Tissue Processing Flashcards
A second routine step when tissues are hard and contains calcium
Decalcification
Ratio of Decalcifying agent-to-Tissue
20:1
Most common Decalcifying agent
Nitric Acid
Process of removing intercellular and
extracellular water to maintain SHAPE of cells and its morphology.
Dehydration
Important function of intracellular water.
Maintain SHAPE of the cells.
True or False. In dehydration, tissue sections are placed in UPGRADED concentration ethanol?
True
Most common dehydrating agent
Ethanol
Why is water removed?
Because it hinder the process for preparation of infiltration and embedding since many embedding media are not miscible with water.
Start/Working Percentage of Ethanol
70%
Ratio of dehydrating agent to tissue
10:1
Increasing Concentration of Alcohol and time Required
70% - 1 hr
80% - 1 hr
90% - 1 hr
95% - 1 hr
Characteristics of Dehydrating Agent
- Dehydrate RAPIDLY
- Should NOT EVAPORATE quickly
- Dehydrate even FATTY tissues
- Doesn’t HARDEN tissues excessively
- Should not be toxic to the body
- Should not be a fire hazard
Why are tissues placed in upgraded concentration?
This allow tissues to ensure gentle removal of water from shrinkage. diffusion currents and osmotic changes because once water is removed rapidly, it results to not maintain the shape and morphology of cells, Therefore, replacing water by alcohol will in upgraded concentration will adjust the cells for aqueous intracellular and extracellular removal for tissues not to produce destruction, distortion, shrinkage and extraction of cellular components.
Results:
Prolonged in High Concentration
Prolonged in Low concentration
A:
High concentration: Make tissues, Brittle, Hard, and Distortion of cellular components causing impregnation unequal and hard cutting of sections.
Low Concentration: Macerate tissues
Factors in Total dehydration time.
- Temperature
- Use of Anhydrous copper sulfate
- Thickness of Tissue specimen.
Dehydrating agent used for FATTY specimen
Acetone
Dehydrating agent which can be stored for months without hardening and distortion.
Cellosolve
Dehydrating agent readily miscible in water, melted paraffin, alcohol and xylol with LESS TISSUE SHRINKAGE.
Dioxane
What is clearing of tissues?
Clearing of tissues is to make of use of clearing agent which is to remove alcohol (de-alcoholization) and is replaced to dissolve the wax which tissues is to be infiltrated and the medium which sections are to be mounted. This process also makes tissues transparent when examined.
True or False. All clearing agent have dual actions.
False. Not all have dual functions because some only remove alcohol without making transparent during staining.
Referred to when clearing agent have DUAL ACTIONS
True Clearing Agent
Index of incomplete dehydration
Milky appearance
Most common and Best clearing agent
Xylene/Xylol
Characteristics of Clearing agent
- Miscible with alcohol
- Miscible with paraffin and/or mounting medium
- Should make tissues transparent
- Doesn’t dissolve in aniline dyes
- Doesn’t evaporate quickly.
Clearing Time
30 minutes to 1 hour
Remedy when milky appearance is demonstrated.
Glycerine/Gum Syrup
It clears both paraffin and celloidin sections and recommended for CNS, Cytological Studies, Smooth muscles and skin.
Cedarwood oil
Clears Embryos, Insects and Delicate specimen
Aniline Dyes
Substitute for xylene and benzen
Toluene
Clears for decalcified tissues
Chloroform
Purpose of Impregnation
To replace clearing agent with a medium that will fill out tissue spaces and cavities in order to have a firm consistency of the tissue for embedding and section cutting.
Three Methods of Infiltration
Paraffin Wax Impregnation
Celloidin Impregnation
Gelatin Impregnation
Most Common and best embedding medium
Paraffin Wax
Ratio of Infiltrating medium-to-tissue
25:1
What will be the result of this factor:
•Low MP
•High MP
Low MP – paraffin is soft
High MP – paraffin is hard
In manual processing, how many changes are required for paraffin?
2-4 changes at 15 mins interval
What is the purpose of 2-4 changes of paraffin?
To ensure complete removal of clearing agent and properly maintain impregnation of specimen.
Precautions in Paraffin wax impregnation
- It should be pure away from dust, water droplets and foreign matters.
- Paraffin should only be used twice.
- Water must be discarded when automatic tissue processing machine .is performed
Paraffin Oven Impregnation
General Rule: 55C - 60C
Methods in Performing Paraffin Wax Impregnation and Embedding
Manual Processing
Automated Processing
Vacuum Embedding
Routine MP if Paraffin Impregnation
56°C MP
Process under NEGATIVE PRESSURE inside and embedding
oven to hasten removal of air bubbles and
clearing agent, NO exposure to HEAT;
recommended for URGENT biopsies, for dense and hard fibrous tissues
VACUUM EMBEDDING
Factors Wax Impregnation
- Nature and Type of Tissue
2. Clearing agent used
What is the result when impregnation is prolonged?
