Sectioning or Cutting Flashcards
Tissue should be surrounded by at least ________ of wax.
2mm
Process of removing of wax after embedding.
Trimming
Trimming using Knife
Coarse Trimming
Usual Thickness for
Paraffin sections
Celloidin Section
Frozen Sections
Ultrathin Sections
4-6 micra
10-15 micra
10 micra
0.5-1.0 micra
After cutting sections are floated out on a water bath ___ set approximately _____ C lower than the melting point of the wax to flatten and prepare for mounting into a slide.
(45-60C)
6-10
use microtome, setting the adjustment at 15mm. Surface is trimmed away until the tissue surface has been exposed.
Fine Trimming
Tissue is cut in uniformly thin sections using microtome to facilitate studies under the microscope.
Sectioning/cutting
for flattening of sections. Temperature is 45ºc.
Water Bath
prolong time of tissue in the water will cause tissue expansion and distortion. Approximately ____ seconds is enough.
30
for drying of sections. Temperature is lower than the melting point of the paraffin wax.
Hot plate or drying oven-
Methods of drying slides
Wax oven 56-60C 2 hours
Incubator 37C overnight
Hot plate 45-55 45 minutes
Bunsen Burner (URGENT)
majority of sections fit comfortably on a 76 x 25 x1.2 mm slide
Slides
most common microtome, for paraffin embeddeed tissues
Rotary microtome
needed to remove folds and creases in sections after floating out.
Brush, seeker, forceps
equiment for specimen labeling
Diamond pencil or pencil
microtome for large paraffin embedded tissue
Rocking microtome
microtome-for celloidin -embedded tissue
Base-sledge microtome
microtome for electron microscopy
Ultrathin microtome
microtome for frozen section
freezing microtome
a chamber that houses the freezing microtome. It maintains a cold temperature.
Cryostat
it is used for paraffin and frozen sections.
Plane Wedge
it is recommended for paraffin section cutting on rotary and sledge type of microtome
Biconcave
used for celloidin section since the blade is thin it will vibrate when for used for other harder materials.
Plane concave
: this is used with a heavy microtome for cutting very hard tissues like undecalcified bone.
Tool Edge
Reason and Remedy for
Brittle or hard tissue
Prolonged fixation, dehydration, clearing, infiltration.
Over heated paraffin oven
Drying of tissue before fixation
Tissue is soak in small dish with detergent, phenol or Molliflex (tissue softener)
Reason and Remedy for
Clearing agent turns milky as soon as tissue is placed in it.
Incomplete dehydration
Repeat dehydration with absolute alcohol then repeat clearing.
Reason and Remedy for
Curved, crooked, or un even ribbon instead of straight
Blunt or dull spot of the knife.
Unparalleled knife edge, knife not parallel to the block, impure paraffin,
Sharpen knife, adjust knife relative to block, re-trim block, repeat impregnation with pure wax.
Reason and Remedy for
Tissue smells of clearing during trimming
Insufficient infiltration
Block trimmed until tissue exposed remaining wax is melted in oven, repeat infiltration
Reason and Remedy for
Tissue is opaque causing difficult in cutting (presence of alcohol)
Insufficient clearing
Repeat clearing, if tissue has bee embedded clear up to 12 hours then re embed.
Reason and Remedy for
Sections roll up adhere and get broken against the edge of the knife.
Blunt knife, dirty knife edge, tilt of knife is too great.
Sharpen the knife, reduce the tilt, clean the knife edge.
Reason and Remedy for
Fail to form ribbons during cutting
Unparalleled surface and edges of the block. Too hard paraffin wax, knife too tilted, too thick sections, dull knife.
re trim the block, re adjust re orient the block, reduce tilt, re adjust thickness of the section, sharpen knife.
Reason and Remedy for
Compressed, wrinkled, jammed sections
Blunt or dull knife, knife edge coated with wax, soft and warm block, too thin sections, microtome set screw is too loose.
