Dehydration (M) Flashcards
What are the steps of conventional tissue processing?
1) Labeling (Numbering)
2) Fixation
a. Washing out
3) Decalcification (optional)
4) Dehydration
5) Clearing
6) Impregnation (Infiltration)
a. Orientation
7) Embedding
a. Trimming
8) Section-Cutting (Microtomy)
a. Floating out
b. Adhesion
9) Staining
10) Mounting
a. Ringing
11) Labeling
True or False
Tissue processing (conventional) starts w/ labeling and end w/ labeling
True
What is dehydration?
It is the removal of fixative and intracellular and extracellular H2O from the sx
Why should H2O be removed (via dehydration)?
Because presence of H2O will impair subsequent steps
What should be done to acquire equal impregnation?
Replacement of fixative and H2O w/ dehydrating agent in preparation for impregnation
What are the characteristics of dehydrating agents?
1) These are of various types of alcohols
2) These alcohols are in increasing strengths
What is the action of dehydrating agents?
These prevents violent osmotic changes
What are the consequences that may be present via the use of various types of alcohols in increasing strengths?
It makes the tissue:
1) Hard
2) Brittle
3) Shrunken
4) Difficult to cut
Dehydration involves what?
It involves slow substitution of the H2O in the tissue w/ an organic solvent
What are the characteristics of an ideal dehydrating solution?
1) Rapid w/out shrinkage or distortion
2) Does not evaporate quickly
3) It should dehydrate fat
4) Does not harden tissues
5) Does not remove stains
6) It should be non-toxic
7) It should be non-flammable
What are the other considerations w/ dehydration?
1) Do not dry out the tissues
2) Use 10 times > the volume of the tissue for complete penetration
3) Dehydration must be brief as possible to minimize extracting the cellular components
4) H2O miscible and anhydrous fluid may be used as long as it does not damage the proteins and miscible to the fluids to be used subsequently
5) Agitation should be done
6) The temp should be 37 DC
7) Signs for H2O content should be observed such as:
a. Macerated tissue
b. Milky xylene
c. Anhydrous copper sulfate
What are the characteristics of anhydrous copper sulfate?
1) Its original color is color white
2) If tissue has H2O content, it is color blue (hence, it is indicative of presence of incomplete dehydration)
What is the typical dehydration sequence for sxs not > 4mm thick?
1) 70% ethanol - 15 mins
2) 90% ethanol - 15 mins
3) 100% ethanol - 15 mins
4) 100% ethanol - 15 mins
5) 100% ethanol - 30 mins
6) 100% ethanol - 45 mins
True or False
In connection w/ a typical dehydration sequence for sxs not > 4mm thick, more delicate tissue (embryo) begins at 30% ethanol
True
What is the processing schedule (stations / station rgnt and their corresponding time) for Leica TP 1020 for general surgical sxs w/c are 3 mm thick or are < or equal to 20 x 20 mm?
1) NBF - 1 hr + hold time
2) 95% alcohol - 1 hr
3) 95% alcohol - 1 hr
4) 95% alcohol - 1 hr
5) 100% alcohol - 1 hr
6) 100% alcohol - 1 hr
7) 100% alcohol - 1 hr
8) Xylene - 1 hr
9) Xylene - 1 hr
10) Xylene - 1 hr
11) Melted Paraffin - 1 hr
12) Melted Paraffin - 1 hr
What is the time for the processing schedule for TP Leica 1020 for processing general surgical sxs (w/c are 3 mm thick or are < or equal to 20 x 20 mm)?
Overnight (12 hrs)
What are the different dehydrants?
1) Ethanol
2) Methanol
3) Butanol (117.7 DC)
4) Tertiary butanol (82.8 DC)
5) Isopropanol
6) Pentanol (Amyl alcohol)
7) Acetone
8) Dioxane
9) Cellosolve (Ethylene glycol monoethyl ether)
10) Triethylphosphate
11) Tetrahydrofuran
What are the advantages of using ethanol as a dehydrant?
1) It is the best dehydrating agent
2) It is recommended for routine dehydration
3) It penetrates easily
4) It may be used for eyes and embryo
5) It is non-toxic
What are the disadvantages of using ethanol as a dehydrant?
1) It is only slightly miscible to wax / resins
2) It extracts methylene blue and other thiaxine dyes from section
3) It extracts more lipids > acetone
4) It may cause more shrinkage of sx and hardening for long periods in ethanol
5) It may react w/ unreduced osmium tetroxide (OsO4)
What is the advantage of using methanol as a dehydrant?
It can be used in blood touch preparations and smear preparations
What is the disadvantage of using methanol as a dehydrant?
It is toxic (it irritates the eye)
What are the advantages of using butanol as a dehydrant?
1) Plant and animal micro-techniques
2) It produces less shrinkage and distortion > ethanol
3) It is recommended for tissues w/c do not require rapid processing
4) It is miscible w/ paraffin
What are the disadvantages of using butanol as a dehydrant?
1) It is odorous
2) It is slow-acting
3) Long periods of infiltration is necessary
4) It has low dehydrating power
What are the advantages of using tertiary butanol as a dehydrant?
1) It is a universal solvent (hence, it acts as a dehydrating and clearing agent)
2) It mixes w/ H2O, ethanol, xylene, and paraffin in all
3) It may be used in staining series as a dehydrating agent
What are the disadvantages of using tertiary butanol as a dehydrant?
1) It is odorous
2) It is more expensive > butanol
3) Primary infiltration must be done in half tertiary butanol and half paraffin, prior to paraffin impregnation
4) Rgnt tends to solidify at room temp / below 25 DC
What are the advantages of using isopropanol as a dehydrating agent?
