Hey poly peeps, Desmond here. It's week 16 and just another 4 more weeks before we go back to campus. Having covered Cytopathology and Histopathology routine (embedding and microtomy) in my 2 previous posts, I've decided to touch on some of the special stains performed in Histopathology.
GRAM STAIN
Function:
This stain differentiates Gram positive and Gram negative bacteria
Principle:
Both bacteria’s, positive and negative cell wall is composed of peptidoglycan, (the gram positive has a thicker wall) and both will take up the crystal violet. The gram-negative has a layer of lipopolysaccharide external to the peptidoglycan wall, which is disrupted in the acetone rinse, allowing the crystal violet to be differentiated out. This allows the gram-negative bacteria to take up the fuchsin stain.
Control:
An infected appendix, or any tissue containing both negative and positive gram rods.
Fixative:
1. 10% Buffered Neutral Formalin
Reagents required:
1% aqueous crystal violet solution
1% Basic fuchsin
Iodine
Potassium iodide
Formalin 37-40%
Glacial acetic acid
Picric acid
Acetone
Xylene
Reagent Preparation:
1. Gram iodine solution
Iodine 1g
Potassium iodide 2g
Type II water 300ml
Allow iodine and potassium iodide to dissolve completely. Mix before use. Solution is stable for 6 months at room temperature.
2. Gallego’s differentiating solution
Type II water 100ml
Formalin 37-40% 2ml
Glacial acetic acid 1ml
Mix before use. Solution is stable for 6 months at room temperature
3. Picric acid-acetone solution
Picric acid 1g
Acetone 100ml
Solution is stable at room temperature for 6 months.
4. Acetone-Xylene solution
Equal parts of acetone and xylene.
Solution is stable at room temperature for 6 months.
5. 1% Crystal violet
Crystal violet 1g
Type II water 100ml
Filter into bottle. Solution is stable for 1 year at room temperature.
6. 1% Basic Fuchsin
Basic Fuchsin 1g
Type II water 100ml
Filter into bottle. Solution is available for 6 months at room temperature.
Staining Procedure:
1. Dewax and bring section to water.
2. Place in 1% crystal violet solution for 1 minute.
3. Rinse in tap water.
4. Place in Gram iodine solution for 1 minute
5. Rinse in tap water.
6. Decolourize in acetone until background is clear.
7. Immediately wash in tap water.
8. Place in 1% Basic Fuchsin solution for 5 minutes.
9. Rinse in tap water.
10. Place in Gallego’s differentiating solution, 2 changes, 1 minute each.
11. Rinse in tap water.
12. Transfer to a staining dish.
13. Treat with acetone for 30 seconds.
14. lace in picric acid-acetone solution for 2-3 minutes.
15. Place in acetone-xylene solution for 2 changes.
16. Clear in xylene, 2 changes.
17. Mount in DPX.
Results:
Gram positive - Blue
Gram negative - Red
Background - Yellow
Ziehl Neelsen Stain
Function:
To demonstrate acid fast bacteria belonging to the genus mucobacterium
Principle:
The lipid capsule of the acid-fast organism takes up carbol-fuchsin and resists decolourization with a dilute acid rinse. The lipoid capsule of the mycobacteria is of such high molecule weight that it is waxy at room temperature and is not stained by bluing solutions such as methylene blue.
Control:
Any tissue containing acid-fast organisms
Fixative:
1. 10% Buffered Neutral Formalin
Reagents required:
Commercial TB stains (Merck)
Loeffler’s Methylene Blue
1% Potassium Hydroxide
1% acid alcohol
Reagent Preparation:
1. Stock Loeffler’s Methylene Blue solution
Methylene Blue 1gm
95% Alcohol 100ml
2. 1% Potassium Hydroxide
Potassium Hydroxide 1gm
Type II water 100ml
3. Working solution
1% Loeffler’s Methylene Blue 30ml
Type II water 99ml
1% Potassium Hydroxide 1ml
4. 1% Acid Alcohol
Concentrated Hydrochloric Acid 20ml
70% Alcohol 1980ml
Staining Procedure:
1. Dewax and bring sections to water.
2. Commercial TB colour – 5 minutes (or hot carbol fuchsin solution for 30 mins)
3. Differentiate with 1% alcohol
4. Wash in water
5. Couterstain with 1% Loeffler’s methylene blue.
6. Wash in water and go straight to 95% alcohol to control blue colour.
7. Dehydrate clear and mount in DPX.
Results:
Tubercle bacilli - Red
Background - Blue
LENNERT GIEMSA STAIN
Function:
Stains Bone marrow lymph node. Differentiates cells present in hematopoietic (lymph nodes).
Principle:
The neutral dyes combining the basic dyes methylene blue and dye eosin, give a wide colour range when staining. The more acid pH levels give more selective chromatin staining and less cytoplasmic basophilla. Less acid pH levels give denser nuclei and increased cytoplasmic basophilla.
Control:
Spleen
Fixative:
1. 10% Buffered Neutral Formalin
Reagents required:
Commercial Giemsa Stain
Reagent Preparation:
Working Giemsa solution (for 5 slides or less)
Giemsa Solution 3ml
Type II water 12ml
Mix and use in plastic slides mailer. Discard after use.
Staining Procedure:
1. Dewax and bring section to water.
2. Rinse section in Type II water
3. Place sections in working Giemsa solution for 1 hour at room temperature.
4. The sections are removed from the Giemsa solution and put into 100ml Type II water, to which 3-4 drops of undiluted glacial acetic acid have been added. The sections are agitated gently in this solution for a few seconds, slightly differentiated and then immediately put into 96% ethyl alcohol, in which they are differentiated further until the desired staining is achieved (microscopic control).
