Gem 201 Flashcards
(15 cards)
What is tissue processing?
How do you do it ?
How to mount a slide (go and look at the cards on the microscope from micro b plus the ones you have in the materials. Go over your anatomy from the access course too)
You do tissue processing(cut some of the tissue and fix them using formalin or a fixative and in this case, formalin is the fixative. Then you dehydrate the tissue in graded alcohol
. Then you infiltrate and probably clear the alcohol you used in the dehydration and then you stain. Most common dye used is H and E)
before you can get sample to put on slide for viewing.
. Cutting
• Fixation: (the aim of fixation is to do the three D’s”decomposition, digestion, drying/dehydration”), crosslink proteins, preserve structures. E.g. formalin (formaldehyde), glutaraldehyde, osmium teraoxide, methanol, ethanol.
• Dehydrate: graded alcohol to absolute alcohol
• Clearing: xylene (miscible with alcohol/wax)
• Infiltration: wax
• Embed: wax
• Sectioning: thin slices are cut
• Stain: dyes e.g. haematoxylin and eosin
Graded alcohol is in different percentages. 70-100%.
Infiltrate the tissue with wax or paraffin but this wax isn’t miscible with alcohol so you have to first clear the acholol with xylene which is miscible with alcohol and wax and this is called clearing.
Then you Embed with wax. Embedding is giving it weight or a body and you do that with wax.
Then you section into thin slices sometime sim micrometer
Then you stain.
Two main types of fixation:
Émersion fixation; you put the tissue you want to fix in it. You immerse it in it
Perfusion fixation: you make an entrance with a needle into the left ventricle of the animal that you want to fix and the heart should still be beating so you put the animal in a semi active state using chloroform then you cut a portion of the right atrium after fixing into the left ventricle. The fixative travels through all of the tissue and is pumped out of the body from the hole you created with the cut.
Perfusion Fixation Process:
1. Anesthetize the Animal – The animal is placed in a semi-active state using chloroform or another anesthetic. The heart must still be beating to allow circulation of the fixative.
2. Insert a Needle into the Left Ventricle – A needle is introduced into the left ventricle of the heart, ensuring it is properly positioned within the circulatory system.
3. Cut the Right Atrium – A small incision is made in the right atrium to create an exit point for the blood and fixative.
4. Perfuse the Fixative – A fixative solution (e.g., formalin or glutaraldehyde) is introduced via the needle. The heart pumps the fixative throughout the circulatory system, ensuring uniform fixation of all tissues.
5. Drainage Through the Right Atrium – As the fixative spreads, blood and excess solution exit through the right atrium incision, preventing buildup and ensuring proper penetration into tissues.
6. Complete Fixation – The process continues until the entire tissue is fixed, preserving its structure for microscopic analysis.
This technique ensures deep tissue fixation, making it ideal for brain, vascular, and whole-body preservation in research.
Tissue Processing in Histology
Tissue processing prepares biological tissues for microscopic examination by preserving their structure and composition. The main steps include fixation, dehydration, clearing, infiltration, embedding, sectioning, and staining.
- Fixation
• Purpose: Preserves tissue structure and prevents decay.
• Fixative Used: Formalin (10% neutral buffered formalin is the most common).
• Mechanism: Cross-links proteins, stopping autolysis and bacterial decomposition. - Dehydration
• Purpose: Removes water from the tissue to prepare it for embedding.
• Method: Uses graded ethanol (70% → 80% → 90% → 95% → 100%) to gradually remove water.
• Why Gradual? Sudden dehydration can cause shrinkage or tissue damage. - Clearing
• Purpose: Removes alcohol and prepares the tissue for infiltration.
• Common Clearing Agents: Xylene, toluene, chloroform (Xylene is most commonly used).
• Why Necessary? Alcohol and paraffin wax are not compatible, so a clearing agent is used to replace alcohol before embedding. - Infiltration
• Purpose: Fills tissue spaces with a medium (usually paraffin wax) to provide support.
• Process: The tissue is placed in molten paraffin wax so that the wax replaces the clearing agent. - Embedding
• Purpose: Hardens the tissue so it can be cut into thin sections.
