Chapter 3: Instrumentation Flashcards
The ability of a microscope to separate small details is defined as:
Resolution.
Sections 90nm thick are commonly cut with a/an:
The ultramicrotome.
Tissues embedded in glycol methacrylate are commonly cut with a/an:
A regular, rotary microtome but with a glass knife to cut 1-2nm sections.
When cutting sections from paraffin blocks, the most common cause of unsatisfactory sections is:
A poor blade edge, it has become dulled.
The regular lab incubator maintains a temperature of:
37C (body temperature).
When the magnification can be changed without the need to refocus, the microscope objectives are said to be:
Parfocal.
If a ribbon splits when cutting paraffin sections, the most likely cause is:
Nicks in the blade edge.
Care must be taken when using the automatic coverslipper to ensure that:
The mounting medium applied is correct for long term storage.
The temperature of the oven used to maintain a supply of melted paraffin for embedding tissue is most commonly about:
60C.
Doubly refractive particles are examined using what instrument?
A polarized light microscope (uric acid crystals).
Crooked paraffin ribbons may be caused by:
Nonparallel block edges, they need to be straight in order to have a non-crooked ribbon.
When using a microscope with a 10X ocular and a 40X objective, what is the total magnification?
400x
What is the routine clearance angle of a microtome blade?
3-8 degrees.
The ordinary freezer when operating normally has an internal temperature of:
-4C and should never exceed 10C.
When a lens for a light microscope has been corrected for 2 colors, it is said to be:
Achromatic (red and blue).
When using paraffin with a melting point of 55-57C, the most common temperature for floating sections would be:
45-50C, water bath temps are typically set 5-10C below the melting point.
What causes compressed or wrinkled sections?
The wrong blade tilt will cause compressed, wrinkled or jammed sections.
The scanning objective on the light microscope is found:
At the lower end of the body tube.
Various sized holes are noted in sections of paraffin embedded liver on the flotation bath. With further ribboning, these holes disappear after decreasing in size. What happened?
The block was faced too aggressively and small pieces of tissue were torn off during rough facing, leaving a hole.
Birefringent substances are best examined with which type of microscope?
The polarizing microscope.
During microtomy, the sections lift from the blade when the block is raised. The most likely cause is:
The blade is either dull or there is not enough blade tilt.
Microscopic evaluation of an H&E reveals chatter, especially at the edges of the tissue. What happened?
The tissue was overdehydrated and was not “soaked” at microtomy.
During cryotomy, sections of varying thickness are obtained. This is most likely the:
Too little blade tilt, it needs to be increased and the parts of the cryostat need to be examined for looseness.
When checking the pH of a staining solution, the pH meter should be calibrated using a standard solution with a pH value:
Closest to that of the staining solution.
The microwave oven creates heat in staining solutions by:
Using nonionizing radiation to produce heat.
Tissue components can be measured with the light microscope in a process known as:
Micrometry.
A microscope that has two eyepieces is called a:
Binocular microscope.
When a lens for a microscope has been corrected for three colors, it is said to be:
Apochromatic.
The “high dry” lens for a light microscope has a magnification of about:
45X.
Tissues are subject to a series of different reagents in a closed processor by:
Fluid transfer.
When processed on a short cycle, tissue must be first:
Cut thin at the grossing station so the reagents can adequately penetrate.
What are the three types of microscopy associated with histotechnology?
Light, polarizing and fluorescent microscopy.
What are the two most common types of EM?
Transmission electron microscopy (TEM) and Scanning electron microscopy (SEM) are the two most common types.
T/F: Living cells are usually examined with a dark field microscope.
False, they are examined with phase-contrast microscopy, not darkfield.
T/F: a 3-D image is obtained with the TEM.
False, the SEM yields a 3-D image, not the TEM.
T/F: Another name for the ocular of a microscope is the eyepiece.
True.
T/F: The analyzer of the polarizing microscope is placed between the specimen and the eye.
True.
T/F: Steel microtome blades are commonly used to section glycol methacrylate.
False, glass knives are usually used for this.
T/F: The angle formed by the block face and the cutting facet of the blade is known as the bevel angle.
False, it is known as the clearing angle.
T/F: When examined microscopically, 8 micron sections will show all nuclei in the same plane of focus.
False. Sections must be cut less than 6 microns in order for the nuclei to all be on the same plane of focus.
T/F: Slow freezing of tissue in the cryostat leads to the formation of ice crystals.
True.
T/F: An overheated cryostat motor is frequently caused by the buildup of ice in the cryostat chamber.
False, it is caused by the buildup of dust on the refrigerant coils.
T/F: If the histotech is skilled, the most critical factor in the lab becomes the care and maintenance of lab instruments.
True.
T/F: Dust collecting on the cooling coils of the cryostat will cause the motor to run more slowly.
False, the dust collection will only cause the motor to overheat.
T/F: Routine maintenance of the microtome requires a thorough cleaning and oiling every two weeks.
False, it should be cleaned every day and oiled per the manufacturer’s instructions.
T/F: The temperature of the room is very important when cutting frozen sections.
False.
T/F: Paraffin sections will not adhere well to uncharged, untreated glass slides.
True.
T/F: Poly-L-lysine is a common additive in the flotation bath.
False, it is used to coat the slides, not add to the flotation bath.
T/F: Water in the flotation bath is a possible source of contamination on sections to be stained for microorganisms.
True.
T/F: New instruments are automatically validated by the manufacturer and may be put into use immediately.
False, they have to be validated by the lab first.
T/F: Household microwaves cannot be used in the lab for processing.
True.
