FIXATIVES AND THEIR SPECIFIC PURPOSE/FUNCTION Flashcards
memorization (88 cards)
1
Q
Mos widely used fixative of all
A
Formaldehyde (formalin)
2
Q
Formalin is a saturated solution of ____ w/v formaldehyde in water
A
37-40% w/v
3
Q
The usual 10% formalin used in fixation of tissues is a 10% solution of formalin, containing about ____% weight to volume of formaldehyde
A
4% weight to volume of formaldehyde
4
Q
Formaldehyde fixation is performed at what temperature?
A
Room temperature
5
Q
NOTE: Formaldehyde fumes are irritating to the nose and eyes and may cause ALLERGIC RHINITIS, SINUSITIS, EXCESSIVE LACRIMATION
A
6
Q
Concentrated solutions of formaldehyde must _____________ since this might precipitate and cause violent explosions
A
MUST NEVER BE NEUTRALIZED
7
Q
FORMALDEHYDE WASTES
1. Can be _____ by distillation or by drain disposal
2. Can be _____ by a commercial product
3. Can be _____ by a licensed waste hauler
A
FORMALDEHYDE WASTES
1. Can be RECYCLED by distillation or by drain disposal
2. Can be DETOXIFIED by a commercial product
3. Can be DISPOSED by a licensed waste hauler
8
Q
Recommended for fixation of central nervous tissue and GENERALPOST-MORTEM tissues for histochemical examination
A
10% FROMOL SALINE
9
Q
Formaldehyde fixative -The BEST GENERAL-PURPOSE TISSUE FIXATIVE
A
10% NEUTRAL BUFFERED FORMALIN
10
Q
Formaldehyde is usually buffered at ________ with _______ buffer
A
Formaldehyde is usually buffered at pH 7 with PHOSPHATE buffer
11
Q
In order to prepare 10% solution of formalin:
A
Mix 1 PART (100 mL) of 37-40% formaldehyde to 9 PARTS (900 mL) of distilled /tap water
12
Q
Composition of 10% Neutral Buffered Formalin:
A
- Distilled/tap water - 900 mL (9parts)
- Formalin (37-40% formaldehyde solution) - 100 mL (1part)
- Sodium phosphate, monobasic, monohydrate - 4 grams
- Sodium phosphate, dibasic, anhydrous - 6.5 grams
13
Q
Formalin is the fixative of choice for:
A
- FATS
- SURGICAL SPECIMENS
- ENZYME HISTOCHEMISTRY
- IMMUNOFLOURESCENCE
- IMMUNOHISTOCHEMISTRY (IHC)
- COLORED TISSUE PHOTOGRAPHY
14
Q
Formalin is the BEST fixative for
A
NERVOUS SYSTEM
15
Q
Formalin is the best fixative for tissues containing:
A
IRON PIGMENTS
16
Q
Formaldehyde fixative recommended for ROUTINE POST-MORTEM tissues
A
FORMOL-CORROSIVE/ FORMOL-SUBLIMATE
**GENERAL Post-Mortem tissues - 10% FORMOL SALINE
**ROUTINE Post-Mortem tissues - FORMOL CORROSIVE/FORMOL-SUBLIMATE
17
Q
Formaldehyde fixative used to fix SPUTUM since it coagulates mucus
A
GENDRE’s (ALCOHOLOC FORMALIN)
18
Q
Formalin pigment; WHITE Crystalline precipitate due to prolonged storage
A
PARAFORMALDEHYDE
19
Q
Removal of paraformaldehyde:
A
- Removed by addition of 10% METHANOL or FILTRATION
*** Methanol is used as a preservative/stabilizer to retard formaldehyde and prevent its decomposition to formic acid or precipitation to paraformaldehyde
20
Q
Prevention of paraformaldehyde:
A
Store in LOW temperature
21
Q
Formalin pigmen; BROWNISH-BLACK pigment granules produced by unstable formaldehyde fixatives as a result of the reaction between formic acid and hemoglobin
A
ACID FORMALDEHYDE HEMATIN
22
Q
Removal of Acid formaldehyde hematin
A
- Kardasewitsch’s method - specimen is placed in mixture of 70% ethyl alcohol and 30% ammonia water then wash with water
- Picric acid method - specimen is placed in saturated picric acid then wash with running water
- Alcoholic KOH (Potassium hydroxide)
- Lillie’s Method - specimen is placed in mixture of acetone, hydrogen peroxide and ammonia water then wash in 70% alcohol and water
23
Q
Prevention of acid formaldehyde hematin:
A
Formalin is BUFFERED to a neutral pH of 7.0 with phosphate
24
Q
Aldehyde fixative made up of TWO formaldehyde residues, linked by THREE carbon chains
A
GLUTARALDEHYDE FIXATVES
25
Glutaraldehyde fixatives are excellent fixative for ______________________, followed by secondary fixation in osmium tetroxide
ELECTRON MICROSCOPY
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NOTE: GLUTARALDEHYDE is the PRIMARY, MOST WIDELY USED ADN EXCELLENT FIXATIVE FOR ELECTRON MICROSCOPY
27
Fixatives of ELECTRON MICROSCOPY
1. GLUTARALDEHYDE
2. Osmium tetroxide
3. Paraformaldehyde
4. Karnovsky's (Glutaraldehyde + Paraformaldehyde)
5. Zamboni fluid
28
How many hours is the fixation time of tissues for EM?