• Process: Tissue is placed in a mold with melted paraffin wax, which solidifies to form a tissue block. - Sectioning
• Purpose: Cuts thin slices of tissue for microscopy.
• Tool Used: Microtome
• Typical Thickness: 4–5 micrometers (µm) - Staining
• Purpose: Enhances contrast for microscopic examination.
• Most Common Stain: Hematoxylin and Eosin (H&E)
• Hematoxylin → Stains nuclei blue/purple (binds to DNA and RNA, which are acidic).
• Eosin → Stains cytoplasm and extracellular matrix pink (binds to basic proteins).
• Other Stains:
• PAS (Periodic Acid-Schiff) → Highlights carbohydrates (e.g., glycogen, mucins).
• Masson’s Trichrome → Stains collagen blue/green and muscle red.
• Silver Stain → Used for reticular fibers and some nervous tissue.
Summary of Tissue Processing Steps
Step |Purpose| Common Agents Used
1.Fixation |Preserve tissue|10% formalin
2.Dehydration|Remove water| Graded ethanol
3.Clearing| Replace alcohol| Xylene
4. Infiltration Support tissue Molten paraffin wax
Embedding Solidify tissue Paraffin mold
Sectioning Cut thin slices Microtome
Staining Contrast enhancement H&E stain
Would you like me to expand on any step?
How to mount slides
Cover slip covers the specimen on the slide.
Mounting means placing the tissue on a slide, adding a medium, and covering it with a coverslip to preserve it permanently. It’s not just putting it under the microscope.
mounting = slide + mounting medium + coverslip — that’s what makes it a permanent slide.
Mount slide on a stage by pulling the caliper (A caliper on the microscope stage helps secure and position the slide firmly and precisely for observation. It is part of the mechanical stage and has a spring-loaded clip or holder that grips the slide. ) and allowing the slide to slide jnto the aperture. You then leave the caliper to hold the slide slowly in place
You then switch on the light and ensure that it’s the lowest objective lens in place. The lowest objective lens is the shortest.
If you don’t do this, you will break the slide cuz as you move to high powers, the lens increases.
The stained portion of the tissue you’re looking at must be in line with the light or the light must be passing through the stained portion.
You use stage controls to position the slide to make sure what you’re looking for is in line with your eyes.
Use coarse adjustment for focusing. When you move the knob anti clockwise, the stage will be going down and clockwise, it will go up
X10 power gives you the general overview of what the tissue is about.
If you want details of the tissue, move to the next power and make sure you hear a clicking sound when you move to the next power cuz the clicking sound shows it’s in place.
Use control knobs to make sure you see a stained portion from your eyepiece. The anterior posterior control knobs move the stage left and right (X axis movement controlled by the horizontal knob) or backwards and forwards (Y axis movement controlled by the vertical knob)
When you shift to a higher objective lens power, it distorts the focusing you did initially so you have to use the coarse and fine adjustments again to make sure the focusing is done well.
But be careful cuz as you increase the power, the lens becomes longer so if you adjust it too much, the lens can hit the slide and break it or spoil the specimen I guess
Great question!
In malaria parasite diagnosis, the slides used for thick and thin blood films are usually not permanently mounted — they are typically temporary preparations.
Here’s the difference:
• You stain directly on the slide (e.g., using Giemsa stain).
• After staining and drying, you count the parasites directly under the microscope.
• But no coverslip and mounting medium are usually added, especially in routine diagnosis.
So technically, that slide is not a mounted (permanent) slide, even though the sample is on the slide. It’s more of a temporary stained slide used for immediate diagnosis.
Summary:
Malaria-stained slides are not typically mounted slides — they are stained and read without a coverslip or mounting medium.
If you want to preserve it long-term, you could mount it. But most lab diagnostics don’t do that for malaria slides.
Want me to give you a line to easily remember this difference?
How is dehydration done
Why is it done?
Why must you dehydrate tissues?
Because water and paraffin wax do not mix.
• The tissue contains water, but wax (used for embedding) is hydrophobic.
