Chapter 3: Instrumentation

Question 1

The ability of a microscope to separate small details is defined as:

Answer 1

Resolution.

Question 2

Sections 90nm thick are commonly cut with a/an:

Answer 2

The ultramicrotome.

Question 3

Tissues embedded in glycol methacrylate are commonly cut with a/an:

Answer 3

A regular, rotary microtome but with a glass knife to cut 1-2nm sections.

Question 4

When cutting sections from paraffin blocks, the most common cause of unsatisfactory sections is:

Answer 4

A poor blade edge, it has become dulled.

Question 5

The regular lab incubator maintains a temperature of:

Answer 5

37C (body temperature).

Question 6

When the magnification can be changed without the need to refocus, the microscope objectives are said to be:

Answer 6

Parfocal.

Question 7

If a ribbon splits when cutting paraffin sections, the most likely cause is:

Answer 7

Nicks in the blade edge.

Question 8

Care must be taken when using the automatic coverslipper to ensure that:

Answer 8

The mounting medium applied is correct for long term storage.

Question 9

The temperature of the oven used to maintain a supply of melted paraffin for embedding tissue is most commonly about:

Answer 9

60C.

Question 10

Doubly refractive particles are examined using what instrument?

Answer 10

A polarized light microscope (uric acid crystals).

Question 11

Crooked paraffin ribbons may be caused by:

Answer 11

Nonparallel block edges, they need to be straight in order to have a non-crooked ribbon.

Question 12

When using a microscope with a 10X ocular and a 40X objective, what is the total magnification?

Answer 12

400x

Question 13

What is the routine clearance angle of a microtome blade?

Answer 13

3-8 degrees.

Question 14

The ordinary freezer when operating normally has an internal temperature of:

Answer 14

-4C and should never exceed 10C.

Question 15

When a lens for a light microscope has been corrected for 2 colors, it is said to be:

Answer 15

Achromatic (red and blue).

Question 16

When using paraffin with a melting point of 55-57C, the most common temperature for floating sections would be:

Answer 16

45-50C, water bath temps are typically set 5-10C below the melting point.

Question 17

What causes compressed or wrinkled sections?

Answer 17

The wrong blade tilt will cause compressed, wrinkled or jammed sections.

Question 18

The scanning objective on the light microscope is found:

Answer 18

At the lower end of the body tube.

Question 19

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?

Answer 19

The block was faced too aggressively and small pieces of tissue were torn off during rough facing, leaving a hole.

Question 20

Birefringent substances are best examined with which type of microscope?

Answer 20

The polarizing microscope.

Question 21

During microtomy, the sections lift from the blade when the block is raised. The most likely cause is:

Answer 21

The blade is either dull or there is not enough blade tilt.

Question 22

Microscopic evaluation of an H&E reveals chatter, especially at the edges of the tissue. What happened?

Answer 22

The tissue was overdehydrated and was not “soaked” at microtomy.

Question 23

During cryotomy, sections of varying thickness are obtained. This is most likely the:

Answer 23

Too little blade tilt, it needs to be increased and the parts of the cryostat need to be examined for looseness.

Question 24

When checking the pH of a staining solution, the pH meter should be calibrated using a standard solution with a pH value:

Answer 24

Closest to that of the staining solution.

Question 25

The microwave oven creates heat in staining solutions by:

Answer 25

Using nonionizing radiation to produce heat.

Question 26

Tissue components can be measured with the light microscope in a process known as:

Answer 26

Micrometry.

Question 27

A microscope that has two eyepieces is called a:

Answer 27

Binocular microscope.

Question 28

When a lens for a microscope has been corrected for three colors, it is said to be:

Answer 28

Apochromatic.

Question 29

The "high dry" lens for a light microscope has a magnification of about:

Answer 29

45X.

Question 30

Tissues are subject to a series of different reagents in a closed processor by:

Answer 30

Fluid transfer.

Question 31

When processed on a short cycle, tissue must be first:

Answer 31

Cut thin at the grossing station so the reagents can adequately penetrate.

Question 32

What are the three types of microscopy associated with histotechnology?

Answer 32

Light, polarizing and fluorescent microscopy.

Question 33

What are the two most common types of EM?

Answer 33

Transmission electron microscopy (TEM) and Scanning electron microscopy (SEM) are the two most common types.

Question 34

T/F: Living cells are usually examined with a dark field microscope.

Answer 34

False, they are examined with phase-contrast microscopy, not darkfield.

