PART 3 - HP - PT -fr Sir Ed Flashcards
Four Major Groups of Fixatives
It act by cross-linking proteins
Aldehydes (formaldehyde, glutaraldehyde)
Four Major Groups of Fixatives
act by cross-linking proteins
Oxidizing agents (osmium tetroxide, potassium permanganate)
Four Major Groups of Fixatives
act by denaturing proteins
Alcohol-based fixatives (methanol, ethanol, acetic acid)
Four Major Groups of Fixatives
act by forming insoluble metallic precipitates
Metallic fixatives (mercuric chloride and picric acid)
Aldehyde Fixatives
-Best for CNS and Gen. post-mortem tissues, ideal for silver impregnation
10% Formol-Saline
Aldehyde Fixatives
Best Gen. tissue fixative; Frozen section, Iron and elastic fibers; Immunohistochemistry and interphase FISH
10% Neutral-Buffered Formalin
Aldehyde Fixatives
for Immunohistochemistry
Zinc Formalin (unbuffered)
Aldehyde Fixatives
for Routine post-mortem tissues, Silver Reticulum methods; Neutral fats and
phospholipids
Formol-Corrosive (F-Sublimate)
Aldehyde Fixatives
EM; Immunocytochemical analysis; Paraffin embedding and sectioning
Paraformaldehyde
Aldehyde Fixatives
EM; Resin embedding and sectioning
Karnovsky’s Fixative
Aldehyde Fixatives
Electron Microscopy
Acrolein
Aldehyde Fixatives
Lipids (frozen section); Fixation & decalcification of calcium-rich tissues
Formol-calcium (Baker’s)
Aldehyde Fixatives
Sputum cytology
Gendre’s
Aldehyde Fixatives
for Phospholipids
Lillie’s (Calcium acetate formalin)
Aldehyde Fixatives
For Nervous tissue (astrocytes)
Cajal’s Formol-ammonium bromide
Aldehyde Fixatives
Wet and dry smears, blood smears, bone marrow tissues and cell cultures; Diluent
in Wright’s stain
Methyl Alcohol (100%)
Aldehyde Fixatives
Fixing touch preparations (impression smears)
Isopropyl Alcohol (95%
Aldehyde Fixatives
Histochemistry especially enzyme studies; Sodium urate crystals in patients with
gout; DNA fragments (PCR)
Ethyl Alcohol (70-100%)
Aldehyde Fixatives
(most rapid fixative)
Chromosomes, lymph glands, urgent biopsies; Brain tissues for rabies diagnosis
(Negri bodies); Nissl and cytoplasmic granules
Carnoy’s Fixative (most rapid)
Aldehyde Fixatives
Frozen sections and smears; Nucleic acids
Clarke’s Solution
Aldehyde Fixatives
Fixation or post-fixation of large fatty specimens (breast); Lymph nodes detection
Alcoholic Formalin
Aldehyde Fixatives
Diagnostic cryostat sections
Formol-Acetic Alcohol
Aldehyde Fixatives
Sputum; Frozen sections; Immunoperoxidase studies and EM; Glycogen and
Micro-incineration technique
Gendre’s Fixative
Aldehyde Fixatives
Mucopolysaccharides and nuclear proteins
Newcomer’s Fluid
Mercuric chloride Fixatives
Tissue photography; Renal tissues, fibrin, CT and muscles; Metachromatic staining
and Trichrome staining; Hematopoietic & RES
Mercuric chloride
Mercuric chloride Fixatives
Good general fixative; Recommended for PTAH & Trichrome staining; Congested
specimens (lung, heart and blood vessels)
Zenker’s Solution
Mercuric chloride Fixatives
Blood-containing organs (pituitary gland, BM, liver and spleen); Extramedullary
hematopoiesis and intercalated discs of cardiac muscle
Zenker-Formol (Helly’s) Solution
Mercuric chloride Fixatives
BM biopsies (hematopoietic tissues); Immunohistochemical staining
Lillie’s B-5 Fixative
Mercuric chloride Fixatives
Tumor biopsies (skin)
Heidenhain’s Susa Solution
Mercuric chloride Fixatives
Wet smears for cytologic examinations
Schaudinn’s (Sublimated alcohol)
Mercuric chloride Fixatives