• So, you must remove the water first—this is done by dehydrating the tissue using increasing concentrations of alcohol (ethanol).
Simple summary:
“Tissues are dehydrated to remove water, because water prevents wax from infiltrating the tissue.”
There’s 50% graded alcohol.
For dehydration: alcohol replaced water in the cells
Then xylene dissolved the alcohol and then paraffin displaces xylene. Specimen is now ready to be embedded
So if it was a skin tissue, you cut it into small pieces, put it in a container called the cassette. You put the cassette in automated tissue processor machine if you have which contains the graded alcohol and xylene and do it for you. It then brings the tissue out and you do embedding with paraffin then you block the tissue and put in the micro tube and you section the tissue and this will make it come out as very flat thin slices and you then put them on top of water to stretch the sections. Then you pick them and put them on the slide then you mount it and stain and cover with a cover slip.
Tissue Processing Overview
1. Fixation:
• Tissue is cut into small pieces and placed in a cassette.
• Fixed using formalin or another fixative to preserve structure.
2. Dehydration:
• Tissue is placed in graded alcohol (e.g., 50% alcohol first, then higher concentrations).
• Alcohol replaces water in the cells.
3. Clearing:
• Xylene dissolves the alcohol, making the tissue compatible with paraffin.
4. Infiltration:
• Paraffin displaces xylene, preparing the specimen for embedding.
5. Embedding:
• The tissue is embedded in paraffin wax to provide support.
• The block is formed for sectioning.
6. Sectioning:
• The block is placed in a microtome, where it is cut into thin slices.
• The sections are placed on warm water to stretch them.
7. Mounting & Staining:
• Sections are transferred onto slides.
• Staining is done, usually using H&E (Hematoxylin & Eosin).
• A cover slip is placed over the section to protect it.
This process ensures tissue is properly prepared for microscopic examination.
Why Do We Need to Embed the Tissue?
Embedding is done to provide support and stability to the tissue before sectioning. Without embedding, the tissue would be too soft and fragile to cut into very thin slices.
• Paraffin embedding: The tissue is infiltrated with paraffin wax, which hardens and allows for precise slicing.
• Alternative methods: Some use resins for harder tissues like bone.
What is a Microtome?
A microtome is a machine used to cut the embedded tissue into very thin slices (usually 3–5 micrometers thick).
• The paraffin block (containing the tissue) is placed in the microtome.
• A sharp blade moves across the block, cutting thin sections.
• The sections float on warm water to flatten before being transferred to a slide for staining.
Without embedding, the tissue would be too delicate to cut properly.
How is brain tissue processes?
Why do you have to keep putting the tissue in increasing concentrations of alcohol for dehydration?
The tissue is placed in increasing concentrations of alcohol (graded alcohol) during dehydration to gradually remove water from the cells without causing damage.
Why Not Use 100% Alcohol Immediately?
• If you put the tissue directly into 100% alcohol, the sudden removal of water would cause shrinkage and distortion.
• A gradual increase (e.g., 50% → 70% → 90% → 100%) allows for controlled dehydration, preventing damage to delicate structures.
What Happens After Dehydration?
Once the water is fully replaced by alcohol, the next step is clearing (e.g., with xylene), which removes the alcohol and makes the tissue compatible with embedding media like paraffin wax.
Why do you have to keep putting the tissue in increasing concentrations of alcohol for dehydration?
The tissue is placed in increasing concentrations of alcohol (graded alcohol) during dehydration to gradually remove water from the cells without causing damage.
Why Not Use 100% Alcohol Immediately?
• If you put the tissue directly into 100% alcohol, the sudden removal of water would cause shrinkage and distortion.
• A gradual increase (e.g., 50% → 70% → 90% → 100%) allows for controlled dehydration, preventing damage to delicate structures.
What Happens After Dehydration?
Once the water is fully replaced by alcohol, the next step is clearing (e.g., with xylene), which removes the alcohol and makes the tissue compatible with embedding media like paraffin wax.
