Instrumentation

Question 1

light microscope

Answer 1

most commonly used in biology labs, also referred to as brightfield microscope.
light seen is transmitted through object being viewed

Question 2

parts of a light microscope

Answer 2

oculars, objetives, condensor, iris diaphragm, filed diaphragm, light source, body tube, base, arm, binocular head, vernier scale, stage.

Question 3

polarizing microscope

Answer 3

used for the ID of crystals and fiction artifacts
reading of some histology stains (amyloid & calcium) are made more specific when this stain is employed

a light microscope may be converted for this is use

Question 4

phase contrasts microscope

Answer 4

used to examine unstained specimens
allows living cells to be seen clearly although almost transparent
a standard microscope can be converted to a phase contrast by changing the condenser and objectives

Question 5

dark field microscope

Answer 5

used for the study of unstained microorganisms
all directly transmitted light is blocked
light which reaches the specimen must be oblique ( on an angle to specimen)
objects appear to be lit against a dark background
used in histopathology

Question 6

fluorescent microscope

Answer 6

used in histo and micro

a substance is bombarded with UV light

used for acid fast bacilli or amyloid fluorescent dyes
used in immunofluorescence

stained preparations are not permanent, photographs are needed for permanent record

requires a special microscope, specific dyes and training

Question 7

electron microscopes (EM) (2 )

Answer 7

transmission : very thin tissue sections transmit or deflect electrons to produce a black and white detailed image

scanning: electric beam scans specimen to release secondary electrons & produces 3-D image

Question 8

resolution of microscope

Answer 8

ability to distinguish objects as separate

light microscope = 0.2 microns
electron microscope = 25 angstroms

Question 9

microtome

Answer 9

instrument for cutting tissue
routine histo labs use rotary microtomes at room temp
can be manual or semi-automatic
block moves ups and down and advances forward by micrometers

Question 10

cryostat

Answer 10

a microtome inside a refrigerator cabinet around -20 degrees
allows specimens to be sectioned in approx. 20 mins; can provide timely info to a surgeon while patient is on the OR table

Question 11

microtome maintenance

Answer 11

cover when not in use
clean after use to remove excess paraffin ( use mineral oil bc xylene is more toxic)
lubricate weekly

Question 12

microtome safety

Answer 12

safety lock stops movement of block holder
always remove blade before cleaning
place guard over knife before leaving machine
clean away debris with brush

Question 13

sliding microtome

Answer 13

not used routinely
used for sectioning celloidin
used for large paraffin blocks

Question 14

clinical freezing microtome

Answer 14

forerunning of cryostat which we use today
not used routinely
a sliding microtome which was clamped to bench top, CO2 was carried with it for freezing

Question 15

microtome blades

Answer 15

condition has more effect on quality of sections than any other single factor

sharp and free of defects

may be high or low profile ( distance from share edge to blunt edge, discard after use

heavy knifes requiring sharpening were stringer than disposables and are used for some specialized procedures

Question 16

what blade is used for Electron microscopy

Answer 16

glass or diamond knives, these specimens are embedded in plastic resin rather than paraffin

Question 17

microtome quality factors

Answer 17

blocks are chilled

• cold plate, ice with water ( need for moisture indicate over dehydration)
• sometimes warm water helps with cutting especially CNS

knife edge
- free of nicks, gouges, scratches or debris

alignment
- block and knife edge must be parallel

tight clamp on knife and clock
- vibrations will cause imperfections

correct thickness
- dependent of procedure

speed of cutting
- generally one turn per second

clearance angle
- proper angle prevents compressions in cut sections as the knife edge passes through the block

hardness of tissue

• very hard tissue may need to be softened
• friable ( easily fractured) tissue may be soaked in water or 10% acetic acid

rough “facing” of block
- trimming block

hardness of wax
- wax can be purchased with different additives

temperature of room
- extremes of hold or cold will affect quality

hard spots in tissue
- calcified areas or staples may tear tissue and damage knife edge

stability

• tighten clamps, use stable surface for microtome
• ensure block holder is not too far advanced

Question 18

what is the correct thickness of a section

Answer 18

generally 4-5 microns

Question 19

recommended clearance angle

Answer 19

2-4 degrees for paraffin sections ( we start at 2.5)

5-7 for resin or frozen sections ( set to 5.0)

Question 20

cryostat

Answer 20

frozen sections ( tissue hardens from freezing)

• urgent : patient in OR results required for surgical procedure
• substance destroyed by processing (enzymes for example are rendered inactive by tissue processing)

unfixed tissue carries infectious risk
- mask, gloves,disinfectant, autoclaving of contaminated materials

anti-roll device

tissue firmness related to water/lipid

• colder for fat containing tissues ( tissue collects on blade if too warm)
• fractures or splits if too cold

usually set at -20 degrees

freezing artifacts may affect the quality of spections produced

Question 21

tissue processors (4)

Answer 21

open ( tissue transfer), closed ( fluid transfer); microwave or continuous throughput
- open involves moving tissue from one solution to the next

• closed involves keeping tissue stationary and moves solutions in and out ( most common)
• microwave fixation speed the process and uses less reagent ( usually manual reagent changing is required)
• continuous throughput processors, allows more specimens to be added continuously during the work day and includes microwaves

temp and pressure can accelerate the process

fluids must be higher than tissue in cassettes

cassettes can’t be packed too tightly; fluid needs to flow though freely

reagent must be monitored and rotated

wax is maintained at 2-4 degrees above melting point

use of microwave processing can be used to improve workflow and TAT but guidelines for processing tissue for breast cancer receptor testing have not been validated using microwave processing

Question 22

automatic stainers

Answer 22

amalgamation of services has created very high volume laboratories. automation is more and more prevalent

staining is more constant, slower and expensive

two general types: linear and robotic

linear staining moves slides from one container to the next but time fora solution can only be increased by adding additional containers

robotic stainers allow for exact staining times

most Hematoxylin & Eosin ( H&E) is preformed using automation

depending on laboratory volumes, automated stainers may be used for special stains and immunohistochemistry staining

fume control is often incorporated

Question 23

microwave staining

Answer 23

staining often requires use of heat
microwaves is one way to provide heat
may be used to speed up: fixation, processing , decalcification, drying of slides, antigen retrieval

Decreases TAT by may be worse processed slides
not suitable for all staining method
use of an agitator prevents “hot spots”

Question 24

microwave safety

Answer 24

**avoid heating formalin or xylene !

keep clean

don’t use flammable or explosive materials in microwave

glass must be tempered

check periodically for radiation leakage