Methods of Histology Flashcards
Branch of science dealing with external form and internal organization of plants and animals. What are the 2 branches?
Anatomy; Gross anatomy & Microscopic anatomy (histology)
Branch of science dealing with microscopic structure and function of plant and animal tissues
Histology
Branch of biology that deals with cell formation, structure and function
Cytology
Histology did not acquire status of a separate science until the discovery of _____ by ______.
Cell Theory; Schwann & Schleiden
Cell Theory states that cells are potentially _________.
Independent organisms
Cell Theory states that plants and animals are ________ of these living units and __________.
Aggregations; their secretions
All cells originate from _________ by cell division
Pre-existing cells
Methods of Direct Observation: Dynamics of blood circulation in organs, secretory properties of pancreatic cells, ovulation in rat
Exteriorization and Transillumination
Methods of Direct Observation: Growth of capillaries and nerves, emigrations of leukocytes, development of adipose tissue; Extension: transplant of ovarian cells in monkey cornea
Transparent Chamber
Methods of Direct Observation: Growth of fibroblasts, bone primordium
Cell and Organ Culture
Methods of Direct Observation: Role of nucleus – microneedles, micropipettes
Mechanical Micromanipulation
Methods of Direct Observation: Cell division
Radiation Probes
Methods of Direct Observation: Certain dyes and stains selectively stain cells and organelles
Vital and Supravital Staining
Homogenate left at gravity for 20 min
- Pellet containing broken cells
- Supernatant
Supernatant from step one centrifuged at 1000 X g for 20 min
- Pellet containing nuclei, broken cells and intact cells
- Postnuclear supernatant
Postnuclear supernatant centrifuged at 20,000 X g for 20 min
- Pellet containing mitochondria, lysosomes and peroxisomes
- Postmitochondrial supernatant
Postmitochondrial supernatant centrifuged at 100,000 X g for 1 hr
- Pellet containing microsomes (fragments of plasma membranes and smooth and rough endoplasmic reticulum)
- Postmicrosomal supernatant
Postmicrosomal supernatant centrifuged at 100,000 – 500,000 X g for a few hours
- Pellet containing free ribosomes, viruses, large macromolecules such as proteins
- Cytosol (soluble cellular components)
_____ stops dynamics of protoplasm.
Fixation
Fixatives
- Neutral formalin (37% formaldehyde solution)
- Osmic acid
- Mercuric chloride
Embedding of soft tissue can take place in _____, ______, ______ & sectioning using ______.
- Paraffin, Gelatin, Celloidin
- Microtome
Most common stains
Hematoxylin and eosin (H&E)
H&E Stain
Nuceli: Blue
Muscle: Pink
Collagen: Pink
Elastic fibers: Pink
Phsphotungstic Acid - hematoxylin (PTAH) Stain
Nuceli: Blue-Black
Muscle: Blue-Black
Collagen: Colorless-Pink
Elastic fibers: Colorless-Pink
Masson Trichome Blue Stain
Nuceli: Blueish-red
Muscle: Red
Collagen: Blue
Elastic fibers: Blue
Azan Stain
Nuceli: Blueish-red
Muscle: Red
Collagen: Blue
Elastic fibers: Blue
Periodic Acid Schiff (PAS) Stain
Specific for insoluble carbohydrates (polysaccharides) - gives a red or magenta (deep purplish red) color
Chemical Procedures: Lipids
Uptake of fat soluble dyes – black color
Chemical Procedures: Carbohydrates
Periodic Acid Schiff (PAS) Reaction – magenta (deep purplish red) color
Chemical Procedures: DNA
Feulgen Reaction – green color
Chemical Procedures: Proteins & Enzymes
Biuret Reagent – blue color
Physical Procedures
UV and X rays
Chemical Basis of Histological Staining
- Amphoteric properties of proteins
- Isoelectric point
- pH
- Acids and bases
- Acidophilic vs. basophilic dyes
______ and ______ are the most commonly used dyes in histology
Hematoxylin and eosin
An _____ dye, such as _____, carries a net negative charge on its colored portion (Na+ dye-)
Acidic; Eosin
A _____ dye, such as ____________, carries a net positive charge on its colored portion (dye+ Cl-)
Basic; Methyl green
__________ does not meet the definition of a strict basic dye but has properties that closely resemble those of a basic dye.
Hematoxylin
_____ dyes react with cell and tissue components that carry a negative charge such as phosphate groups of ____________ and carboxyl groups of proteins.
Basic; Nucleic Acids
______ dyes react with cell and tissue components that carry a positive charge such as ___________ in proteins.
Acidic; Amino groups
Distinguishing objects separated by very minute distances
Resolution
Enlargement
Magnification
Degree of difference between light and dark
Contrast
Most common, principle is differential absorption of light, improved by staining
Light Microscope
Has optical device for converting minute distances
Phase Contrast Microscope
Utilizes two beams of light and has a quantitative feature
Interference Microscope
Light of specific wavelengths is used to illuminate specimens – example UV microscope
Fluorescent Microscope
Instead of light a beam of electrons emitting from a tungsten filament
Transmission Electron Microscope (TEM)
Image projected on a TV monitor and scanning is possible
Scanning Electron Microscope (SEM)
Specimen Preparation for Electron Microscopy: Only ______ specimens suitable
Dehydrated
Specimen Preparation for Electron Microscopy: Fixatives include _______, _______, _______, _______, and ______.
- Neutral formalin
- Osmium tetroxide
- Osmium dichromate
- Acrolein
- Glutaraldehyde
Specimen Preparation for Electron Microscopy: Most common fixative is _________.
1 or 2 % osmium tetroxide, pH 7.2-7.8
Specimen Preparation for Electron Microscopy: Electrons do not register ______ and hence the electron micrographs are black and white - coloring after a picture is taken is possible
Color
One of the most powerful tool for surface topography at molecular and atomic resolution
Atomic Force Microscopy (AFM)
It is a nonoptical microscope
Atomic Force Microscopy (AFM)
Works as finger tip by touching and feeling
Atomic Force Microscopy (AFM)
A very sharp, pointed probe, scans the specimen.
Atomic Force Microscopy (AFM)
