Microorganisms
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Carbol-fuchsin: Kinyoun, Ziehl-Neelsen, Fite
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Gram modified (Brown-Brenn)
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Giemsa
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Hotchkiss-McManus PAS for fungi
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CAS for fungi
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Grocott methenamine silver for fungi
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Mayer mucicarmine for C. neoformans
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Warthin-Starry for spirochetes
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Dieterle for spirochetes, Legionella
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Steiner/Steiner for spirochetes, Legionella, H. pylori
Carbol-fuchsin methods:
Kinyoun
Purpose: demo AFB, namely M. tuberculosis, M. avium
Principle:
After staining with carbol-fuchsin, the waxy lipid-rich capsules of AFB are resistant to decolorization with acid alcohol, so they retain the red stain. Non-AFB do not possess these waxy capsules, and so acid alcohol will decolorize the stain from them.
The waxy capsules are also very heavy in molecular weight, and remain waxy at room temp - so aqueous stains (like Gram) are not able to penetrate. Only alcoholic stains like carbol-fuchsin can penetrate these thick waxy lipidic capsules.
We use alcoholic acid (not aqueous acid) to decolorize, because alcohol provides more uniform decolorization.
The phenol crystals and alcohol in the carbol-fuchsin solution help enhance the stain, and also act as solvents.
Fixative:
- 10% NBF
- DO NOT USE CARNOY - this will turn AFB into non-AFB
Control:
- Sections must contain AFB
- Do not use overwhelmingly positive controls, or controls with too many organisms, as this can cause cross-contamination and over-decolorization
Ingredients:
-Kinyoun carbol-fuchsin solution:
- basic fuchsin - dye
- 95% alcohol - dye enchancer; solvent
- phenol crystals - dye enhancer; solvent
- Millipore water - solvent
-Acid alcohol - decolorizer; washes away carbol-fuchsin from non-AFB
- 70% alcohol
- HCl
-Methylene blue - counterstain
Results:
- AFB = BRIGHT RED
- Background = LIGHT BLUE
Notes:
- NO TAP WATER before carbol-fuchsin. Tap water contains AFB, which can create false-positives. Only use Millipore-filtered water before carbol-fuchsin stain. After the staining step, then you can use tap water.
- Rinse sections very well in water after acid alcohol, or else methylene blue will not stain.
- Time methylene blue counterstain very carefully, it's just a few quick dips. Do not overstain, or you will mask the AFB.
- if you do overstain:
- bring slides back to acid alcohol to remove methylene blue
- rinse very well in tap water to remove acid alcohol
- redo methylene blue counterstain
- Do not use overwhemingly positive controls, or controls with too many organisms. This can cause over-decolorization and cross-contamination during microtomy and staining.
- DO NOT let sections dry after carbol-fuchsin, or else a compound resistant to decolorization will form. If you try to remove this compound, eventually AFB will be decolorized too.
- Phenol aka carbolic acid - that's why it's called carbol-fuchsin. Penetration of phenol is enhanced by heat and wetting agents.
- Phenol is also highly toxic if ingested, inhaled, or skin absorbed. TWA 5ppm. Use gloves, goggles, fume hood.
- DO NOT use Carnoy! It will turn AFB into non-AFB.
Ziehl-Neelsen
Purpose: demo AFB, namely M. tuberculosis, M. avium
Principle:
After staining with carbol-fuchsin, the waxy lipid-rich capsules of AFB are resistant to decolorization with acid alcohol, so they retain the red stain. Non-AFB do not possess these waxy capsules, and so acid alcohol will decolorize the stain from them.
The waxy capsules are also very heavy in molecular weight, and remain waxy at room temp - so aqueous stains (like Gram) are not able to penetrate. Only alcoholic stains like carbol-fuchsin can penetrate these thick waxy lipidic capsules.
We use alcoholic acid (not aqueous acid) to decolorize, because alcohol provides more uniform decolorization.
The phenol crystals and alcohol in the carbol-fuchsin solution help enhance the stain, and also act as solvents.
Fixative:
- 10% NBF
- DO NOT USE CARNOY - this will turn AFB into non-AFB
Control:
- Sections must contain AFB
- Do not use overwhelmingly positive controls, or controls with too many organisms, as this can cause cross-contamination and over-decolorization
Ingredients:
-Ziehl-Neelsen carbol-fuchsin solution:
- basic fuchsin - dye
- 100% alcohol - dye enchancer; solvent
- phenol crystals - dye enhancer; solvent
- Millipore water - solvent
-Acid alcohol - decolorizer; washes away carbol-fuchsin from non-AFB
- 70% alcohol
- HCl
-Methylene blue - counterstain
Results:
- AFB = BRIGHT RED
- Background = LIGHT BLUE
Notes:
- This is the preferred method for AFB.
- All other notes same as Kinyoun.
Fite for leprosy
Purpose: demo AFB of M. leprae - only method that can demo leprosy
Principle:
After staining with carbol-fuchsin, the waxy lipid-rich capsules of AFB are resistant to decolorization with acid alcohol, so they retain the red stain. Non-AFB do not possess these waxy capsules, and so acid alcohol will decolorize the stain from them.
The waxy capsules are also very heavy in molecular weight, and remain waxy at room temp - so aqueous stains (like Gram) are not able to penetrate. Only alcoholic stains like carbol-fuchsin can penetrate these thick waxy lipidic capsules.
We use alcoholic acid (not aqueous acid) to decolorize, because alcohol provides more uniform decolorization.
