Tissue preparation Flashcards
What is the purpose of a floatation bath in histology?
Used to float paraffin ribbons cut on microtome
Floatation baths help in the proper handling and positioning of tissue sections for further processing.
What temperature should the floatation bath be maintained at?
About 5°C to 10°C below the melting point of embedding paraffin wax
This is typically around 55°C to 58°C for common paraffin.
What are common artifacts that may occur if the floatation bath is too hot?
- Artifactual separation of tissue components resembling edema
- Overstretching of ribbon
- Floating ribbons for too long
What is the ‘parched earth appearance’ artifact in tissue preparation?
An artifact that may result from a water bath that is too hot or improper processing
This appearance can also be caused by chilling blocks with fluorocarbon spray.
What is the best practice for transferring ribbons to the floatation bath?
Pull the ribbon with a pair of forceps and remove from blade with a second tool such as a brush or dissecting needle.
What should be done between specimens in the floatation bath to avoid contamination?
Skim the bath with lintless tissue paper
This prevents tissue contamination from the bath and ensures clean processing.
What can cause sections to wash off untreated slides during staining procedures?
Improper adhesion of tissue sections to slides.
What type of water should be used in the floatation bath?
Deionized water only.
What should be done with the floatation bath daily?
Dump, clean, and refill with deionized water.
What is the main reason for using adhesives in histology?
To prevent tissue sections from floating off slides during subsequent procedures.
Name a common protein adhesive used in histology.
Mayer’s Egg Albumin.
What is the disadvantage of using egg albumin as an adhesive?
Prone to bacterial growth or heavy staining.
Why is celloidin rarely used today in histology?
It is not really an adhesive technique but an entrapment substance.
It is porous, allowing stains to penetrate easily.
What is the purpose of the formalin vapour method in gelatin application?
To treat gelatin, rendering it irreversible for firmer sections.
What are chrome gelatin slides effective for?
Frozen sections of fixed tissues and special stains.
What is the purpose of poly-L-lysine treated slides?
To provide excellent adhesive qualities for various tissue sections.
What is a key characteristic of APES treated slides?
They are positively charged, enhancing tissue adhesion.
What type of adhesive is araldite?
A high-performance adhesive that is not affected by subsequent dyes and solvents.
Why should starch not be used for carbohydrate demonstrations?
Because starch is a carbohydrate itself.
What happens if slides are not completely dried before deparaffinization?
Sections may wash off during staining, leading to ‘white spots’ in tissue sections.
What is the recommended drying temperature for slides after sectioning?
Above the melting point of paraffin, commonly around 60°C.
What is the typical use for microwave ovens in histology?
Performing special staining, fixation, and improving staining reproducibility.
What is a possible consequence of overheating in the microwave oven?
Nuclear bubbling in tissue sections.
At what temperature are incubators typically maintained for enzyme procedures?
37°C.
What creates heat in a microwave oven during the staining process?
Electrical field of microwave creates friction as it passes through bipolar (water) molecules
This process causes the molecules to reverse themselves, generating heat.
What is a significant advantage of using a microwave oven for staining techniques?
Saves marked time for numerous staining techniques
However, microwaves should never be used for certain techniques.
How can overheating be minimized when using a microwave oven?
Use the LOW setting to slow down the reaction
This reduces the likelihood of overheating.
What must be done to paraffin before staining?
Paraffin must be removed to allow penetration of water soluble dyes
This process is known as deparaffinization.
What is the first step in the deparaffinization process?
Immerse tissue section in xylene for 2 minutes
This step is crucial for removing wax.
List the steps involved in deparaffinization after immersing in xylene.
- Immerse in xylene for 2 minutes
- Immerse in 100% alcohol for 2 minutes
- Immerse in 95% alcohol for 2 minutes
- Immerse in 70% alcohol for 2 minutes
- Rinse well in running tap water
Each step is essential for preparing tissue for staining.
What is the primary purpose of mounting media?
To permanently adhere coverslip over tissue section
This protects the specimen from physical damage and prevents drying out.