T/F: Bar codes may be linear or 3-D.
False, bar codes are linear and 2-D.
T/F: Most contamination from floaters occurs in the deparaffinization steps of the staining process.
True.
T/F: Side-by-side comparisons are the best form of instrument validation.
True, we do cross lots at work all the time.
T/F: Some staining kits may have the problem of disproportionate solution volumes.
True, like the Steiner kit.
What is the main cause of Venetian blinds/ washboarding/ section undulations?
A loose component in the microtome. The blade clamp and block need to be tightened.
How can you prevent air bubbles in a water bath?
Let the bath stand out overnight to release all the bubbles, or “sweep” them out with a brush.
What can cause “parched earth”?
A ribbon that is floated on a water bath that is too hot or an improperly processed block.
What are the two types of electron microscopy?
Transmission and scanning.
What is a pro for using darkfield microscopy?
Objects appear much larger than they actually are so it is easier to see fine details than with regular light microscopy.
What are two stains that can be demonstrated by staining with fluorescent dyes?
AFB and Congo Red.
When are glass microtome knives useful?
When cutting plastic embedded tissue.
What is a Ralph knife?
A special conformation of glass that is used to cut glycol methacrylate material.
What will cause small irregular holes in the section?
Facing too aggressively.
What will happen to the section if it is excessively dehydrated?
Small holes in the tissue and chatter.
What will cause washboarding or Venetian blinds?
The block or blade wasn’t adequately clamped in or the microtome has to be serviced.
What will cause fragments and tears in the tissue section?
Inadequate processing.
What will cause compressed sections?
A dull blade, too little knife tilt, cutting too rapidly or paraffin accumulation on the blade.
What causes incomplete sections in a cryostat?
The blade is not sharp or the cryostat temperature is not appropriate.
What causes parched earth?
If either the tissue has been improperly fixed or the water bath is too hot.
What happens if too much egg albumin is used?
It can show up as additional staining on the slide.
What causes extreme separation in a tissue section?
Too much time floating on the waterbath.
What can cause a crooked ribbon?
If the block face is not square and straight, the chuck holder needs to be readjusted.
What is a clinical freezing microtome?
An old-school cryostat that isn’t really used anymore. It clamps to the tabletop and is used for certain special stains.
What is the general purpose and benefit of recycling reagents?
They are beneficial for the environment, provide less waste and require the lab to spend less money on additional reagent.
How should muscle biopsies be submitted for diagnosis?
Unfixed, frozen and sectioned at 8-10 microns.
What is the microtome used for EM called?
An ultramicrotome.
During the sectioning of plastic blocks with an ultramicrotome, scratches are seen in the sections. What happened?
The sections are paraffin embedded and cut at 4-5microns and the grit or dirt in the plastic block caused these scratches.
The supervisor noticed that a tech is sectioning by rocking the hand wheel on the microtome. Is this right?
No, rocking the hand wheel on the microtome can lead to carpal tunnel syndrome.
A section of skeletal muscle tissue has been frozen in isopentane and liquid nitrogen but multiple holes are seen in the H&E stained sections. How can this problem be avoided in the future?
Making complete sure that the isopentane is at -150C.
How can sectioning with a cryostat be improved?
Infiltrating the tissue with 30% sucrose before freezing.
When cutting frozen sections, the tissue is frequently becoming detached from the chuck. What is the MOST likely cause of this?
The chuck was too cold when the embedding medium was applied.
“thick” sections for EM or sections to be viewed with the light microscope should be cut at what width?
0.5-1.0 microns.
If chatter is noted on several microscope sections, what all should be checked?
Overdehydrated tissue, an overextended block holder shaft or a dull microtome blade will all cause this.
What is the preferred blade for cutting thin sections of epoxy imbedded tissue?
Diamond knives.
What is a possible con of using coolant sprays during sectioning?
It will cause folds or wrinkles.
What is the most serious artefact in the histology lab?
Contaminant from another tissue source, because it can lead to a false positive or negative.
What is the most common cause of tears and section fragmentation?
Incomplete fixation and processing.
According to the Histology Quality Improvement Program, what is the most common artefact?
Wrinkles and folds.
A fingernail has been fixed in formalin, routinely processed and embedded in paraffin. The tissue is very hard and sections are difficult to obtain. Sectioning quality will be improved and tissue components will be BEST preserved by gently facing the block and then doing what?
Soaking the block in a solution that softens keratin.
When a femoral head is sectioned, the marrow portion sections satisfactorily, but the cortex fragments. What happened?
The bone fragment was underdecalcified.
What is the material of choice for immunoflourescence microscopy and enzyme histochemical studies?
Unfixed frozen sections.
An H&E stained lymph node section reveals overlapping nuclei. What happened?
The section was cut too thick.
The tissue block fails to advance during the preparation of frozen sections. How can this be corrected?
The cryostat needs to be cleaned and oiled.
For good demonstration of myelin sheaths, how should paraffin sections be cut?
10-15 microns.
During microtomy, it is noted that there is a central area that is soft and mushy. What’s wrong?
The tissue needs to be reprocessed, it hasn’t been adequately processed.
Lymph tissue shatters when sectioned in a cryostat maintained at -20C. What happened?
The block needs to be warmed slightly, too cold of a block will shatter.
What problem will be seen in a section that is floated on a waterbath that is too cold?
Wrinkles and folds will be seen.
What is one cause of sections sticking to each other or to parts of the microtome?
Static electricity, get a humidifier.
When frozen sections bunch up at the knife edge and cannot be flattened, what is happening?
A knife that is too warm.
