Dehydrate - Clear - Impreg - Embed Flashcards
Most common processing problem
Incomplete dehydration
Addition of this at the bottom of the container accelerates dehydration & a __ discoloration will indicate full saturation
Copper sulfate (1/4 in thick) w/ filter paper
Blue
Alcohol dehydrating agents
Most common:
Toxic:
Versatile:
Slow
Most common: Ethyl alcohol
Toxic: Methyl
Versatile: Isopropyl
Slow: Butyl
Excellent dehydrating & clearing agent
Dioxane
Uses pure dioxane & paraffin
Graupner
Tissues wrapped in a gauze & submerged in dioxane w/ calcium oxide/quicklime
Weisberge
Best universal solvent dehydrating & clearing agent
Tetrahydrofuran (THF)
Dehydrating agents for EM
Dehydrating agent:
Transition fluid:
- substitute:
Dehydrating agent: Ethanol
Transition fluid: Propylene oxide
- substitute: Acetonitrile
Clearing is also known as
Dealcoholization
Clearing agent should be __ the volume of the tissue
10x
Must be miscible with alcohol & doesnt evaporate quickly
Good clearing agent
Most commonly used & most rapid clearing agent
Xylene
Not suitable for nervous tissue & lymph nodes
& becomes milky with incomplete dehydration
Xylene
Used for tough tissues, nervous tissues, embryos & lymph nodes
Chloroform
It does not make the tissue transparent & is toxic to liver after prolonged exposure
Chloroform
Rapid acting clearing agent but highly inflammable &
causes aplastic anemia & damage to BM
Benzene
Substitute for benzene & xylene
Toluene
Recommended for CNS & smooth muscle & skin in both paraffin & celloidin sections
Cedarwood oil
Becomes milky after prolonged storage & clears extremely slow
cedarwood oil
Recommended for delicate specimens (insects)
Aniline oil
Quality is not guaranteed due to its tendency to become adulterated
Clove oil
slow clearing agents used during double embedding technique
Methyl benzoate & methyl salicylate (oil of wintergreen)
Earliest transition solvent
Terpene (Limonene)
Xylene substitute & nitrocellulose solvent
N-Butyl acetate
Clearing agent is completely removed & replaced by a medium that completely fills all the tissue cavities
Impregnation/Infiltration
Most important step in embedding
Orientation
Simplest, most common & best embedding for routine tissue processing
Paraffin
Fresh wax must be __ before use
Filtered
Paraffin oven must be maintained at a temp of
56°C
Paraffin wax may be used only
Twice (2x)
Coarse filter paper used for filtering
Green’s No. 904
Manual processing
At least __ changes of wax at __ mins intervals to ensure complete removal
__ hours to ensure complete embedding
4 changes
15 mins
3 hrs
Automatic processing (embed)
__ processing steps
Wax bath temp: __ above the MP of wax
12 steps
3°C above
“FDCI”
Recommended for urgent biopsies, delicate & decalcified bones
Impregnation under negative atmospheric pressure
Vacuum embedding
Highly purified paraffin & synthetic plastic polymer
For large dense tissue blocks
Paraplast (56-57°C)
Synthetic wax subtitute for embedding
Embeddol (56-58°C)
Recommended for embedding eyes
Bioloid
Paraffin with rubber
Tissue mat
Used for impregnation w/o prior clearing of tissue & done using a heavy-duty microtome
Ester wax
Water-soluble wax suitable for enzyme histochemical studies
Carbowax
When using carbowax, you can’t use water for tissues to float on so use:
Pearse soln
Blank & McCarthy soln
When using celloidin
No __ is required & the resultant block has a __ consistency
Heat
Rubbery
Celloidin method used for bones, teeth & whole organs
tissue stored at 70-80% alcohol
Wet celloidin method
Preferred for processing whole eyes
Uses Gilson’s mixture (composition?)
Dry celloidin method
Equal parts of chloroform & cedarwood oil
Another form of soluble celloidin but w/ ether & alcohol that can penetrate tissue quickly
Low Viscosity Nitrocellulose (LVN)
Infiltrated w/ Celloidin
Embedded in Paraffin
Double embedding method
Embedded method where dehydration is avoided
For histochemical & enzyme studies
prevents fragmentation
Gelatin method
Gelatin method
Impregnating medium should be at least __ the vol of tissue
addition of __ prevents the growth of molds
25x
1% phenol
Superior embedding method results for light microscopy (undecalcified bone) but used for EM
Plastic method
Component of epoxy plastic that is known to be a carcinogen
Vinyl cyclohexane dioxide (VCD)
Epoxy plastic:
aka Araldite
Bisphenol A
Epoxy plastic:
aka EPON
Glycerol
Epoxy plastic:
aka SPURR
cyclohexane dioxide
plastic method:
Includes polyglycol methacrylate (GMA) & methyl methacrylate (MMA) used for LM
Acrylic
2 ‘L’ shaped strips w/ heavy brass
Leukhart’s embedding mold
Interlocking plates resting on a flat metal base forming several compartments
Compound embedding units
Disposable embedding molds that gives perfect even block w/o trimming
peel-away
Disposable embedding molds normally for celloidin
paper boat
Disposable embedding molds recommended for busy labs
plastic ice trays