Re sharpen knife, cool block until firm, clean knife edge, re adjust thickness, tighten screw. Reduce tilt
Reason and Remedy for
Hole formed in the section
Bubbles or dirt in the embedding medium, calcium in the tissues.
Re embed with freshly filtered wax, base sledge knife with plane wedge knife.
Reason and Remedy for
Frozen tissue crumbles and comes off the block holder when cut
Inadequate freezing
Re freeze tissue block
Reason and Remedy for
Sections adhere to the knife or other parts of the microtome
Static electricity due to low atmospheric humidity, dirty knife edge, dull knife edge, too great knife great
Breath out or blow gently on the block or knife to break up static electricity, or boil water to increase humidity, clean knife edge, sharpen knife, reduce tilt
Reason and Remedy for
Frozen tissue chips into fragment
Over freezing of tissue
Warm the tissue with finger.
Reason and Remedy for
Horizontal or parallel lines across sections (CHATTERS) are seen resulting to thick and thin zones
Vibration of knife due to hard tissue, knife tilt is too great.
Treat phenol during processing, reduce tilt.
Process of putting cover slid on stained tissue using mounting medium to stick the cover to slide. Its purpose is to facilitate handling and prevent damage to tissue.
Mounting
Components of mounting
Gelatin, Glycerin jelly, gum Arabic
Glycerol
Sugar
Component of mounting used to solidify medium
Gelatin
Glycerin Jelly
Gum Arabic
Component of mounting to prevent cracking and drying of the preparation
Glycerol
Component of mounting to raise the refractive index (RI)
Sugar
Mounting media RI should be equal to RI of glass slides and coverslip which is about ____
1.52.
Composition and Refractive Index of Glycerin Jelly
Gelatin
Glycerol
D. water
Phenol Crystals
1.47
Composition and Refractive Index of apathy’s medium
Pure gum arabic
Cane sugar or sucrose
D. water
Thymol Crystals
1.52
Composition and Refractive Index of Farra’s medium
Gum arabic
D. water
Glycerol
Na methiolate
1.43
Composition of Brun’s Fluid
Glucose
Glycerine
Spirits of camphor
D. Water
Resinous mounting media refractive index of
Canada Balsam
DPX
XAM
Clarite
1.524
1.532
1.52
1.544
Process of sealing the edges of the coverslip to prevent escape of mounting media and evaporation. It is done to immobilize the coverslip and avoid sticking of slides upon storage.
Ringing
it is an effective way to preserve the integrity of the mount.
Ringing
Process of indicating the year and specimen number (accession number) for proper identification.
Labelling
The quality of sections and quality of staining produced by histopathology must be checked before issuing them to pathologist.
Quality control
Quality control for quality of sections
Broken slides
slides stuck together
Restaining of old sections
If the broken is only on the near end far from the tissue, re affix broken fragment with a ___
cellophane tape.
If the slide is broken into several pieces and if enough pieces are present.
Reconstitute the pieces on a blank slide using mounting medium as adhesives
Wipe them gently with xylene
Dry the slide flat on a hot plate
If the cover slip is damaged, removed the broken pieces with ___ and apply new coverslip.
xylene
Soak the slides in a dish of xylene to dissolved the sticky mounting media.
When the slides are separated, clean and replace coverslip as needed.
Quality control- disengage stuck glass slides.
Quality control- old section staining
Slides is immersed in xylene for ____ or gently heated until mounting medium begins to ____
Coverslip maybe removed by lifting it with ___.
Section is xylene for ___ to remove the remaining mounting media and brought to water.
It is placed in _____for ____, rinse with tap water and subsequently immersed in _____ for _____or until section is decolorized.
Wash again in running tap water for another _____, the sections can be re stained with appropriate staining technique.
24 hours
bubble.
dissecting needle
30 minutes
0.5 % K permanganate
5-10 minutes
5 % oxalic acid
5 minutes
5 minutes
