microtome knives; sectioning Flashcards
Plane concave knife (length 25mm, shortest); less concave side for celloidin, more concave for paraffin; used for base sledge rotary or rocking microtome
Plane concave knife
Longest knife (length 120mm); both sides concave; used for cutting paraffin sections in rotary microtome
Biconcave knife
Knife used for rocking microtomes with a fixed handle
Heiffor knife
Knife used for frozen sections or very hard tissues; length 100mm; used with base sledge or sliding microtome
Plane wedge knife
More commonly used type of knife; coated with polytetrafluoroethylene to allow easy ribboning of tissues
Disposable blades
Knives used for ultrathin microtomes; made of glass (Ralph knives) or diamond
Glass and diamond knives
Cutting fault found on the tapered edge of a knife
Bevel
Angle formed between the cutting edge and the knife back; typically between 27-32 degrees
Bevel angle
Angle formed between the surface of the block and the cutting edge of the knife; optimum is 15 degrees for maximum penetration with less distortion
Cutting angle
Angle formed between the upper surface of the facet and surface of the tissue/block; high angles are for soft tissues
Rake angle
Angle formed between the sides of the knife
Wedge angle
Process of sharpening a knife by removing nicks or irregularities; movement from heel to toe; 10-20 strokes per surface
Honing
Stone used for honing; dimensions 8x3 inches; lubricants include soapy water, mineral oil, clove oil, xylene, or liquid paraffin
Honing stone
Type of honing stone that provides the best results
Belgium yellow stone
Honing stone that gives more polishing effect
Arkansas stone
Type of honing stone used for badly nicked knives
Fine carborundum stone
Substitute for stone in honing; dimensions 8x3x1 inches
Plate glass
Method of sharpening knives mechanically using a honing machine
Knife sharpeners
Process of cleaning a honed knife, often done with xylene
Cleaning the honed knife
Process of polishing knives after honing
Polishing (stropping)
Removal of burrs (irregularities) formed after honing; movement from toe to heel; use of paddle strop made of horse leather; 40-120 double strokes
Stropping
Dimensions of a paddle strop
3-4 inches x 18 inches
Substance to oil the back of strops (avoid mineral oil)
Special oil (not mineral oil)
Type of sections cut at 4-6 µm
Paraffin sections
Type of sections cut at 10-15 µm
Celloidin sections
Type of sections for renal biopsies cut at 2 µm
Renal biopsy sections
Type of sections cut using plastic embedding medium
Semi-thin sections
Type of ultra-thin sections for electron microscopy (EM); recommended size is 80nm (silver/straw-colored sections)
Ultra-thin sections
Type of frozen sections prepared at 4 µm when a rotary microtome is used
Frozen sections
Point where frozen tissue begins to thaw and becomes visible to the naked eye
Dew line
Brush used to remove ribbon sticking to the knife blade
Camel hairbrush
Rapid freezing agent with a temperature of -190°C
Liquid nitrogen
Freezing agent liquid at room temperature (-150°C)
Isopentane
Optimum temperature range for cryostat use (-18°C to -20°C)
Cryostat temperature
Freezing agent for conventional freezing microtome (-50°C)
Carbon dioxide
Aerosol sprays made of fluorinated hydrocarbons, not for muscle samples
Fluorinated hydrocarbon sprays
Freezing agent with better thermal conductivity than liquid nitrogen
Freon 2.2
Clearing agents for frozen sections; no need for dealcoholization, increases refractive index
Clearing agents
Used for monitoring clearing agents; synthetic water-soluble glycols and resins
Monitoring agent
Recommended compound for frozen section embedding; optimal cutting temperature
OCT (Optimal Cutting Temperature Compound)
Recommended tissue block size for frozen section
2-4 mm
Section thickness for frozen section cutting
5-10 µm
Fault due to faulty fixation, dehydration, or embedding; tissue becomes brittle and hard
Brittle tissue
Fault due to faulty clearing; clearing agent becomes milky
Milky clearing agent
Fault due to faulty embedding; very hard tissues with tissue shrinkage
Tissue shrinkage
Air pockets in trimmed tissue block caused by improper processing
Air holes
Moist tissue block that crumbles due to faulty embedding
Crumbly tissue block
Tissue block that emits a xylene odor due to improper clearing
Xylene-smelling tissue block