Lipids and proteins; Nervous tissue and brain; Nuclear preservation
Ohlmacher’s Solution
Mercuric chloride Fixatives
Lipids and fatty tissues (liver and adipose); Nuclear preservation
Carnoy-Lebrun Solution
Osmium tetroxide Fixatives
Nuclear structures (Chromosomes); Fixes fats
Flemming’s Solution
Osmium tetroxide Fixatives
Cytoplasmic structures (mitochondria)
Flemming’s w/o Acetic Acid
Chromate Fixatives
Precipitates all proteins and adequately preserves carbohydrates
Chromic Acid
Chromate Fixatives
Chromatin, mitochondria, mitotic figures, golgi bodies, RBCs and colloidcontaining tissues
Regaud’s (Muller’s) Fluid
Chromate Fixatives
Study of early degenerative processes and tissue necrosis; Rickettsia and other
bacteria
Orth’s Fluid
Chromate Fixatives
Preserves mitochondria, fixes lipids
Potassium Dichromate
Picric acid Fixatives
-Embryos and pituitary biopsies; GIT biopsies, animal embryos and endocrine
gland tissue
-GREEN GREGORIOS: Preserving soft and delicate structures (endometrial
curettings); Fragmentary biopsies; Masson’s trichrome; Not suitable for fixing
kidney structures, lipid and mucus
Bouin’s Solution
Picric acid Fixatives
GIT specimens and endocrine tissues; Has decalcifying properties
Hollande’s Solution
Picric acid Fixatives
-Glycogen and other carbohydrates
-BLACK GREGORIOS: Preserving soft and delicate structures (endometrial
curettings); Fragmentary biopsies; Masson’s trichrome; Not suitable for fixing
kidney structures, lipid and mucus
Gendre’s Solution
Picric acid Fixatives
Best routine fixative for glycogen
Brasil’s Alcoholic Picroformol
Other Fixatives
Nucleoproteins (nuclei); Chromosomes and chromatin materials (nuclear
components); Destroys mitochondria and Golgi bodies
Glacial acetic acid (HAc)
Other Fixatives
Acid mucopolysaccharides and CT mucin
Lead Fixatives
Other Fixatives
Proteins and nucleic acids; Weak decalcifying agent
Trichloroacetic Acid (TCA)
Other Fixatives
Water diffusible enzymes (Lipases, Phosphatases); Brain tissues for rabies
diagnosis (Negri bodies); Solvent in freeze-substitution tech
Acetone
Other Fixatives
Transport medium of fresh unfixed tissues; NOT a fixative
Michel’s Solution
Fixative
Recommend for Immunohistochemical techniques, cell smears, nucleic acids, CNS: Formaldehyde
Formaldehyde
Fixative
Recommended for chromaffin tissues, adrenal medulla and mitochondria
Chromate
Fixative
Recommended for Connective tissues and glycogen; Histones & basic proteins; Biogenic and polypeptide hormones Immunostaining; Fragmentary biopsies:
Picric acid
Fixative
Good fixative and excellent stain for lipids:
Osmium tetroxide
Nuclear and histochemical fixative
Newcomer’s fluid
Microanatomical and histochemical
10% Formol saline
Microanatomical and nuclear:
Heidenhain’s susa
Microanatomical and cytoplasmic
Zenker-formol (Helly’s)
10% NBF Composition
✓ 37-40% formaldehyde - 100 mL (10% = equivalent to 3.7-4% formaldehyde)
✓ DW - 900 mL
✓ Sodium phosphate, monobasic - 4 g
✓ Sodium phosphate, dibasic (anhydrous) - 6.5 g
*anhydrous – no water / water-free
Bouin’s Fixative Composition
✓ 2.1% Picric acid saturated aqueous solution - 75 mL
✓ 40% formaldehyde - 25 mL
✓ Glacial Acetic acid - 5 mL
Most commonly used fixative in histology:
Formaldehyde
Most widely used fixative for routine histology:
10% NBF
Used to slow down decomposition if the tissue needs to be photographed and cannot be fixed
immediately
Refrigeration
Used to prevent formation of dark brown artifact pigment granules (hematin) when using formalin fixatives:
