HISTO RA Flashcards
It is a step in tissue processing that involves preservation of tissues as close to original as possible
FIXATION
Primary Goal of Fixation
to preserve cells and tissues as close as to the original as possible
Secondary Goals of Fixation (2)
- to HARDEN tissues to facilitate EASY cutting
- to PROTECT the tissue from the TRAUMA of further HANDLING
FACTORS THAT INFLUENCE FIXATION
- Thickness/ Size
- Hydrogen Ion Concentration
- Osmolality
- Concentration of Fixative
- Volume
- Time and Duration
- Temperature
THICKNESS of tissue in FIXATION
3-5 mm
SIZE of tissue in FIXATION and also in LUNG specimen
3x2 cm; 1-2 cm
Hydrogen Ion Concentration of tissue in FIXATION
Ideal pH between 6-8
IDEAL Osmolality of tissue in FIXATION
SLIGHTLY HYPERTONIC
In OSMOLALITY, HYPERTONIC solution causes
CELL SHRINKAGE
In OSMOLALITY, HYPOTONIC solution causes
CELL SWELLING
BUT in PRACTICE osmolality can be
ISOTONIC SOLUTION
T/F
In Concentration of Fixative, We use concentrated solution as it can be damaging to tissues
FALSE; WE NEVER USE
In Concentration of Fixative, this is diluted to produce 10% formalin
FORMALDEHYDE
In Concentration of Fixative, GLUTARALDEHYDE is diluted to produce and is used for
a. 3% Glutaraldehyde – Electron microscopy
b. 0.25% – Immunoelectron microscopy
The maximum effective or VOLUME of tissue
20 TIMES the volume of specimen (routine)
Fixative to Tissue Ratio:
15-20:1
Penetration Rate of Formalin:
1 mm/ hour
Volume of Specimen for Osmium Tetroxide (Electron Microscopy) and reason for this
5-10 times the volume of specimen as it is quite expensive
For Museum Preparations:
should not be less than 50-100 times the volume of the specimen
Time and Duration of tissues
Usually subjected to fixative within 24-48 hours
Temperature of tissue for MANUAL FIXATION
ROOM TEMPERATURE
FIXATION Temperature of tissue for EM and Histochemistry
0-4 degC
FIXATION Temperature of tissue for AUTOTECHNICON and why?
40 degC (hastens fixation)
T/F
Does application of heat shortens fixation time
TRUE
Temperature range that will ACCELERATE fixation:
37-56 degC
FACTORS TO CONSIDER WHEN CHOOSING THE RIGHT FIXATIVE
- Urgency of the case/ need for immediate examination
- Type of specimen/ tissue to be processed
- Tissue structure to be studied
- Type of section to be made
- Staining technique to be applied
In Urgency of the case/ need for immediate examination, you must use a fixative that is ____
FAST-ACTING
In Urgency of the case/ need for immediate examination, this is used for rapid in action, thus it’s the most commonly used fixative
FORMALIN
Type of specimen/ tissue to be processed that is for preservation of brain tissue for rabies
Carnoy’s & Acetone
Type of specimen/ tissue to be processed that is not used for kidneys
Bouin’s
In Tissue structure to be studied, it is for glycogen
Picric acid fixatives (Brasil’s fluid)
In Tissue structure to be studied, for preservation of fats it is not used as it dissolve fats
ACETONE
In Tissue structure to be studied, it is for enzymes, lipases, phosphatase
ACETONE
Types of section to be made
- Serial
- Individual
Staining technique to be applied that is a ROUTINE STAIN
H&E
This can inhibit HEMATOXYLIN
OSMIUM TETROXIDE
FACTORS THAT WILL RETARD OR PROLONG FIXATION PROCESS
- Cold Temperature
- Presence of blood and mucus
- Presence of Fat/ Fatty Tissues
- Size and thickness
FILL IN THE BLANKS (RETARD OR PROLONG FIXATION)