5. Differentiation is stopped and, at the same time, dehydration is achieved by dipping in 3 changes of isopropanol for 2 minutes each.
6. Dehydrate, clear and mount with DPX.
Results:
RNA, DNA - Blue (Basophillic)
Acidophilic substances - Pink or reddish orange
Acid mucopolysaccharides - Reddish violet
JENNER’S GIEMSA STAIN
Function:
Stains Bone marrow and gastric biopsies. To demonstrate helicobacter. To differentiate cells present in hematopoeietic tissue (lymph nodes). Use in blood smears.
Principle:
The neutral dyes combining the basic dyes methylene blue and dye eosin, give a wide colour range when staining. The more acid pH levels give more selective chromatin staining and less cytoplasmic basophilla. Less acid pH levels give denser nuclei and increased cytoplasmic basophilla. Methylene blue in an alkaline pH solution stains metachromatic.
Control:
Stomach or colon tissue containing helicobacter. Skin for mast cells.
Fixative:
1. 10% Buffered Neutral Formalin
Reagents required:
Commercial Jenner solution
Commercial Giemsa solution
1% acetic acid
Reagent Preparation:
1. Jenner working solution
Stock Jenner Solution 2ml
Type II water 2ml
Mix and use. Discard after use.
2. Giemsa working solution
Stock Giemsa solution 2ml
Type II water 40ml
Mix and use. Discard after use.
Staining Procedure:
1. Dewax and bring sections to water.
2. Place slides in 2 changes of methyl alcohol for 3 minutes,
3. Dip direct into working Jenner solution for 5 to 6 minutes.
4. Transfer direct to working Giemsa solution for at least 45 minutes.
5. Rinse in distilled water. Check under microcope and monitor differentiation
6. Differentiate in 1% acetic acid.
7. Dip in 95% alcohol for 5 to 6 times.
8. Dehydrate clear and mount in DPX.
Results:
Cytoplasm - Pink
Nuclei - Blue
Erythrocytes - Red
Mast Cell granules - Purple
Bacteria - Blue
Malaria Parasite - Blue
Desmond Heng
0503179D
TG02
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10 comments:
Hello Desmond,
It is such a long post! Anyway, here are some of my queries.
T and B cells are present in the lymphnode. HOw can the Lennert Giemsa stain differentiate the T and B cells?
Out of curiosity, what is the Type II water that is use in this procedure and what kind of cells will contain acid mucopolysaccharide that will turn reddish violet upon staining with Lennert Giemsa stain?
=) thanks!
Loh Sharon, tg 01
hey hi desmond!
erm are all the stains mentioned above can be viewed under normal microscopy?
thanks,
natalie
Hi Desmond
What is a infected appendix?
Thanks
Eugene Wong
TG02
Heya...
Just wanna ask ya what is the purpose of testing the presence of acid fast bacteria in the tissues?
Thanks ya
michelle
tg02
Hey desmond,
im quite curious to know whats the difference between safranin and fuchsin. doesnt both of them stain gram negative cells?
thanks alot!
boonching
hey des,
I want to know what troubleshooting steps do you take when you realise the gram stain is not working well.
thanks,
randall
tg02
hihi desmond that was a long list of some type of staining in histo.
as i was wondering, if your control slide is not done well. what actions will be taken?
Juexiu
tg02
It has come to my attention that the answers I posted on the 24th of October got deleted. I only discovered this today, so here are the answers again.
To sharon,
T and B cells are differentiated based on the different pH levels. Type II water is simply distilled water. Tumour/cells cells contain acid mucopolysaccharides that turn reddish violet upon staining.
To Natalie,
All of the mentioned stains can be viewed under electron microscopy.
To Eugene,
An infected appendix refers to appendicitis; Appendicitis is an inflammation of the lining of the appendix spreading to its other portions. When an acute inflammation is involved, severe pain will be felt in the lower right part of the abdomen. By this time the appendix has usually become filled with pus. If not treated right away the infection can spread through the wall of the appendix and can turn into gangrene and rupture.
To Michelle,
Acid-fastness is a physical property of some bacteria referring to their resistance to decolorization by acids during staining procedures.
Acid-fast organisms are difficult to characterize using standard microbiological techniques (e.g. Gram staining), though they can be stained using concentrated dyes, particularly when the staining process is combined with heat. Once stained, these organisms resist the dilute acid and/or ethanol-based de-colorization procedures common in many staining protocols—hence the name acid-fast.
To boonching,
Acid fuchsin distributes more rapidly and more widely than safranin, reflecting differences between the dye molecules in state of ionization.
To Randall,
Problem: Over-decolorization: This is an extremely common problem, often caused by using a strong decolorizer or by leaving the decolorizer on the slide for too long.
Solutions: Use decolorizer manufactured by the same company as your stains,and follow their protocol.
• Run positive and negative controls with your daily stains.
• Test your decolorizer solution using different times (e.g. 5, 10, 15 seconds) with positive, negative and some weakly positive cultures; until you find a method that works well for you.
Problem: Gram variability: This can be due to the organism itself, and not to the staining method.
Solutions: The vast majority of Gram-variable organisms are Gram-positive. Characteristically Gram variable organisms (e.g. Corynebacterium variabilis) or those whose membranes alter with age and appear Gram-variable (e.g. Arthrobacterium spp.) are grouped with the Gram-positive organisms. Therefore, they are treated as Gram-positive organisms.
To Juexie,
If the control slide for a specific batch is not stained properly, that specific batch would be restained.
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