1) It is an excellent substitute for ethanol
2) It produces less shrinkage and hardening > ethanol
3) It is sufficiently H2O-free to use in place of absolute alcohol
4) It is less expensive
5) Microwave processing
What are the disadvantages of using isopropanol as a dehydrating agent?
1) It cannot be used in the celloidin technique since nitrocellulose is insoluble in it
2) It cannot be used for preparing staining solutions (since dyes are not soluble in it)
What are the advantages of using pentanol as a dehydrant?
1) It is miscible w/ 90% alcohol, toluene, and xylene
2) It dissolves paraffin wax
What are the disadvantages of using pentanol as a dehydrant?
1) It is toxic
2) It cannot be used in poorly ventilated rooms
3) It is not miscible w/ H2O
What are the characteristics of acetone?
It is a fixative and dehydrating solution
What are the advantages of using acetone as a dehydrant?
1) It is cheap
2) Its action is rapid
3) It does not extract methylene blue and other dyes from stained sections
4) It may cause less shrinkage of sx > ethanol
5) It is not reactive w/ OsO3 remaining in sx
6) It is miscible w/ most embedding resins
What are the disadvantages of using acetone as a dehydrant?
1) Its volume must be 20 times of the tissue
2) Best processing requires a graded series of a mixture of acetone and xylene before 1 can go in paraffin
3) It is flammable
4) It is volatile
5) Brittleness is present in prolonged immersion
What are the advantages of using dioxane as a dehydrant?
1) It is a universal solvent (hence, it dehydrates and clears)
2) It is miscible w/ the ff:
a. H2O
b. Alcohol
c. Xylene
d. Paraffin
3) It does not harm tissue over long time periods
4) It is a faster dehydrant > ethanol
What are the disadvantages of using dioxane as a dehydrant?
1) It needs large volume
2) It costs about 4x more than absolute alcohol
3) It must be used in well-ventilated rooms
4) It is highly toxic
5) It is odorous
6) It has a risk for explosion
7) It cannot be reused (because it is highly toxic, odorous, and has a risk for explosion)
8) It distorts tissue-containing cavities
9) It ribbons poorly
What are the 2 methods for dehydration via the use of dioxane?
1) Graupner’s method
2) Weiseberger’s method
What is the time schedule of dehydration w/ dioxane (via Graupner’s method)?
(1st) pure dioxane solution - 1 hr
(2nd) pure dioxane solution - 1 hr
(3rd) pure dioxane solution - 2 hrs
(1st) paraffin wax - 15 mins
(2nd) paraffin wax - 45 mins
(3rd) paraffin wax - 2 hrs
Embed in mold and cool in H2O
What is the principle (/ method) of dehydration via Weiseberger’s method?
The tissue is wrapped in a gauze bag and suspended in a bottle containing dioxane and a little anhydrous Ca oxide
What are the advantages of using cellosolve as a dehydrant?
1) It is a rapid dehydrating agent
2) The tissue may remain in it for months w/out injury
3) The tissue may be transferred from H2O or NSS and stored for months w/out hardening or distortion
4) It avoids distortion and does not require graded dilutions
What are the disadvantages of using cellosolve as a dehydrant?
1) It is expensive
2) It rapidly absorbs H2O from the air
3) It requires clearing agent
4) Ethylene glycol ether combust at 110 - 120 DF (43 - 48 DC)
5) It is toxic to the ff:
a. Reproductive system
b. Fetal system
c. Urinary system
d. Blood system
What are the advantages of using triethylphosphate as a dehydrant?
1) It may be used in routine paraffin technic / technique
2) It displaces H2O readily w/ slight distortion
3) It does not harden tissue excessively
4) It may be used as a dehydrating solution in the staining sequence
5) It is soluble in the ff:
a. Alcohols
b. Toluene
c. Xylene
d. Ether
e. Chloroform
What are the disadvantages of using triethylphosphate as a dehydrant?
1) High exposure w/ this dehydrant can cause the ff:
a. Headache
b. Nausea
c. Dizziness
2) It can irritate the ff:
a. Nose
b. Throat
3) Prolonged / repeated exposure to this dehydrant may cause the ff:
a. Chills
b. Fever
c. Chest discomfort
d. Cough
e. Shortness of breath (chemical pneumonitis)
What are the advantages of using tetrahydrofuran as a dehydrant?
1) It is a universal solvent (hence, it dehydrates and clears)
2) It is miscible in all proportions w/ the ff:
a. H2O
b. Ether
c. Chloroform
d. Acetone
e. Thydrocarbons
f. Xylene
g. Toluene
h. Benzene
3) It is rapid w/out excessive shrinkage and hardening (hence, easier cutting of sections can be achieved)
4) It has / produces low toxicity (hence, it has low fire and explosion hazards)
What are the disadvantages of using tetrahydrofuran as a dehydrant?
1) It is odorous (hence, it should be used in a well-ventilated room)
2) It evaporates rapidly
3) Aniline dyes does not dissolve in tetrahydrofuran
4) 6 mos > exposure to this dehydrant produces conjunctival irritations
What are the dehydrating agents (their specificities) for electron microscopy?
Dehydrating solvent: ethanol
Transiction (/ transition) fluid: propylene oxide
Substitute: acetonitrile
What are the additives to dehydrating agents?
1) Glycerol
2) Phenol
3) 4% phenol
What is the action of glycerol (as an additive to dehydrating agents)?
It is a softening agent for hard tissues
What is the action of phenol (as an additive to dehydrating agents)?
It acts as a softening agent for hard tissues like:
1) Tendons
2) Nail
3) Dense connective tissues
4) Keratin masses
What is done to 4% phenol (as an additive to dehydrating agents)?
It is added to 95% ethanol stations