Question 35

T/F: a 3-D image is obtained with the TEM.

Answer 35

False, the SEM yields a 3-D image, not the TEM.

Question 36

T/F: Another name for the ocular of a microscope is the eyepiece.

Answer 36

True.

Question 37

T/F: The analyzer of the polarizing microscope is placed between the specimen and the eye.

Answer 37

True.

Question 38

T/F: Steel microtome blades are commonly used to section glycol methacrylate.

Answer 38

False, glass knives are usually used for this.

Question 39

T/F: The angle formed by the block face and the cutting facet of the blade is known as the bevel angle.

Answer 39

False, it is known as the clearing angle.

Question 40

T/F: When examined microscopically, 8 micron sections will show all nuclei in the same plane of focus.

Answer 40

False. Sections must be cut less than 6 microns in order for the nuclei to all be on the same plane of focus.

Question 41

T/F: Slow freezing of tissue in the cryostat leads to the formation of ice crystals.

Answer 41

True.

Question 42

T/F: An overheated cryostat motor is frequently caused by the buildup of ice in the cryostat chamber.

Answer 42

False, it is caused by the buildup of dust on the refrigerant coils.

Question 43

T/F: If the histotech is skilled, the most critical factor in the lab becomes the care and maintenance of lab instruments.

Answer 43

True.

Question 44

T/F: Dust collecting on the cooling coils of the cryostat will cause the motor to run more slowly.

Answer 44

False, the dust collection will only cause the motor to overheat.

Question 45

T/F: Routine maintenance of the microtome requires a thorough cleaning and oiling every two weeks.

Answer 45

False, it should be cleaned every day and oiled per the manufacturer's instructions.

Question 46

T/F: The temperature of the room is very important when cutting frozen sections.

Answer 46

False.

Question 47

T/F: Paraffin sections will not adhere well to uncharged, untreated glass slides.

Answer 47

True.

Question 48

T/F: Poly-L-lysine is a common additive in the flotation bath.

Answer 48

False, it is used to coat the slides, not add to the flotation bath.

Question 49

T/F: Water in the flotation bath is a possible source of contamination on sections to be stained for microorganisms.

Answer 49

True.

Question 50

T/F: New instruments are automatically validated by the manufacturer and may be put into use immediately.

Answer 50

False, they have to be validated by the lab first.

Question 51

T/F: Household microwaves cannot be used in the lab for processing.

Answer 51

True.

Question 52

T/F: Bar codes may be linear or 3-D.

Answer 52

False, bar codes are linear and 2-D.

Question 53

T/F: Most contamination from floaters occurs in the deparaffinization steps of the staining process.

Answer 53

True.

Question 54

T/F: Side-by-side comparisons are the best form of instrument validation.

Answer 54

True, we do cross lots at work all the time.

Question 55

T/F: Some staining kits may have the problem of disproportionate solution volumes.

Answer 55

True, like the Steiner kit.

Question 56

What is the main cause of Venetian blinds/ washboarding/ section undulations?

Answer 56

A loose component in the microtome. The blade clamp and block need to be tightened.

Question 57

How can you prevent air bubbles in a water bath?

Answer 57

Let the bath stand out overnight to release all the bubbles, or "sweep" them out with a brush.

Question 58

What can cause "parched earth"?

Answer 58

A ribbon that is floated on a water bath that is too hot or an improperly processed block.

Question 59

What are the two types of electron microscopy?

Answer 59

Transmission and scanning.

Question 60

What is a pro for using darkfield microscopy?

Answer 60

Objects appear much larger than they actually are so it is easier to see fine details than with regular light microscopy.

Question 61

What are two stains that can be demonstrated by staining with fluorescent dyes?

Answer 61

AFB and Congo Red.

Question 62

When are glass microtome knives useful?

Answer 62

When cutting plastic embedded tissue.

Question 63

What is a Ralph knife?

Answer 63

A special conformation of glass that is used to cut glycol methacrylate material.

Question 64

What will cause small irregular holes in the section?

Answer 64

Facing too aggressively.

Question 65

What will happen to the section if it is excessively dehydrated?

Answer 65

Small holes in the tissue and chatter.

Question 66

What will cause washboarding or Venetian blinds?

Answer 66

The block or blade wasn't adequately clamped in or the microtome has to be serviced.

Question 67

What will cause fragments and tears in the tissue section?

Answer 67

Inadequate processing.

Question 68

What will cause compressed sections?

Answer 68

A dull blade, too little knife tilt, cutting too rapidly or paraffin accumulation on the blade.