1. Cold Temperature: it can _____ enzymes
2. Presence of blood and mucus: Remedy ___
3. Presence of Fat/ Fatty Tissues: Remedy ___
4. Size if it is ___ and thickness if it is ___
can inactivate enzymes
can be removed by washing the tissue with NSS
cut the specimen thinly to ensure rapid entry of fixative
larger; thicker
FACTORS THAT WILL ACCELERATE OR SHORTENS FIXATION PROCESS
- Heat Application
- Agitation
- Size & Thickness
FILL IN THE BLANKS (ACCELERATE OR SHORTENS FIXATION)
1. Heat Application ____; beyond this temp, it can lead to _____
2. Agitation
3. Size if it is _____ & Thickness if it is ___
37-56°C ONLY; damage
continuous mixing (autotechnicon)
smaller; thinner
Problem in FIXATION:
Loss of substances soluble in fixing agent/ Loss or inactivation of enzyme; Cause
May result from wrong choice of fixative
Problem in FIXATION:
Soft tissues & feather like consistency; Cause
Incomplete washing of fixative
Problem in FIXATION:
Brittle & hard tissues; cell shrinkage & swelling; Cause
Overfixation/ prolonged fixation
Problem in FIXATION:
Presence of artifacts on tissues; Cause
Inadequate or incomplete washing
Problem in FIXATION:
Failure to arrest early autolysis; Cause
Insufficient fixative; failure to fix abruptly
FIXATION METHODS
- Physical Method
- Chemical Method
TYPES of PHYSICAL METHOD
HEAT FIXATION
MICROWAVE TECHNIQUE
Type of Physical Method that involves thermal coagulation of proteins
HEAT FIXATION
FIXATION METHOD that is commonly done and uses chemical fixatives
CHEMICAL METHOD
TYPE OF FIXATIVES
I. According to Mechanism of Action (as to manner of preservation)
II. According to Action (as to purpose)
II. According to Active Component Substance (as the main ingredient)
FILL IN THE BLANKS
I. According to Mechanism of Action: as to ____
II. According to Action: as to ____
II. According to Active Component Substance: as the ____
manner of preservation
purpose
main ingredient
Types of FIXATIVES According to Mechanism of Action (as to manner of preservation)
ADDITIVE FIXATIVES
NON-ADDITIVE FIXATIVES
Type of FIXATIVES According to Mechanism of Action (as to manner of preservation) that when used are combined or absorbed by the tissues
ADDITIVE FIXATIVE
In ADDITIVE FIXATIVES when used are combined or absorbed by the tissues it becomes ____
part of the tissue
ALL the fixatives available are additive except:
Acetone & Alcohol
Type of FIXATIVES According to Mechanism of Action (as to manner of preservation) that do not bind with the tissue
NON-ADDITIVE FIXATIVES
In NON-ADDITVE FIXATIVES it does not bind with the tissue for it is ____
not absorbed or taken in by the tissue
Examples of NON-ADDITIVE FIXATIVES
Acetone and Alcohol
Types of FIXATIVES according to Action (as to purpose)
- Microanatomical
- Cytological
- Histochemical
Type of FIXATIVES according to Action (as to purpose) that allows general microscopic study of tissue structure
MICROANATOMICAL
Type of FIXATIVES according to Action (as to purpose) that preserve parts of the cell (either nucleus or cytoplasm)
CYTOLOGICAL
Types of FIXATIVES in Cytological
- Nuclear
- Cytoplasmic
Fixative that is for nuclear chromatin, chromosomes, etc.
NUCLEAR FIXATIVE
NUCLEAR FIXATIVE
___ glacial acetic acid
pH with ___ 4.6
WITH
LESS THAN
Nuclear Fixative EXAMPLES
Bouin’s
Carnoy’s
Heidenhein’s Susa
Newcomer’s
Flemming’s with HAc
Fixative that is for golgi body, mitochondria, etc.