State the two main types of stains used
STAINING
1. Basic dyes e.g. haematoxylin, toluidine blue, alcian blue, and methylene blue - (stains blue, blue-black).
2. Acidic dyes e.g. eosin (stains purple/pink)
There can also be special stains such as the GIEMSA stain for malaria parasite
The parts in the tissue that will be stained depends on the biochemical structure of the tissue
Basophilic structures (-ve charged/acidic subs.)
e.g. nucleic acids, GAGs: high affinity for basic
dyes (+ve)
• Acidophilic structures (+ve charge sub./basic) e.g. proteins, mitochondria, collagen: high affinity for
acidic dyes (-ve).
Nice question!
Why do nucleic acids stain blue?
Because nucleic acids (DNA and RNA) are acidic (negatively charged), and they attract basic (positively charged) dyes like hematoxylin, which stains them blue or purple.
Simple explanation:
• Nucleic acids = acidic → attract basic dyes.
• Hematoxylin = basic dye → binds to DNA/RNA → stains them blue/purple.
This is part of what we call “basophilic staining” — tissues that attract basic dyes.
One-liner summary:
“Nucleic acids stain blue because they’re acidic and attract basic dyes like hematoxylin.”
Want a quick way to remember it?
“Acid loves base — DNA loves blue.”
Let me know if you want a visual or chart to help you lock this in!
Yes! In the microtome, the tissue is sliced into thin sections, and these sections often form a ribbon-like structure.
Why Do Ribbons Form?
• The microtome knife slices the tissue sequentially, so each new slice remains attached to the previous one, forming a continuous strip or ribbon of sections.
• This makes it easier to handle and transfer multiple sections at once onto a slide for staining and examination.
How Are the Ribbons Handled?
1. The ribbon is floated on warm water to spread and straighten the sections.
2. Individual sections are then picked up onto glass slides for further processing.
The ribbon formation is especially important in paraffin-embedded tissues, where the thin slices (typically 3-5 micrometers thick) need to stay intact for proper staining and microscopic examination.
Periodic acid shape is a type of special stain and are specifically used for goblet cells.
And why?
Why not H and E stain
Periodic Acid-Schiff (PAS) stain is a special histochemical stain used to highlight polysaccharides, mucins, and glycoproteins, which are abundant in goblet cells.
Why is PAS stain used for goblet cells?
• Goblet cells secrete mucus, which contains mucin, a glycoprotein.
• PAS stain reacts with carbohydrate-rich structures, turning them magenta (pink-purple).
• This makes goblet cells easily visible under a microscope, helping to identify and study them in tissues like the intestine and respiratory tract.
Hematoxylin and Eosin (H&E stain) is the standard stain in histology, but it does not specifically highlight goblet cells well because:
1. Hematoxylin stains nuclei (blue-purple) but does not strongly react with mucins.
2. Eosin stains cytoplasm and extracellular proteins (pink), but mucins in goblet cells are weakly eosinophilic or may appear almost clear.
3. Goblet cells contain polysaccharides and glycoproteins, which require a carbohydrate-specific stain like PAS to be clearly visualized.
Thus, PAS stain is preferred for goblet cells because it specifically reacts with the mucins, making them bright magenta, while H&E may not differentiate them clearly from the surrounding tissue.
Is xylene miscible with alcohol and wax?
It’s miscible with wax which will help with embedding the tissue with the wax but it is not miscible with alcohol cuz it’ll need to displace the alcohol since the alcohol will prevent the wax from embedding in the tissue cuz wax and alcohol are immiscible. So alcohol will be displaced by xylene and then xylene will come and mix with the wax for the tissue to embed with the wax easily
Xylene is not miscible with alcohol because its function is to displace it, not mix with it. But it is miscible with wax because it helps the wax penetrate the tissue effectively.
Autopsy and biopsy difference
Why must fixatives be used to fix?(talk about cross linking of proteins?
Fixatives are reagents that cross link proteins. Cross linking is important because it binds the proteins together to make them more stable and this prevents the tissue from decaying
Cross-linking reagents bind proteins to each other, making them more stable and preserving the tissue structure.”
For example, formaldehyde forms covalent bonds between amino groups in protein molecules — this is what we mean by cross-linking.