THREE (3) Hours is the fixation time of tissues for EM
29
Tissue fixation for EM is performed at what temperature:
0 to 4C (REF. TEMP)
30
Embedding medium for EM:
PLASTIC medium (such as Epoxy resin)
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Type of microtome used for EM:
Ultrathin microtome
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Knife to be used for EM:
Diamond or Glass knives
33
Stains for EM:
1. LEAD CITRATE - BEST STAIN FOR EM
2. Uranyl acetate
3. Phosphotungstic acid
34
MOST COMMON METALLIC FIXATIVE; it permits brilliant metachromatic staining of cells:
MERCURIC CHLORIDE
35
Mercuric chloride is most commonly used metallic fixative recommended for fixing _____ biopsies
RENAL BIOPSIES
36
Mercuric chloride is the routine fixative of choice for preservation of:
CELL DETAIL IN TISSUE PHOTOGRAPHY
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NOTE:
Mercurial fixatives and reagents used in dezenkerization MUST NOT go through drain disposal
38
Removal of black mercuric deposits:
Dezenkerization
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Mercuric deposits may be removed by immersing tissues in _______ solution prior to staining, through the process known as DEZENKERIZATION treated together with sodium thiosulfate
ALCOHOLIC IODINE
40
MERCURIC CHLORIDE FIXATIVES:
1. B-5 FIXATIVE
2. HEIDENHAIN'S SUSA
3. ZENKER'S FLUID
4. ZENKER'S FORMOL (HELLY'S FLUID)
BHZZe
(ang mga tao sa Malaysia ay mga BHZZe 'busy')
41
Mercuric chloride fixative that is used for cytology of BONE MARROW BIOPSIES
B-5 FIXATIVE
42
Recommended mainly for TUMOR BIOPSIES OF THE SKIN and an excellent cytologic fixative
Heindenhain's Susa
43
The ONLY metallic fixative that DOES NOT produce mercuric chloride black deposits
Heidenhain's susa
44
Recommended for fixing SMALL PIECES OF LIVER, SPLEEN, CONNECTIVE TISSUE FIBERS and NUCLEI; Contains mercuric chloride, potassium dichromate, sodium sulfate, distilled water and GLACIAL ACETIC ACID
ZENKER'S FLUID
REMEMBER: Glacial acetic acid goods na goods sa nucleus, bad na bad sa cytoplasm
45
Excellent microanaotomic fixative for PITUITARY GLAND, BONE MARROW and BLOOD-CONTAINING ORGANS such as spleen and liver; contains mercuric chloride, potassium dichromate, sodium sulfate, distilled water and STRONG FORMALDEHYDE (40%)
**FIXATIVE OF CHOICE FOR INTERCALATED DISCS**
ZENKER'S FORMOL (HELLY'S)
46
Chromate fixatives:
1. Chromic acid
2. Regaud's fluid (Muller's)
3. Orth's fluid
4. Potassium dichromate
47
Chromate fixative that precipitates all proteins and adequately preserves CARBOHYDRATES
Chromic Acid
48
Chromate fixative recommended for demonstration of chromatin, mitochondria, mitotic figures, Golgi bodies, RBCs and colloid-containing tissues
Regaud's fluid (Muller's0
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Chromate fixative recommended for the study of early degenerative processes and tissue necrosis; demonstrates RICKETTSIAE and other bacteria
Orth's fluid
50
Chromate fixative for mitochondria; if acid is added, this fixes mucleoproteins
Potassium Dichromate
51
4% aqueous solution of basic lead acetate; fixes tissue mucin and is recommended for ACID MUCOPOLYSACCHARIDE
LEAD FIXATIVE
NOTE:
LEAD - ACID MUCOPOLYSACCHARIDE
NEWCOMER'S - MUCOPOLYSACCHARIDE
52
Excellent fixative for GLYCOGEN demonstration; YELLOW STAIN taken in by tissues prevents small fragments from being overlooked
PICRIC ACID FIXATIVE
53
PICRIC ACID is highly explosive when _____
DRY
picric acid is highly explosive when dry therefore must be kept moist with distilled water or saturated alcohol during storage
54
NOTE: PICRIC ACID can FIX, DIFFERENTIATE, and STAIN TISSUE ALL BY ITSELF!
