DEHYDRATION Flashcards
What are the main steps involved in tissue processing?
Dehydration
What is the purpose of dehydration in tissue processing?
Removal of water from aqueous-fixed tissue.
Why is clearing necessary after dehydration?
Because most alcohols and paraffin are not miscible.
List the steps in tissue processing in order.
- Numbering
- Fixation
- Decalcification
- Dehydration
- Clearing
- Impregnation
- Embedding
- Blocking
- Trimming
- Sectioning
- Staining
- Mounting
- Labelling
What is the pre-eminent method of tissue processing for routine preparation?
Paraffin Wax method.
Why is the Paraffin Wax method preferred for routine tissue processing?
It is suitable for routine preparation
What does the Paraffin Wax method utilize as dehydrating fluid?
A series of alcohols.
Name the characteristics of an ideal dehydrating solution.
- Rapid dehydration without significant shrinkage
- slow evaporation
- ability to dehydrate fatty tissues,
- minimal hardening
- does not remove stains
- non-toxic,
- not a fire hazard.
List commonly used dehydrating agents.
- Alcohol (most common)
- Acetone
- Dioxane
- Cellosolve
- Triethyl phosphate
- Tetrahydrofuran
Which alcohol is recommended for routine dehydration of tissues and why?
Ethyl alcohol (ethanol) because:
- it is fast-acting
- mixes well with water and inorganic solvents
- penetrates tissues easily
- not poisonous
- not expensive
What is a disadvantage of using methyl alcohol (methanol) as a dehydrating agent?
It is toxic to the body.
Why is butyl alcohol (butanol) not suitable for rapid tissue processing?
Because it is a slow dehydrating agent.
Describe the dehydration process in tissue processing.
Removal of water from tissue using dehydrating agents, often a series of increasing alcohol concentrations to prevent tissue distortion.
What is the role of anhydrous copper sulfate in alcohol dehydration?
It serves as an indicator to ensure the final alcohol bath is free of water by turning blue if alcohol gets diluted.
What are the advantages of using acetone as a dehydrating agent?
- Cheap
- rapid-acting
- more miscible with epoxy resins than alcohol.
What are the disadvantages of using acetone?
- Highly flammable
- penetrates tissues poorly
- causes brittleness
- removes most lipids
- extremely volatile.
What is dioxane used for in tissue processing?
As a dehydrating and clearing agent.
List the advantages of using dioxane.
- Produces less tissue shrinkage
- , tissues can be left in it for long periods without affecting consistency or staining
- can be placed directly after washing.
List the disadvantages of using dioxane.
- Expensive
- extremely dangerous
- poor ribboning of tissue sections
- highly toxic vapors
- increases risk of explosive peroxides.
What is Graupner’s Method in dioxane dehydration?
A time schedule involving multiple pure dioxane solutions followed by paraffin wax infiltration and embedding.
What is Weiseberg’s Method?
Tissues are wrapped in gauze and dehydrated in a bottle containing dioxane and anhydrous calcium oxide
What is Cellosolve and its primary use in tissue processing?
Ethylene glycol monoethyl ether used as a rapid dehydrating agent that does not harden or distort tissues.
What precautions should be taken when using Cellosolve?
It is combustible at 110-120°F and toxic; use propylene-based glycol ether if possible.
What is triethyl phosphate used for in tissue processing?
Removes water readily with minimal distortion and hardening
What is tetrahydrofuran (THF) used for?
Both dehydrates and clears tissues
What are the disadvantages of using tetrahydrofuran (THF)?
It is toxic
How does the dehydration schedule vary based on the fixative used?
Different fixatives require different alcohol concentrations and exposure times to prevent tissue shrinkage and ensure proper dehydration.
What is the typical dehydration sequence for specimens not more than 4mm thick?
70% ethanol for 15 min
Why is the strength of initial alcohol concentration important in dehydration?
It depends on tissue size and nature; smaller
What happens if tissues are stored in concentrated alcohols for too long?
They produce shrinkage and make the tissue hard
What is the effect of prolonged storage in lower concentrations of alcohol (below 70%)?
It tends to macerate the tissue.
How does temperature affect the dehydration process?
Higher temperatures
What indicates that the final alcohol bath is free of water during quality check?
Anhydrous copper sulfate turns blue if alcohol is diluted with water.
What is the main purpose of fixation in tissue processing?
To preserve the tissue by using aqueous fixatives.
What concentration of ethanol is typically used during fixation in the Paraffin Wax method?
60-70% ethanol.
How many changes of absolute alcohol are used in the Paraffin Wax method clearing step?
2-3 changes.
What factors influence the strength of initial alcohol required in dehydration?
Size and nature of the tissue and the type of fixative used.
Why should tissues treated with chromate fixatives be washed before using dioxane?
To remove chromate
What is the advantage of using Cellosolve over other dehydrating agents?
It dehydrates rapidly and can be stored without causing hardening or distortion.
What safety precautions are necessary when handling dioxane?
Proper ventilation
What does embedding involve in tissue processing?
Encasing the dehydrated tissue in a solid medium like paraffin wax to allow for thin sectioning.
What is the purpose of sectioning in tissue processing?
To cut the embedded tissue into thin slices for microscopic examination.
Why is mounting important in preparing tissue sections for microscopy?
It secures the tissue sections onto slides and preserves them for staining and viewing.
What should be done if tissues do not penetrate deeply during dehydration?
Use lower concentrations of alcohol and shorter intervals between steps.
What is the consequence of unequal impregnation in tissue processing?
Poor sectioning and inconsistent staining.
How does the Paraffin Wax method aid in the storage of tissue samples?
Allows for long-term storage without significant degradation.
What is the role of impregnation in tissue processing?
To infuse the tissue with wax or another embedding medium after clearing.
What happens during the clearing step in tissue processing?
Removal of alcohol and other dehydrants
Why should dioxane not be used routinely despite its advantages?
Due to its high cost
What is the effect of concentrated alcohol on tissue during dehydration?
It causes tissue shrinkage
Which dehydrating agent is considered the best for routine dehydration and why?
Ethyl alcohol (ethanol) because of its effectiveness and safety profile.
What is the main disadvantage of using butyl alcohol in dehydration?
Its slow action makes it unsuitable for rapid processing.
What property of tetrahydrofuran (THF) improves staining procedures?
It dissolves many substances and clears tissues effectively.
How does triethyl phosphate contribute to minimal tissue shrinkage?
It removes water readily with very little distortion and hardening.
What should be used instead of ethylene-based glycol ethers if they cannot be avoided?
Propylene-based glycol ethers.
What is the advantage of using a graded alcohol series in dehydration?
Prevents tissue shrinkage and distortion by gradually removing water.
What is the purpose of blocking in tissue processing?
To organize the embedded tissue into a manageable shape for trimming and sectioning.
What happens during trimming in tissue processing?
Excess embedding medium is removed to expose the tissue for sectioning.
Why is labeling important in the final steps of tissue processing?
To identify and organize tissue samples for future reference and study.