Would you like a super simple line to remember it?
“Cross-linkers lock proteins together to preserve the tissue.”
Diff between embedding and infiltration
Great question!
Difference between Infiltration and Embedding:
1. Infiltration
• This is the process where molten wax (or embedding medium) enters the tissue after clearing. • The goal is to replace the clearing agent (like xylene) inside the tissue with wax. • It prepares the tissue to be solidified in wax. “Infiltration = Wax enters the tissue.” 2. Embedding • This is the step after infiltration, where the infiltrated tissue is placed in a mold and surrounded with molten wax, which is then allowed to cool and harden. • It gives the tissue a solid block form for easy cutting (sectioning). “Embedding = Tissue is placed in a block of wax.”
Simple way to remember:
Infiltration fills the tissue. Embedding surrounds the tissue.
Want a one-line analogy?
“Infiltration is soaking a sponge with wax; embedding is placing the sponge into a wax block.”
Let me know if you’d like a diagram-style summary too.
Great! The full meaning of DPX is:
Distrene Plasticiser Xylene
What it is:
DPX is a synthetic resin-based mounting medium used in histology to permanently mount slides (after staining), especially with a coverslip.
Breakdown:
• D = Distrene (a polystyrene resin)
• P = Plasticiser (helps keep the resin flexible)
• X = Xylene (the solvent that keeps everything in liquid form until it dries)
Simple summary:
DPX = a clear, quick-drying mounting medium used with a coverslip to make slides permanent.
Let me know if you’d like a short memory trick for that too!
What are artifacts
Great! Here’s a clear and simple explanation:
Artifacts in histology are unwanted or artificial changes that appear in tissue sections — they are not part of the normal tissue but happen due to errors or effects during tissue processing, sectioning, or staining.
Examples of artifacts include:
• Folds or wrinkles in the tissue section
• Air bubbles under the coverslip
• Knife marks or lines (from microtome blades)
• Shrinkage or distortion of tissues
• Staining precipitates (extra dye particles)
• Cracks or tears in tissue
• Tissue separation from the slide
Causes of artifacts:
• Poor fixation
• Improper dehydration or clearing
• Rough handling during sectioning
• Overheating during embedding
• Contaminated staining reagents
• Incorrect mounting technique
Simple one-liner to remember:
Artifacts are false features caused by handling or processing errors, not by the actual tissue itself.
Want a short table to help you memorize causes and examples side by side?
Excellent thinking — that’s a smart question!
What if it’s not an artifact but a new discovery?
That’s why identifying artifacts requires experience, context, and comparison with known tissue patterns. If something looks unusual:
1. Check if it repeats in other slides or samples.
• Artifacts often appear in multiple areas due to handling, while a real discovery is consistent in structure and location in biological context.
2. Compare with normal histology.
• If the change matches a biological pattern (e.g., a new type of cell, structure, or staining behavior), it might be a real finding, not an artifact.
3. Consider reproducibility.
• A new discovery should consistently appear in similar tissues or under the same conditions, while artifacts tend to be random or inconsistent.
4. Consult others or literature.
• If you suspect something new, you can compare with references or ask experienced pathologists.
Bottom line:
Artifacts usually look messy, irregular, or technical in origin. A true discovery fits biology, has structure, and shows a pattern — not random distortion.
Would you like a short checklist to help you distinguish artifact vs possible real finding?
Type of Artifact | How to Identify It |
|—————————-|—————————————————————————————–|
| Tissue folds/wrinkles | Tissue appears doubled over itself, twisted, or uneven in thickness. |
| Air bubbles | Clear, round, empty circles under the coverslip. |
| Knife marks (chatter lines)| Repeated parallel lines or ridges across the tissue section from the microtome blade. |
| Staining precipitates | Granular dye clumps or spots not part of the tissue structure. |
| Tissue shrinkage | Gaps between tissue and slide, or distorted shrunken appearance. |
| Tears or cracks | Breaks, holes, or splits in the tissue section. |
| Tissue separation from slide| Tissue lifts or floats away from the slide in some areas. |