✓ Add buffer
- Sat. alcoholic Picric acid
- Others: Acidified potassium phosphate buffer, Sodium acetate buffer and Citrate buffer
✓ Add Alcoholic KOH (1% KOH in 80% alcohol)
Fixative and decalcifying agent
Trichloroacetic acid
Picric acid
Chromic acid
Fixative and dehydrating agent:
Alcohol (Methanol, Carnoy’s, Gendre’s)
Acetone
Fixative and stain:
Picric acid, Osmium tetroxide
Decalcifying agent and tissue softener:
Perenyi’s fluid
Dehydrating agent and clearing agent (universal rgt):
Tetrahydrofuran
Dioxane,
3° Butanol
Oil of Bergamot
May cause excessive hardening or brittleness of tissues:
✓ Prolonged fixation
✓ Prolonged dehydration
✓ Prolonged clearing
✓ Prolonged infiltration
✓ Prolonged embedding (overheating)
Airholes found in the tissue during trimming is due to:
Incomplete infiltration
Prolonged dehydration in the higher grades of alcohol will render the specimen:
Hard and brittle
Makes the tissue opaque and difficult to cut due the presence of alcohol:
Incomplete clearing
The most common (routine) and most rapid decalcifying agent used so far:
Nitric acid
Best for cellular preservation and staining; Recommended for routine decalcification of post-mortem research tissues:
Formic acid
Recommended for Surface decalcification of tissue blocks if used in 1% solution with 70% alcohol:
Hydrochloric acid
Permits good nuclear staining; Does not require washing out; Not recommended for urgent examinations (very slow acting):
Trichloroacetic acid
A good clearing agent must be ____________ with dehydrating agent (alcohol), melted paraffin wax and mounting medium.
Miscible
An excellent and true clearing agent; most rapid:
Xylene
Substitute for xylene or benzene:
Toluene
It is recommended for tough (skin, fibroid and decalcified tissues) and large tissue specimens;
Chloroform
Clearing agent
Rapid acting, recommended for urgent biopsies and routine purposes
Benzene
Dehydrates and clears at the same time since it is miscible in both water and paraffin:
Tetrahydrofuran
Recommended for CNS tissues and cytological studies (esp. Smooth muscles and skin):
Cedarwood oil
Clears both Paraffin and Celloidin sections; Quality is not always uniform and good and is extremely slow
Cedarwood oil
Properties are very similar to chloroform but it is cheaper; Toxic on prolonged exposure:
Carbon tetrachloride
Slow-acting clearing agents that can be used when double embedding techniques are required:
Methyl-benzoate/Methyl-salicylate
The clearing agent that becomes milky on prolonged storage and its quality is not always good and uniform:
Cedarwood oil
The clearing agent that becomes milky when dehydration is not complete:
Xylene
▪ Infiltrating and embedding media:
✓ Paraffin
✓ Paraffin substitutes
✓ Celloidin
✓ Gelatin
✓ Resin
The simplest, most common, and by far the best embedding medium for routine use:
Paraffin wax
The impregnation method used for delicate specimens and frozen tissue sections because it prevents fragmentation of tough and friable tissue:
Gelatin Impregnation
Specimens with large cavities or hollow spaces:
Celloidin wax impregnation
EM; Hard tissues (undecalcified bone); renal and BM biopsies:
Resin/Plastic Impregnation
Preferred for processing of whole eye sections.
Material embedded with the dry method can be cut without alcohol due to the presence of the cedarwood oil in the block.
DRY Celloidin Method
equal parts of chloroform and cedarwood oil, what mixture it is?
Gilson’s mixture