Instrumentation

Question 1

Describe Polarizing Microscopy

Answer 1

Used to examine tissue for substances exhibiting the phenomena of double refraction, anisotropism, and birefringence

Question 2

Describe Phase-contrast microscopy

Answer 2

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

Question 3

Describe Darkfield Microscopy

Answer 3

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

Question 4

Describe Fluorescence microscopy

Answer 4

A substance is bombarded with short-wavelength light in the UV, violet, or blue range and visible light is emitted

Question 5

State the most common objectives used in light microscopy and the approximate magnification of each.

Answer 5

○ Scanning lens: x2.5 to x4
○ Intermediate lens: x10 to x20
○ High-powered dry lens: x40 to x45
○ Oil immersion lens: x90 to x100

Question 6

Describe the method of determining total magnification.

Answer 6

Multiply the magnification of the ocular and the objective
x10 oculars and x45 objective = x450 total magnification

Question 7

Define: clearing agent

Answer 7

Removes alcohol from tissues and is replaced by fluid which is miscible with wax with which tissue must be impregnated

Question 8

Define: Bevel angle

Answer 8

Angle made when the 2 cutting facets of a microtome knife intersect. Standard 28-32 degrees

Question 9

Define: Resolution

Answer 9

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)

Question 10

Define: Ocular

Answer 10

The eye piece of the microscope, or the lens found at the upper end of the barrel or body tube

Question 11

Define: Objective

Answer 11

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

Question 12

Define: Micrometry

Answer 12

The measurement of minute distances with the microscope

Question 13

Define: Microtomy

Answer 13

The cutting of thin sections of tissue for microscopic examination

Question 14

Define: Microscopy

Answer 14

Study of the microanatomy of cells, tissues, and organs as seen through a microscope

Question 15

Define: Achromatic

Answer 15

Without color, or not easily stained. Microscopy - achromatic lenses are corrected for 2 color, red and blue = helps produce images free from chromatic aberrations.

Question 16

Define: Apochromatic

Answer 16

Corrected for both spherical and chromatic aberration.

Question 17

Define: Binocular

Answer 17

Having 2 oculars (2 eye pieces on a microscope)

Question 18

Define: Parfocal

Answer 18

In the same plane of focus. Microscope objectives are said to be parfocal when they can be changed without the need to refocus.

Question 19

List 3 types of microtomes.

Answer 19

Rotary, sliding, and clinical freezing

Question 20

Identify the use of the rotary microtome.

Answer 20

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

Question 21

Identify the use of the sliding microtome.

Answer 21

Used for sectioning celloidin and large paraffin blocks
Not used for routine histopathology

Question 22

Identify the use of the clinical freezing microtome.

Answer 22

(has been replaced by the cryostat)
It is easier to obtain free-floating sections required for some special stains than with the cryostat

Question 23

Describe routine microtome maintenance.

Answer 23

1. 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
2. If the model requires, apply microtome oil or grease to all sliding parts as indicated by the manufacturer
3. Document the service, repair, or routine preventative maintenance performed.
4. Cover the microtome when it is not in use.

Question 24

Describe glass knives (microtome blades).

Answer 24

Used for cutting sections of plastic embedded material (Ralph knives are used to section glycol methacrylate embedded material, not discussed further)

Question 25

Describe disposable microtome blades.

Answer 25

Standard in labs, made of austenitic stainless or carbon steel with or without various coatings and found in high and low profile form.

Question 26

Define and describe clearance angle on the microtome.

Answer 26

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

Question 27

What is the cause & corrective action of crooked ribbons?

Answer 27

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

Question 28

What is the cause & corrective action of block face unevenly sectioned?

Answer 28

○ 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

Question 29

What is the cause & corrective action of holes in the section?

Answer 29

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

Question 30

What is the cause & corrective action of failure to form ribbon?

Answer 30

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

Question 31

What is the cause & corrective action of lifting of the section from the blade as the block is raised?

Answer 31

Cause: dull blade, too little blade tilt Corrective Action: increase blade tilt (greater clearance angle), change to harder paraffin (higher melting point)

Question 32

What is the cause & corrective action of water trapped underneath tissue during section pick-up onto slide?

Answer 32

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

Question 33

What is the cause & corrective action of washboarding or undulations in the section?

Answer 33

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

Question 34

What is the cause & corrective action of chatter in the section?

Answer 34

Cause: Corrective Action:

Question 35

What is the cause & corrective action of thick and thin sections?

Answer 35

Cause: Corrective Action:

Question 36

What is the cause & corrective action of compressed, wrinkled, or jammed section?

Answer 36

Cause: Corrective Action:

Question 37

What is the cause & corrective action of lengthwise scratches or splits in the ribbon?

Answer 37

Cause: Corrective Action:

Question 38

What is the cause & corrective action of fragmented or torn sections?

Answer 38

Cause: Corrective Action:

Question 39

Identify 4 artifacts that may occur during section flotation.

Answer 39

○ Parched Earth (exploding) ○ Wrinkles/folds ○ Bubbles ○ Contamination with a tissue from another source ○ Squamous cell contamination from an ungloved operator

Question 40

Describe the mechanisms of closed tissue-processing systems

Answer 40

○ 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

Question 41

Identify critical factors in processing tissue on a short cycle

Answer 41

Only very thin, small, well fixed tissue sections of a standard size will process well on a short cycle.

Question 42

Describe the role of vacuum in processing tissue.

Answer 42

Vacuum can speed up processing

Question 43

Correlate maintenance of all laboratory instruments with quality control and/or quality assurance.

Question 44

Discuss the principle of pH and .

Answer 44

○ 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

Question 45

Discuss the use of pH meter.

Answer 45

○ 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

Question 46

List 3 section adhesives and state why the use of adhesive may be necessary

Answer 46

○ Albumin, poly-L-lysine, aminoalkylsilane, chrome alum solution ○ Use of adhesive may be necessary to avoid section wash-off during staining

Question 47

Describe the principle of microwave-generated heat.

Answer 47

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.

Question 48

Describe linear automatic stainers.

Answer 48

○ 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

Question 49

Describe robotic automatic stainers.

Answer 49

○ 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

Question 50

Poorly adjusted antiroll plate: identify the cause and corrective action.

Answer 50

○ 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

Question 51

Incomplete sections: identify the cause and corrective action.

Answer 51

○ 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

Question 52

Describe the basic function and benefits of recycling reagents.

Answer 52

○ 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.

Question 53

List steps that might decrease tissue carryover in staining baths.

Answer 53

○ 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

Question 54

Describe how a microwave processor can be useful.

Question 55

Describe how a microwave staining oven can be useful.

Question 56

List the equipment in the histopathology lab that employs temp control. Sate the proper temp for each.