Question 69

What causes incomplete sections in a cryostat?

Answer 69

The blade is not sharp or the cryostat temperature is not appropriate.

Question 70

What causes parched earth?

Answer 70

If either the tissue has been improperly fixed or the water bath is too hot.

Question 71

What happens if too much egg albumin is used?

Answer 71

It can show up as additional staining on the slide.

Question 72

What causes extreme separation in a tissue section?

Answer 72

Too much time floating on the waterbath.

Question 73

What can cause a crooked ribbon?

Answer 73

If the block face is not square and straight, the chuck holder needs to be readjusted.

Question 74

What is a clinical freezing microtome?

Answer 74

An old-school cryostat that isn't really used anymore. It clamps to the tabletop and is used for certain special stains.

Question 75

What is the general purpose and benefit of recycling reagents?

Answer 75

They are beneficial for the environment, provide less waste and require the lab to spend less money on additional reagent.

Question 76

How should muscle biopsies be submitted for diagnosis?

Answer 76

Unfixed, frozen and sectioned at 8-10 microns.

Question 77

What is the microtome used for EM called?

Answer 77

An ultramicrotome.

Question 78

During the sectioning of plastic blocks with an ultramicrotome, scratches are seen in the sections. What happened?

Answer 78

The sections are paraffin embedded and cut at 4-5microns and the grit or dirt in the plastic block caused these scratches.

Question 79

The supervisor noticed that a tech is sectioning by rocking the hand wheel on the microtome. Is this right?

Answer 79

No, rocking the hand wheel on the microtome can lead to carpal tunnel syndrome.

Question 80

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?

Answer 80

Making complete sure that the isopentane is at -150C.

Question 81

How can sectioning with a cryostat be improved?

Answer 81

Infiltrating the tissue with 30% sucrose before freezing.

Question 82

When cutting frozen sections, the tissue is frequently becoming detached from the chuck. What is the MOST likely cause of this?

Answer 82

The chuck was too cold when the embedding medium was applied.

Question 83

"thick" sections for EM or sections to be viewed with the light microscope should be cut at what width?

Answer 83

0.5-1.0 microns.

Question 84

If chatter is noted on several microscope sections, what all should be checked?

Answer 84

Overdehydrated tissue, an overextended block holder shaft or a dull microtome blade will all cause this.

Question 85

What is the preferred blade for cutting thin sections of epoxy imbedded tissue?

Answer 85

Diamond knives.

Question 86

What is a possible con of using coolant sprays during sectioning?

Answer 86

It will cause folds or wrinkles.

Question 87

What is the most serious artefact in the histology lab?

Answer 87

Contaminant from another tissue source, because it can lead to a false positive or negative.

Question 88

What is the most common cause of tears and section fragmentation?

Answer 88

Incomplete fixation and processing.

Question 89

According to the Histology Quality Improvement Program, what is the most common artefact?

Answer 89

Wrinkles and folds.

Question 90

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?

Answer 90

Soaking the block in a solution that softens keratin.

Question 91

When a femoral head is sectioned, the marrow portion sections satisfactorily, but the cortex fragments. What happened?

Answer 91

The bone fragment was underdecalcified.

Question 92

What is the material of choice for immunoflourescence microscopy and enzyme histochemical studies?

Answer 92

Unfixed frozen sections.

Question 93

An H&E stained lymph node section reveals overlapping nuclei. What happened?

Answer 93

The section was cut too thick.

Question 94

The tissue block fails to advance during the preparation of frozen sections. How can this be corrected?

Answer 94

The cryostat needs to be cleaned and oiled.

Question 95

For good demonstration of myelin sheaths, how should paraffin sections be cut?

Answer 95

10-15 microns.

Question 96

During microtomy, it is noted that there is a central area that is soft and mushy. What's wrong?

Answer 96

The tissue needs to be reprocessed, it hasn't been adequately processed.

Question 97

Lymph tissue shatters when sectioned in a cryostat maintained at -20C. What happened?

Answer 97

The block needs to be warmed slightly, too cold of a block will shatter.

Question 98

What problem will be seen in a section that is floated on a waterbath that is too cold?

Answer 98

Wrinkles and folds will be seen.

Question 99

What is one cause of sections sticking to each other or to parts of the microtome?

Answer 99

Static electricity, get a humidifier.

Question 100

When frozen sections bunch up at the knife edge and cannot be flattened, what is happening?

Answer 100

A knife that is too warm.