Shrinkage
Hard
Brittle
Difficulty in cutting
What is the result when impregnation is inadequate?
Soft
Retention of Clearing agent
Shrinkage
Tissue blocks will be crumbled and break up in bath
Substitute for Paraffin Wax
Paraplast
Ester Wax
Embeddol
Carbowax
Paraplast
More Elastic, Resilient and Better ribboning of sections
Recommended for embedding Eyes
Boiloid
Embedding medium that is insoluble in water but soluble in 95% ethanol and other clearing agent
Ester Wax
True or False. Ester wax is used with prior clearing of tissues
False.
Ester Wax can be used without prior clearing of tissues.
Most commonly used water soluble wax which do not require dehydration and clearing of tissues.
Carbowax
It is a purified form of NITROCELLULOSE
Celloidin
Celloidin is most suitable for;
Large hollow cavities
Hard & Dense tissues
Large Tissues (embryo)
Neurological tissues.
Two Celloidin Impregnation Method
Wet Celloidin: Bones, Brain sections, Whole Organ
Dry Celloidin: Whole Eye
Gilson’s mixture
Cedarwood oil + Chloroform
Difference of Dry with Wet Method
Dry method does not use 70% alcohol upon
storage, it utilizes Gilson’s mixture (Chloroform and
cedarwood oil)
When is Gelatin Impregnation used?
If dehydration is to be avoided and when tissues are to be subjected to histochemical and enzyme studies.
Other terms for tissue Embedding
Tissue Casting/Tissue Blocking
True or False. The medium used in infiltration is DIFFERENT from the medium utilized in embedding.
False. The medium used to infiltrate the tissue is
usually THE SAME medium utilized for embedding. –
known as embedding medium
What is Embedding process?
Impregnated tissue is placed in a molder or block that forms the shape of the tissue and require proper arrangement/position of tissues (orientation) containing a medium that is allowed to solidify.
What is Orientation?
The proper arrangement or specific position of tissue in the mold, during embedding, on the microtome before cutting and slide before staining.
General Rule in embedding
Infiltrating and Embedding medium must be the same.
Blocking Out Molds
Enumerate:
3 Permanent Molders
3 Disposable Molders
Permanent Molders
- Leuckhart’s embedding mold
- Compound embedding unit
- Plastic embedding rings and base molds
Disposable Molders
- Peel away molds
- Plastic ice tray
- Paper Boats
It is most routinely used mold, can be adjusted and with 2 L-shaped pieces of metal
Leuckhart embedding mold
Plastic Embedding Rings and base molds
Special stainless base mold fitted with a plastic embedding ring (block holder)
Embedding method using both celloidin (1st) and paraffin (2nd) media are used.
DOUBLE EMBEDDING METHOD
Trimming
Removal of excess wax in all sides and cutting-off pointed edges of the tissue block.
IDEAL Trimmed Tissue Block
FOUR SIDED PRISM/TRUNCATED PYRAMID
Microtome Knives
Plane-Concave Knife (25 mm)
Biconcave Knife (120 mm)
Plane Wedge Knife (100 mm)
Plane Concave Knife
25 mm
One side - Flat
Other - Concave
Less concave recommended for CELLOIDIN embedded tissue on SLIDING microtome
Biconcave Knife
120 mm
Both sides are concave
Used in Rotary Microtome
Plan wedge Knife
100 mm
For Frozen and Hard tissues
For base-sledge or SLIDING microtome
Microtome Knife Angles
Bevel Angle (27-32) - angle between CUTTING EDGE of knife Wedge Angle (15) - angle between BACK SIDES of knife Clearance Angles (0-15) - Angle between CUTTING FACET and tissue block
Two processes of sharpening Microtome Knives
HONING
STROPPING
Honing
Removal of GROSS NICK and GRINDING the edge of microtome knife on a hone.
Stropping
Removing the BURRS produced during honing that polishes the sides of the cutting edge; POLISHING
Results when knives are not sharpened
- Torn tissue sections and Tissue ribbons will split or have LENGTHWISE vertical scratches
- Tissue sections are lifted from the knife on upstrokes
- Uneven thickness of tissue sections, thick or thin.
Honing Direction
CUTTING EDGES FIRST, with HEAL TO TOE DIRECTION
Stropping Direction
CUTTING EDGE LAST, with HEAL TO TOE DIRECTION
Process which embedded tissues are trimmed, or cut into UNIFORMLY thin slices or “sections” in microtome which produces a predetermined thickness by sliding the block into a cutting tool to facilitate microscopic study of tissues.
MICROTOMY
Three essential parts of Microtomy
- Block Holder
- Knife carrier and Knife
- Pawl , Rachet feed wheel, and adjustment screws
Number of STROKES:
Honing
Stropping
Honing: 20 - 30 double-strokes
Stropping: 40 - 120 double strokes
Purpose of Float-out-bath
To smoothen-out, straighten-out, or flatten out wrinkles in tissue sections produced during sectioning.
