Instrumentation Flashcards
light microscope
most commonly used in biology labs, also referred to as brightfield microscope.
light seen is transmitted through object being viewed
parts of a light microscope
oculars, objetives, condensor, iris diaphragm, filed diaphragm, light source, body tube, base, arm, binocular head, vernier scale, stage.
polarizing microscope
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
phase contrasts microscope
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
dark field microscope
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
fluorescent microscope
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
electron microscopes (EM) (2 )
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
resolution of microscope
ability to distinguish objects as separate
light microscope = 0.2 microns
electron microscope = 25 angstroms
microtome
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
cryostat
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
microtome maintenance
cover when not in use
clean after use to remove excess paraffin ( use mineral oil bc xylene is more toxic)
lubricate weekly
microtome safety
safety lock stops movement of block holder
always remove blade before cleaning
place guard over knife before leaving machine
clean away debris with brush
sliding microtome
not used routinely
used for sectioning celloidin
used for large paraffin blocks
clinical freezing microtome
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
microtome blades
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
what blade is used for Electron microscopy
glass or diamond knives, these specimens are embedded in plastic resin rather than paraffin
microtome quality factors
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
what is the correct thickness of a section
generally 4-5 microns
recommended clearance angle
2-4 degrees for paraffin sections ( we start at 2.5)
5-7 for resin or frozen sections ( set to 5.0)
cryostat
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
tissue processors (4)
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
automatic stainers
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
microwave staining
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”
microwave safety
**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