55
Picric acid fixatives:
1. Bouin's solution
2. Brasil's alcoholic picroformol
56
Picric acid fixative recommended for fixation of EMBRYOS and pituitary biopsies; excellent in preserving soft and delicate structures such as endometrial curetting
BOUIN'S SOLUTION
NOTE:
BOUIN'S SOLUTIOON is NOT SUITABLE for fixing kidney structures, lipid and mucus
57
Bouin's solution contains:
- FORMALDEHYDE
- PICRIC ACID
- GLACIAL ACETIC ACID
mnemonic:
anga mga BATA (EMBRYO) mahilig s FPG, ang probinsyano
F -formaldehyde
P - picric acid
g - glacial acetic acid
58
BETTER and LESS MESSY than Bouin's solutions; excellent fixative for GLYCOGEN
BRASIL'S ALCOHOLIC PICROFORMOL
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Yellow stain produced by picric acid can be removed though the use of:
TAP WATER
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Used in concentrations 70-100% because less concentrated solutions will produce lysis of cells
ALCOHOL FIXATIVES
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ALCOHOL FIXATIVES:
1. 95% Isopropyl alcohol
2. Carnoy's fluid
3. 70-100% Ethyl alcohol
4. 100% Methyl alcohol
5. Newcomer's fixative
mnemonic: ICE MuNa bago ALCOHOL
62
Used in fixing TOUCH PREPARATIONS for certain special staining procedures such as Wright-Giemsa
95% Isopropyl alcohol
63
Considered to bethe "MOST RAPID FIXATIVE" with a fixation time of 1-3 hours; fixes and dehydrates at the same time
CARNOY'SFLUID
64
Carnoy's fluid is recommended for fixing:
- Chromosomes
- Urgent biopsies
- Rabies (fix brain tissues for diagnosis for rabies)
- Lymph glands
mnemonic = CURL/ kulot si Carnoy
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Carnoy's fluid contains:
- Glacial acetic acid
- Absolute alcohol
- Chloroform
66
Used for histochemistry especially for enzyme studies; preserves nucleopeptides and nucleic acids and give the most DNA fragments for PCR
70-100% Ethyl Alcohol
67
Excellent for fixing dry and wet smears, blood smears, and bone marrow smears
100% Methyl alcohol
68
Recommended for fixing mucopolysaccharides and nuclear proteins; BOTH a NUCLEAR and HISTOCHEMICAL fixative
Newcomer's fixative
REMEMBER:
LEAD - ACID MUCOPOLYSACCHARIDE
NEWCOMER's - MUCOPOLYSACCHARIDE
69
Major disadvantage of OSMIUM TETROXIDE:
NOT COMPATIBLE with H&E STAINING because it inhibits hematoxylin
NOTE:
- Osmium tetroxide can also cause CONJUNCTIVITIS or BLINDNESS
- Kept in dark colored, chemically clean bottle to prevent evaporation and reduction by sunlight
- Exposure to sunlight can also cause black deposits to buildup ---> remedy: tap water
70
Osmium tetroxide (osmic acid) fixatives:
1. Flemming's solution with glacial acetic acid
2. Flemming's solution without glacial acetic acid
71
Most common osmium tetroxide fixative; Recommended for NUCLEAR section preparation; excellent fixative for nuclear structures such as CHROMOSOMES
Flemming's solution WITH GLACIAL ACETIC ACID
REMEMBER: Glacial acetic acid; goods na goods sa Nucleus, bad na bad sa cytoplasm
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recommended for CYTOPLASMIC structures particularly the MITOCHONDRIA
Flemming's solution WITHOUT Glacial acetic acid