Paraffin embedding machine with sections for paraffin reservoir, warm plate, tissue tank, and cold plate
Tissue-Tek Embedding Center
Temperature of floatation water bath (10°C below wax melting point); overheating causes ‘parched earth’ artifact
Floatation bath temperature
Dimensions of floatation water bath (diameter = 11 inches, height = 4 inches, capacity = 2 L)
Floatation bath dimensions
Floatation bath water level should be filled to 1/2 to 1 cm from the top
Water bath level
Drying oven temperature (5°C higher than the melting point of wax)
Drying oven temperature
Type of brush used for cleaning slides in the drying process
Squirrel hair brush
Dimensions of clean slides for section mounting (76 x 25 mm; 1-1.2 mm thick)
Clean slide dimensions
Labeling frosted slides; tool used is
pencil
Labeling non-frosted slides; tool used is a
diamond pencil or glass etcher
Process of placing ribbons into a water bath to flatten; takes 30 seconds
Fishing out
Maximum time ribbons should stay in the water bath before removal
1-2 minutes
Tool used to separate and lift individual sections from the ribbon in the water bath
Forceps
Direction of slide immersion for fishing out a section
Vertically
Reason why the first one or two sections in a ribbon are not picked up on slides
Avoid defects in initial sections
Angle for draining slides after fishing out sections
60-85 degrees
Time for draining slides after fishing out sections
2-5 minutes
Hot plate drying temperature and duration for sections
45-55°C for 30-45 minutes
Incubator drying duration for sections; best for nervous tissue
Overnight
Blower-type electric slide dryer drying temperature and duration
50-55°C for 20-30 minutes
Urgent drying method using a heat source to melt wax quickly
Above a Bunsen burner
Cause of mushy tissue block leading to crumbling and feathering during sectioning
Faulty processing or embedding
Cause of sections failing to form ribbons
Improper knife adjustment or dull blade
Problem when sections roll up or wrinkle during cutting
Adhesion to the knife or static electricity
Horizontal or parallel lines across sections caused by static electricity
Chatters
Thick and thin areas in a section caused by improper knife clearance angle
Venetian blinds
Foreign particles left on the water bath that adhere to sections
Floaters and contaminants
Most common tissue adhesive, consisting of egg white, glycerol, and thymol crystals; background staining may occur
Mayer’s Egg Albumin
Component of Mayer’s Egg Albumin that prevents mold growth
Thymol crystals
Component of Mayer’s Egg Albumin that increases viscosity and prevents complete drying
Glycerol
Tissue adhesive that provides firmer attachment than albumin, requires heating before use, and may stain with many dyes
Gelatin
Concentration of gelatin used as a tissue adhesive
0.01
Tissue adhesive in the form of 1% methyl cellulose; does not stain significantly with most commonly used reagents
Cellulose
General-purpose section adhesive widely used in immunohistochemistry, with no background staining
Poly-L-lysine
Commercial syrup adhesive diluted 1:10 with strong adhesive properties; has minimal staining with most dyes and is unaffected by mild alkaline solutions; blackens in silver impregnation techniques and stains red in methyl green pyronin technique
Sodium Silicate
Greatest adhesion tissue adhesive made of epoxy resin, diluted 1:10 with acetone; minimally affected by most fluids during section treatments
Resins
Specific epoxy resin adhesive known for its strong adhesion and compatibility with most fluids during section processing
Araldite
Preservation method using rapid freezing at -160°C (quenching) followed by desiccation in a vacuum at -40°C for sublimation; time-consuming and expensive; typically used for enzyme studies
Freeze Drying
Process of stopping chemical reactions and diffusion in tissue by rapid freezing at -160°C, followed by sublimation to remove water vapor through tissue surface
Quenching
Time-consuming stage where heat is applied to the frozen tissue causing sublimation, removing water vapor from ice crystals through the tissue
Drying (Freeze Drying)
Fixation method where formalin plays a critical role; used in combination with freeze-drying and freeze substitution techniques
Vapor Fixation
Technique similar to freeze drying but using a vacuum procedure, typically involving Rossman’s formula or 1% acetone for dehydration before embedding in paraffin wax
Freeze Substitution