CYTOPLASMIC FIXATIVES
CYTOPLASMIC FIXATIVE
___ glacial acetic acid
pH with ___ 4.6
WITHOUT
MORE THAN
Cytoplasmic Fixatives EXAMPLES
Regaud’s/ Moller’s
Orth’s
Formalin with post chroming
Flemming’s without HAc
Type of FIXATIVES according to Action (as to purpose) that preserve the chemical components of tissues like enzyme
HISTOCHEMICAL
EXAMPLES of Histochemical
Acetone
Absolute ethyl alcohol
Newcomer’s
10% Formol Saline
Types of Fixatives According to Active Component Substance (as the main ingredient)
- Aldehyde Fixatives
- Metallic Fixatives
- Picric Acid Fixatives
- Alcoholic Fixatives
- Glacial Acetic Acid
- Acetone
- Osmium Tetroxide
- Trichloacetic Acid
Types of Aldehyde Fixatives
A. FORMALDEHYDE/ FORMALIN FIXATIVES
B: GLUTARALDEHYDE FIXATIVES
C: GLYOXAL
Types of FORMALDEHYDE/ FORMALIN FIXATIVES
a. 10% Formalin
b. 10% Formol Saline
c. 10% Neutral Buffered Formalin
d. Formol Corrosive/ Formol Sublimate
e. Alcoholic Formalin/ Gendres
ROUTINELY USED Formaldehyde/ Formalin Fixative
10% Formalin
10% Formalin preparation:
1 L or 1000 mL Formalin:
Combine 100 ml of 37-40% formalin + 900 ml distilled water
Formaldehyde/ Formalin Fixative that is to preserve CNS and post mortem tissues
10% Formol Saline
10% Formol Saline uses ____ as diluent
NSS
Formaldehyde/ Formalin Fixative that is for tissues with iron pigments
10% Neutral Buffered Formalin
10% Neutral Buffered Formalin preparation
Same with 10% formalin, but sodium phosphate was added as buffer
Formaldehyde/ Formalin Fixative that is best for lipids & neutral phospholipids
Formol Corrosive/ Formol Sublimate
Formol Corrosive/ Formol Sublimate preparation
Formaldehyde + Mercuric Chloride
Formaldehyde/ Formalin Fixative that is for sputum and microincineration techniques
Alcoholic Formalin/ Gendres
Alcoholic Formalin/ Gendres preparation
Combination of 95% Ethanol + Glacial Acetic Acid (HAc) + Formaldehyde
Disadvantage of Formalin:
It may precipitate upon longer storage
To prevent precipitation of formaldehyde to white paraformaldehyde:
Add 10% Methanol
Aldehyde Fixative that is for Electron Microscopy
GLUTARALDEHYDE FIXATIVE
Types of GLUTARALDEHYDE FIXATIVES
a. 2-5% Solution
b. 4% Solution
Glutaraldehyde fixative that is for small tissue fragments/ needle biopsies
2-5% Solution
Glutaraldehyde fixative that is for larger tissues less than 4 mm thick
4% Solution
Aldehyde Fixative that is SMALLEST ALDEHYDE
GLYOXAL
Types of Metallic fixatives
A. MERCURIC CHLORIDE
B. LEAD FIXATIVES
C. CHROMATE FIXATIVES
It is the most common metallic fixative
MERCURIC CHLORIDE
Metallic Fixative that is excellent for trichrome staining & tissue photography
MERCURIC CHLORIDE
Types of MERCURIC CHLORIDE
a. Zenker’s Fluid
b. B5
c. Heidenhein’s Susa
d. Zenker Formol/ Helly’s Fluid
MERCURIC CHLORIDE fixative that is for Liver, spleen, connective tissue fibers & nuclei
Zenker’s Fluid
MERCURIC CHLORIDE fixative that is for bone marrow
B5
MERCURIC CHLORIDE fixative that is for Tumor skin biopsies and an excellent cytologic fixative
Heidenhein’s Susa
MERCURIC CHLORIDE fixative that is for Pituitary gland, bone marrow, spleen, and liver
Zenker Formol/ Helly’s Fluid
DISADVANTAGE of MERCURIC CHLORIDE:
They produce black mercury deposits
REMEDY for MERCURIC CHLORIDE:
Alcohol Iodine is used to wash out excess mercuric fixatives
