin, small, well fixed tissue sections of a standard size will process well on a short cycle.
Describe the role of vacuum in processing tissue.
Vacuum can speed up processing
Correlate maintenance of all laboratory instruments with quality control and/or quality assurance.
Discuss the principle of pH and .
○ Used to measure the acidity, or H+ ion concentration of a solution
○ pH scale goes from 1-14 (acidic - basic, respectively)
○ The addition of acid increases the hydronium/hydrogen (H+) ion concentration and the pH decreases, is a base is added the hydroxyl ion concentration increases and the pH increases
Each unit on the pH scale represents a 10-fold change in ion concentration
Discuss the use of pH meter.
○ Measures pH with a voltage output in proportion to the active acid concentration using a sensing electrode and a reference electrode
○ pH meters are standardized with buffer solutions whose composition has been defined by the US National Bureau of Standards
○ Necessary for enzyme histochemistry techniques because most solutions require a specific pH before use
List 3 section adhesives and state why the use of adhesive may be necessary
○ Albumin, poly-L-lysine, aminoalkylsilane, chrome alum solution
○ Use of adhesive may be necessary to avoid section wash-off during staining
Describe the principle of microwave-generated heat.
Microwave radiation (a nonionizing radiation) is produced through the interaction of strong magnetic and electrical fields. The microwave electrical field that is produced creates friction as it passes through dipolar molecules (water) and polar side-chains of proteins, causing them to reverse themselves 2.45 billion times per second - creating heat.
Describe linear automatic stainers.
○ Transfer slides from 1 container to the next with the same time allowed in each container
○ The time in each different solution can be changed only by varying the number containers holding that particular reagent, or some modules provide smaller buckets to reduce time
○ Slides may be continuously loaded
Describe robotic automatic stainers.
○ Allows total computerized programming; allows for exact staining times in any given station
○ Reduce chemical exposure by using filters and by the ability to be vented
Poorly adjusted antiroll plate: identify the cause and corrective action.
○ Cause: Plate that is too warm will cause the sections to stick; if the plate is too far above the blade edge sections will rub the plate
○ Corrective Action: Ensure the antiroll plate is parallel to and very slightly above the blade facet
Incomplete sections: identify the cause and corrective action.
○ Cause: a dull blade or incorrect cryostat temperature for the tissue being sectioned
○ Corrective Action: Ensure blade is sharp and the cryostat is at the appropriate temperature
Describe the basic function and benefits of recycling reagents.
○ The recycling system works by boiling off the impurities, such as water or other chemicals, separating contaminants from the solvent being recycled, and restoring it to its original composition .
○ Can be used for alcohol, xylene, and xylene substitutes to prepare them for reuse multiple times.
○ Recycling can be a cost-saving measure for the lab by reducing both expense of reagent purchases and the disposal of used solvents, while having a positive impact on the environment.
List steps that might decrease tissue carryover in staining baths.
○ Ensure complete fixation
○ Mount sections on quality plus (+) slides
○ Drain and wick slides before drying
○ Ensure mounted sections are completely dry before staining
○ Change deparaffinization solutions frequently
○ Change or filter other solutions daily
Describe how a microwave processor can be useful.
Describe how a microwave staining oven can be useful.
List the equipment in the histopathology lab that employs temp control. Sate the proper temp for each.