What are the two types of mounting media?
- Aqueous
- Resinous
Resinous is generally preferred unless incompatible with dehydrating or clearing agents.
What characteristic of mounting media helps fill tissue spaces without forming air bubbles?
Viscosity of the solution
This allows the medium to flow readily between the slide and cover glass.
What happens to mounting media as the diluent evaporates?
It hardens
This is desirable on the slide but not in the stock solution.
What is the refractive index (RI) of most resinous mounting mediums?
1.51 to 1.55
Tissues generally have a RI of 1.53 to 1.54.
What is Canada Balsam and why is it significant?
First introduced in 1832, it is an oleoresin from the bark of fir trees
Canada Balsam is transparent and adheres to glass, but has been largely replaced by synthetic resinous mounting mediums.
What are some detrimental characteristics of natural resins?
- Set very slowly
- Acidic, causing fading in certain stains
- Yellow with age
- Poor preservatives of basic aniline dyes
These factors limit their use in modern histology.
Name three commonly used synthetic mounting mediums.
- DPX
- Clarite
- Permount
These mediums are preferred for their neutral properties and quick setting.
What is a characteristic of aqueous mounting media?
Used when dehydrating and clearing will adversely affect the stain
They are also required for preservation of certain tissue elements such as fat.
What are the typical sealants used for coverslips with aqueous media?
- Melted paraffin
- Glue
- Clear fingernail polish
These sealants help adhere the coverslip and prevent drying.
What must be done to tissue sections before coverslipping?
They must be completely dehydrated and cleared
This involves using three alcohols and three clearing agents.
What are the consequences of allowing tissue sections to dry before coverslipping?
Artifacts such as cracks in medium and brown stippling may occur
This can lead to poor microscopic examination.
What is a common issue with automated coverslipping systems?
Occasionally, two coverslips can get through, leading to double coverslipping
Regular maintenance and cleaning can help prevent this issue.
How can water bubbles in mounted sections be corrected?
Change dehydrating and clearing solutions, then rehydrate and remount
Ensuring sections are completely dehydrated before staining is essential.
What is the refractive index (RI) of aqueous mounting media?
1.41 to 1.43
This is significantly different from tissue, affecting microscopic examination.
What is the first step in the process of removing coverslip and mounting medium?
Removing coverslip and mounting medium with xylene and returning to fresh abs alcohol
This process is essential to clear the sections before mounting with synthetic resin.
What causes all areas of a section to not be brought into focus?
Mounting medium on top of cover glass
This issue can be corrected by modifying or changing the technique for applying the cover glass.
How can one correct the issue of all areas of a section not being in focus?
Ensuring mounting medium is NOT present on top of cover glass
Alternatively, remove the cover glass and remount the section with a clean cover glass.
What is cornflaking in mounted sections?
A drying artifact seen on mounted sections
It appears as small glossy black nuclei or large areas of granular brown stippling resembling pigment.
What causes cornflaking in mounted sections?
Sections are allowed to air dry before mounting
This leads to the creation of a drying artifact in the tissue section.
How can cornflaking be corrected?
Ensuring slides do NOT DRY before mounting
If severe, remove coverslip and mounting medium with xylene, rehydrate to water, then re-dehydrate and clear.
What happens if mounted stained sections are not crisp or transparent when viewed microscopically?
Mounting medium is too thick
This can be corrected by thinning out with xylene.
What is the routine process for slide labeling?
Slides labeled after floatation complete with a paper label
The label includes assigned lab number, staining method, and date.
What should be avoided when labeling slides?
Using labels that cannot endure chemical processes
For example, paper labels may not survive certain chemical treatments.
How should slides be stored after staining?
Allowed to dry FLAT for at least 2 weeks in a slide folder
After drying, they may be stored standing on their short side in metal or plastic racks.
What happens if slides are stored before 2 weeks of drying?
Slides tend to be stuck together and require re-coverslipping
In rare cases, slides may NOT be unstuck.