It is proposed as mercuric chloride substitute as it is toxic to man
ZINC SULFATE
METALLIC FIXATIVE that is for acid mucopolysaccharide
LEAD FIXATIVE
METALLIC FIXATIVE that have potassium
dichromate
CHROMATE FIXATIVES
Types of CHROMATE FIXATIVES
a. 1-2% Chromic Acid
b. 3% Potassium Dichromate
c. Regaud’s/ Moller’s
d. Orth’s Fluid
CHROMATE FIXATIVE that is for carbohydrates
1-2% Chromic Acid
CHROMATE FIXATIVE that is for lipids &
mitochondria
3% Potassium Dichromate
CHROMATE FIXATIVE that is for mitochondria, RBC & colloid containing tissues
Regaud’s/ Moller’s
CHROMATE FIXATIVE that is for Rickettsiae & other bacteria, tissue necrosis
Orth’s Fluid
Type of Fixative According to Active Component Substance (as the main ingredient) that is an excellent for glycogen demonstration
PICRIC ACID FIXATIVES
This is used as fixative, decalcifying agent, and stain
PICRIC ACID FIXATIVES
Types of PICRIC ACID FIXATIVES
a. Bouin’s Solution
b. Brasil’s Alcoholic Picformol
c. Hollande’s Solution
Type of Picric Acid Fixative that is for embryos, pituitary biopsies & endometrial curetting
Bouin’s Solution
T/F
Bouin’s Solution is for KIDNEY STRUCTURES
FALSE; IT IS NOT
T/F
Bouin’s Solution is compatible with feulgen’s stain
FALSE; IT IS NOT
This is used to wash out excess amount of picric acid fixatives
50-70% Alcohol
This acts as both fixative and dehydrating agent
ALCOHOL
Types of ALCOHOL FIXATIVES
a. 95% Isopropyl Alcohol/ IPA
b. Methyl Alcohol (100%)
c. Ethyl Alcohol (70-100%)
d. Carnoy’s
e. Newcomer’s
Type of ALCOHOL FIXATIVE that is for touch preparations
95% Isopropyl Alcohol/ IPA
Type of ALCOHOL FIXATIVE that is the MOST RAPID tissue fixative
CARNOY’S
Type of ALCOHOL FIXATIVE that is for fixing chromosomes & lymph glands
CARNOY’S
Type of ALCOHOL FIXATIVE that is for mucopolysaccharides (histochemical) & nuclear protein
NEWCOMER’S
Type of Fixative According to Active Component Substance (as the main ingredient) that solidifies at 17 degC
GLACIAL ACETIC ACID
It is Contraindicated for cytoplasmic fixation as it is a fixative for nucleus
GLACIAL ACETIC ACID
This is used in combination with other fixatives to produce a compound fixative (but can be used alone)
GLACIAL ACETIC ACID
Type of Fixative According to Active Component Substance (as the main ingredient) that is used both fixative & dehydrating agent
ACETONE
This must be used at ICE COLD TEMPERATURE between -5 to 4 degC
ACETONE
This is for phosphatases & lipases enzymes
ACETONE
DISADVANTAGE of ACETONE
Evaporates easily & dissolves fats
Type of Fixative According to Active Component Substance (as the main ingredient) that can be used for myelin & peripheral nerves
OSMIUM TETROXIDE
Type of Fixative According to Active Component Substance (as the main ingredient) that is a fixative & a weak and slow decalcifying agent
TRICHLOROACETIC ACID
Fixatives for brain tissues for the diagnosis of rabies
- Carnoy’s Fluid – MOST RAPID FIXATIVE
- Acetone
Fixatives for Enzyme Histochemistry
- 4% Formaldehyde
- Formol Saline
Fixatives for Electron Microscopy
- Osmium Tetroxide
- Glutaraldehyde
- Paraformaldehyde
Fixatives for Enzyme Histochemistry & Electron Immunocytochemistry
- Karnovsky’s
- Glutaraldehyde
- Paraformaldehyde
Fixatives often used in conjunction with other fixatives to produce a compound fixative
- Glacial acetic acid
- Trichloroacetic acid/ TCA
