basic histologic techniques Flashcards
12 Steps of Histologic Techniques
numbering receiving of specimens fixation dehydration clearing infiltration embedding blocking and trimming sectioning staining mounting labelling
first and most important step
logging in a log book the details of the tissue
numbering
tissue specimens should be properly labeled and with a corresponding request
tissue s will be described by the pathologist (gross description)
tissues will be cut by the pathologist
receiving of specimens
tissues are immersed in a fixative to prevent decomposition and preserve structure
fixation
removal of excess water using ascending grades of alcohol
dehydration
removal of excess alcohol
makes tissues clear/transparent
prepares tissues for paraffin impregnation
clearing/dealcoholization
tissue cavities are saturated w/ paraffin wax
infiltration
impregnated tissue is oriented and embedded in paraffin block to form a tissue block
embedding
separation of embedded tissues into blocks
removal of excess paraffin wax using knives
blocking and trimming
very thin sections of tissue are cut using microtome
sectioning
using H& E (routine stain)
staining
stained tissue sections on a slide are added w/ mounting medium and covered w/ coverslip
mounting
final step in tissue preparation
labeling
Responsible for transcribing the dictations of the pathologist
Medical technologist
Used in labeling slecimens
Pencil
Provides the gross description
Cuts of sections the specimen when received
Pathologist
Technique done to kill microorganism in the specimen
Fixation
Routinely used fixative
Formalin (10%) or formaldehyde
Stock solution of formalin that must be diluted
37% formalin
Agent used in dehydration
Increasing grades of Ethanol or ethyl alcohol
Effect of clearing to tissues
Tissues become translucent or transparent
Most common clearing agent
Xylol or xylene
Melting point of paraffin wax
56-57C
Ways of performing infiltration
Manual
Using automatic tissue processor
Another name for infiltration
Impregnation
Performs fixation to infiltration (steps 2-5)
Has a basket wherein tissue cassette will be placed
Automatic tissue processor
How long do cutting to infiltration take?
1 day
The act of placing impregnated tissue in the embedding mold
Orientation
Examples of embedding mold
Paper boat
Plastic mold
Ice cube tray
Separation of embedded tissues into blocks
Separating small block from big block
Blocking
Removal of excess paraffin wax using knives until a truncated pyramid is made
Trimming
Thickness required when sectioninf
4-6 nm
Where tissue section is placed before in the microtome
Surface is colored black
Removes wrinkles
Flotation Water bath
After flotation, perform ____
Fishing
Act of scooping the tissue from the flotation water bath
Fishing out
Another term for staining
Coloring
A nuclear stain
Color is blue or dark blue
Hematoxylin
A cytoplasmic stain
Reddish or pinkish
Eosin
Also serves as a counterstajn
Eosin
Provides contrast and background
Eosin
2 types of mounting medium
Aqueous
Resinous
Primary mounting medium used in histology
Resinous
Ex of aqueous mounting medium
Water
Example of resinous mounting medium
Eukitt and Canada balsam
Final step
Use of gum labels
May be computerized or bar coded
Labelling
– fixative often used for EM; reinforces fixation by being a dialdehyde capable also of cross-linking proteins
• Gluteraldehyde
– preserves and stains membrane lipids and proteins
Osmium tetroxide
In electron microscopy, a _____ procedure is done.
double-fixation
basic histologic technique
• ethanol is then replaced by an organic solvent miscible with both alcohol and embedding medium
• Alcohol is removed in toluene or other agents in which both alcohol and paraffin are miscible
- Clearing
- Fully cleared tissue is placed in melted paraffin in an oven at 52 – 60C
- At 52 – 60C, clearing solvent evaporates and tissue is filled with paraffin wax
- Infiltration
- The paraffin-infiltrated tissue is placed in a small mold with melted paraffin and allowed to harden
- Impregnated tissue hardens in a small container of paraffin at RT
Embedding
SPATIAL UNITS in Histology:
micrometer, nanometer, angstrom
– cell components with a net negative charge (anionic) stain readily with basic dyes (eg. Nucleic acids)
-have acids in their composition
• Basophilic
- cell components that are cationic have affinity for acidic dyes (eg. Proteins with many ionized amino groups)
• Acidophilic
– toluidine blue, alcian blue, methylene blue, hematoxylin
• Basic Dyes
– eosin, orange G, acid fuchsin
• Acid dyes
type of stain
– used in more complex histologic procedures
-help to distinguish extracellular tissue components better than H&E
• Trichrome (Mallory stain, Masson stain)
chromatin with active dna
stains lightly
euchromatin
inactive dna
stains darkly
heterochromatin
euchromatin that becomes a heterochromatin
facultative heterochromatin
whole process of an X chromosome becoming a barr body or sex chromatin
lyonization
according to lyon’s hypothesis, at the _____ cell stage, one of the X in the female gets turned off
100
specimen used for determining sex chromatin
peripheral blood (neutrophil) tongue and buccal cavity
first blood must be wiped off as it contains
tissue juices
methanol number of seconds
5 seconds, air dry
eosin number of seconds
3
methylene blue number of seconds
6 seconds