MICROTOMY PRINCIPLE
A spring –balanced teeth or pawl is brought in
contact with an turns a rachet feed wheel
connected to a micrometer screw, which is in
turn rotated moving the tissue block at a pre-determined distance towards the knife for
cutting sections at uniform thickness.
5 Types of Microtome
- Rocking Microtome (serial sections & large blocks of paraffin embedded tissues)
- Rotary Microtome (paraffin embedded tissues)
- Sliding Microtome (celloidin embedded tissues)
- Freezing Microtome (unembedded frozen tissues)
- Cryostat or Cold microtome (cutting frozen sections)
- Ultrathin microtome (for section for EM)
Microtome for cutting PARAFFIN embedded tissues.
Rotary Microtome
Microtome for cutting CELLOIDIN embedded tissues.
Sliding Microtome
Microtome for cutting UNEMBEDDED FROZEN sections
Freezing Microtome
Give the functions of each part of Microtome.
- Tissue Bloch Holder
- Knife carrier, and Knife
- Pawl, Rathchet feedwheel and adjustment
- Tissue Bloch Holder. Hold Tissues in Position
- Knife carrier, and Knife. For actual cutting of sections
- Pawl, Rathchet feedwheel and adjustment. Lines up the tissue block in proper position and adjusting the proper thickness of tissue.
Why float-out-bath is demonstrated in black color?
To facilitate visualization of tissue sections during section and fishing out of ribbon.
Give 3 problems in sectioning due to tissue Processing.
- Hard and Brittle Tissues
- Tissue is soft when block is trimmed
- Difficulty in cutting due to presence of alcohol
Give the CAUSES:
- Hard and Brittle Tissues
- Tissue is soft when block is trimmed
- Difficulty in cutting due to presence of alcohol
- Prolonged Fixation, Dehydration, Clearing and Infiltration. Drying out of tissues.
- Incomplete fixation.
- Incomplete clearing
Give the Causes:
- Sections Fail to form Ribbons
- Sections are wrinkled, jammed, crumbled.
- A hole is formed in the sections.
Causes
- Horizontal surface of the block is not parallel to the knife.
- Paraffin wax is warmed or soft.
- Bubble or Dirt is formed in the embedding medium.
Tissue section size of Paraffin sections
4-6 um
Tissue section size of Celloidin sections
10-15 um
Why are adhesives used?
Adhesives are viscous mixture that are used for tissue ribbons from the float-out-bath adhere/stick to the slide for easy fishing out of tissue.
Most common Adhesive used.
Mayer’s Egg Albumin (1:1 mixture - Egg albumin:Glycerine)
What are tissue ribbons?
Tissue ribbons are series tissue sections in length with uniform sizes formed during microtomy and is placed in the float-out bath.
Length of Tissue ribbons produced?
9-12 inches and only 2-6 are used to be fished-out and placed in float-out bath
Fishing out Materials
Camel’s Hair brush
Needle probe
Index finger
Temperature set in Float-out bath
40 C - 45 C or 5 C - 10 C below the MP of paraffin
Mounting
Process which a syrup fluid is applied between tissues and the slide which keeps the slide to its permanent position.
Purpose of Mounting
- Protect specimen from damage which may lead to distortion of image during examination.
- Protect tissues from scratches, bleaching and deterioration due to oxidation.
- Preserve slide for permanent keeping and teaching purposes
- Easy handling and Storage
Characteristics of A Good Mounting Medium
▪ It should NOT dry quickly
▪ It should not dissolve out of fade tissue sections
▪ It should not cause shrinkage and distortion of
tissues
▪ It should set HARD, thereby producing PERMANENT mounting of sections.
▪ To avoid distortion of the image, the refractive index
of the mountant should be as near as possible to that
of the glass slide (1.518)
Two main groups of Mounting Medium
AQUEOUS MEDIA
RESINOUS MEDIA
Mounting medium used to mount water miscible preparations and for TEMPORARY MOUNTING.
AQUEOUS MEDIA
Mounting medium used for preparations that is dehydrated and cleared in xylene. For PERMANENT mounting.
RESINOUS MEDIA
Enumerated Aqueous mounting media and its RI
Glycerin Jelly (Kaiser’s 1880): 1.47
Farrant’s medium: 1.43
Apathy’s medium: 1.52
Enumerated Resinous mounting media and its RI
Canada Balsam: 1.524
DPX: 1.532
XAM: 1.52
CLARITE: 1.544
Types of Mounting
Dry Mount (inanimate objects) Wet Mount (cold living organism)
Process of sealing the margins of coverslip
Ringing
Difficulties in Mounting
- Tissue sections are cloudy or milky
2. Bubble formation
How bubbles are removed?
Gently press using a pointed forceps or a mounting needle. In case with many air bubbles, put slide back into the xylene so that coverslip is separated and demount the section without air bubbles.
Benzene is recommended for ________ and routine purposes
Urgent
Benzene can cause _____________
aplastic anemia