REMEMBER: Glacial acetic acid; goods na goods sa nucleus, bad na bad sa cytoplasm
73
OTHER SIGNIFICANT FIXATIVES TO REMEMBER:
- GLACIAL ACETIC ACID
- TRICHLOROACETIC ACID (TCA)
- ACETONE
Glacial acetic acid
- normally used in conjunction with other fixatives to a compound solution
- Solidifies at 17C
- Precipitates chromosomes and chromatin materials; hence is useful in the study of NUCLEAR components of the cell
- It causes tissues to SWELL
Trichloroacetic acid (TCA)
- Used for the precipitation of proteins and nucleic acids
- A fixative and decalcifying agent; a 'WEAK" decalcifying agent
Acetone
- used in fixing brain tissue for the diagnosis of rabies
74
REMEMBER:
FIXATIVE and DEHYDRATING AGENT USED TO FIX BRAIN OF RABIES PATIENTS:
- Carnoy's fluid
- Acetone
75
IMPORTANT PROCEDURES RELATED TO FIXATION
- Washing-out
- Secondary fixation (Post-mordanting)
- Post-chromatization
76
Process of removing excess fixative from tissue after fixation to improve staining, and to remove artifacts from the tissues
WASHING-OUT
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Removes excess FORMALIN, OSMIUM TETROXIDE, CHROMATE FIXATIVE
TAP WATER
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Removes excess PICRIC ACID FIXATIVE (Boiun's and Brasil's)
50 to 70% ALCOHOL
79
Removes excess MERCURIC CHLORIDE FIXATIVES
ALCOHOLIC IODINE
80
Process of placing an already fixed tissue in a second fixative
SECONDARY FIXATION (POST-MORDANTING)
81
A form of secondary fixation which uses a 2.5-3.0% potassium dichromate solution for 24 hours to act as mordant for better staining effects and to aid in cytologic preservation of tissues
POST-CHROMATIZATION
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PHYSICAL METHODS OF FIXATION
1. Heat fixation
2. Microwave fixation
3. Cryopreservation (freeze-drying)
83
SIMPLEST FORM of physical fixation; usually employed for FROZEN TISSUE SECTIONS and preparation of BACTERIOLOGIC SMEARS
HEAT FIXATION
84
Works as a PHYSICAL AGENT to increase the movement of molecules and accelerate fixation
MICROWAVE FIXATION
Chief advantage: Tissue is heated through the block in a VERY SHORT TIME, thereby potentially allowing the study of cellular processes that proceed VERY RAPIDLY
Disadvantage: Microwaves generated by commercial ovens ONLY penetrate tissue to thickness of 10 to 15 mm
85
A special way of preserving tissues by rapid freezing of fresh tissue 92mm thick) and subsequently removing ice water (desiccation) by a physical process of transferring the still frozen tissue block in a vacuum at a higher temperature
FREEZE-DRYING
86
Freeze-drying temperatures
QUENCHING:
SUBLIMATION:
Freeze-drying temperatures
QUENCHING: -160 to -180C
SUBLIMATION: -30 to -40C
87
Similar to freeze drying, the only variation is that the frozen tissue instead of being subjected to dehydration in an expensive vacuum drying apparatus, frozen tissue is fixed in a ROSSMAN'S FORMULA or in 1% acetone and dehydrated in absolute alcohol
FREEZE-SUBSTITUTION
88
REMEMBER: FREEZE-SUBSTITUTION
Contents of ROSSMAN'S FORMULA:
- Picric acid
- 100% Ethanol
- Formalin
"PEF-peeve si ROSSMAN (rosmar) na mahilig magpalit pag nilalamigan"