The phenol crystals and alcohol in the carbol-fuchsin solution help enhance the stain, and also act as solvents.
Fixative:
- 10% NBF
- DO NOT USE CARNOY - this will turn AFB into non-AFB
Control:
- Sections must contain M. leprae
- Do not use overwhelmingly positive controls, or controls with too many organisms, as this can cause cross-contamination and over-decolorization
Ingredients:
-Xylene-peanut oil solution - to deparaffinize; also helps increase acid-fastness of M. leprae
-Ziehl-Neelsen carbol-fuchsin solution:
- basic fuchsin - dye
- 100% ethanol - dye enchancer; solvent
- phenol crystals - dye enhancer; solvent
- Millipore water - solvent
-Acid alcohol - decolorizer; washes away carbol-fuchsin from non-AFB
- 70% alcohol
- HCl
-Methylene blue - counterstain
Results:
- M. leprae, other AFB = BRIGHT RED
- Background = LIGHT BLUE
Notes:
- Can also stain Nocardia - just use sulfuric acid as decolorizer
- M. leprae and Nocardia are weakly acid-fast and not alcohol-fast - so avoid using alcohol solutions for deparaffinization and dehydration
- air dry sections before mounting instead of dehydrating in alcohol
- Issue with M. leprae is resistance of uptake of stain, not retention of stain.
- Fite is best for M. leprae; not as good for other AFB
- All other notes same as for Kinyoun
Gram+ and Gram- bacteria:
Gram - Brown-Hopps modification
Purpose:
- to demonstrate gram+ and gram- bacteria
- can also screen for:
- actinomycosis
- nocardiosis
- coccidiodomycosis
- aspergillosis
- rhinosporidiosis
- amebiasis
Principle:
Gram+ and gram- bacteria are distinguished by thickness of their cell wall. Gram+ bacteria have thicker cell wall with many more layers of peptidoglycan; this lets them resist decolorization from acetone. Gram- bacteria have thinner cell walls, and after decolorization, they will lose the crystal violet stain and become re-stained by the secondary stain (basic fuchsin).
Fixative:
- 10% NBF
Control:
- Must contain both gram+ and gram- bacteria
Ingredients:
* Crystal violet --> stains both gram+ and gram- bacteria
* Iodine --> mordant; forms dye lake with crystal violet
* Acetone --> decolorizer; will wash CV-iodine complex out of gram- bacteria
* Gallego solution --> differentiator
- formaldehyde
- acetic acid
* Picric acid-acetone --> counterstain; extra decolorizer because of acetone
Results:
Gram+ = BLUE-BLACK
Gram- = RED
Nuclei = LIGHT RED
Background = YELLOW
Notes:
- Weakly gram- bacteria can be stained more intensely by increasing concentration of basic fuchsin from 0.1% to 1.0%
- Picric acid must be anhydrous for better decolorization.
- Do not let sections dry at any point, or else insoluble compounds will form and you will never be able to decolorize them using picric acid-acetone!
- If your lab can't use dry picric acid, then use this intead: 95% ethanol, saturated aqueous picric acid
- If patient is on antibiotics, this will affect the cell walls of gram+ bacteria and they will not stain properly
Giemsa methods
Giemsa stains are used to demonstrate:
- H. pylori = peptic ulcer disease, gastritis
- Rickettsiae
- T. gondii
Modified Diff-Quik Giemsa for H. pylori
Purpose:
To demonstrate H. pylori
Principle:
The Giemsa method is polychromatic (aka Romanowsky). It is a "neutral" stain because it contains basic/cationic methylene blue and acid/anionic eosin. This color combo produces a range of colors in tissues and blood smears - the range of colors is due to impurities in the dyes. And at alkaline pH, methylene blue forms new metachromatic substances.
Diff-Quik is propprietary dye solution. This is the preferred method for H. pylori because it is the most sensitive, easy to carry out, and easy to reproduce.
Fixative:
- 10% NBF
Control:
- Sections must contain H. pylori
Ingredients:
* Diff-Quik Solution I --> stains cytoplasm
- xanthene (aka eosin) (-)
* Diff-Quik Solution II --> stains H. pylori, other bacteria, nuclei
- methylene blue, azure A (+)
* Acetic acid --> differentiator
Results:
H. pylori, other bacteria = BLUE
Nuclei = BLUE
Background = PINK
Notes:
- Diff-Quik I is buffered eosin Y (anionic, stains cytoplasm)
- Diff-Quik II is buffered mix of methylene blue and azure A (cationic, stains bacteria and nuclei)
- Do not leave sections too long in final water rinse of in dehydrating alcohols, or else it it further decolorize
- H. pylori means peptic ulcer disease, gastritis, gastric carcinoma, gastric lymphoma
Fungus methods
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CAS (chromic acid Schiff)
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Hotchkiss-McManus with PAS
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Grocott's methenamine-silver
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Mayer mucicarmine for C. neoformans
CAS (chromic acid Schiff)
Purpose:
To demonstrate fungi
Principle:
Chromic acid oxidizes polysaccharides in fungal walls into aldehydes, which are then stained by Schiff.
Chromic acid is much stronger oxidizer than periodic acid. It will remove all aldehydes except in the places with greatest concentration: glycogen, mucins, fungal cell walls. This means periodic acid provides a much cleaner background than periodic acid.
Fixative:
- 10% NBF
Control:
- Sections must contain fungi
Ingredients: