Module 1 [cont]: Methods of cytology and histology Flashcards
How many lenses does compound light microscope hae.
Two or more
Light source for compound LM?
Transmitted light
T/F, compound light mic. Uses live specimens such as examination of stool for parasites
True
Total magnification of CLM?
Not more than 2000x
Max magnification of CLM?
400x
T/F: eye piece only enlarges the images obtain by the objective and doesnt improve the resolution of the image
True
3 objectives of CLM & their magnifications
a. Scanning objective- 4x
b. Low power objective- 10x
c. High power objective -40x (blue line)
Part of CLM where it serves as a platform upon which the slide rests
Stage
A transparent lens through which light reveals. Size and brightness can be adjusted
Iris diaphragm
Light source of the clm
Lamp
Steps on using microscope (shortcut)
a. Power on, make sure light is working; objective is set to LPO or scanning
b. Always start at scanning objective
c. Place glass slide on stage; fix using specimen holder
d. Move the slide to focus on image you want to see
e. Adjustment of course adjustment & fine adjustment knob
f. Adjust light intensity
g. Once image is clear, you can switch to a higher magnification to see individual details
h. If finished, switch back to LPO
i. Remove slide carefully and put it in proper box
J. Turn off microscope
Knob that lowers the objective lenses
Course adjustment knob
Knob that gives clearer image
Fine adjustment knob
T/F, to preserve the morphologic and chemical integrity of the cell in as life-like manner as possible is the primary aim of fixatives
True
Size of a tissue block used in light microscopy
2 x 0.4 sq. cm
Size of a tissue block used in electron microscopy
1-2 sq. mm
Main factors involved in fixation
a. Hydrogen ion concentrion (6-8)
b. Temperature
c. thickness of sxn
d. Osmolality
e. Concn. of Fixative
f. Duration of fixative (6-24hrs)
Process of removing intracellular and extracellular water
Dehydration
What percent of alcohol is used to dehydrate delicate tissues (i.e embryonic tissues)
30%
Most recommended alcohol for dehydration and best dehydrating agent
Ethanol
Why do we need to dehydrate tissue samples?
To remove water from previous fixation where in melted paraffin that will be used to infiltrate the tissue subsequently is Hydrophobic
Most used clearing agent
Xylene
Most common embedding medium being used
Paraffin
It is used to adhere tissue sections in an untreated glass slide
Bonding Agent/ Adhesive
Two example of bonding agents
a. albumin
b. poly-l-lysine
Most common bonding agent
Albumin
Two methods of staining
a. direct staining
b. indirect staining
A process of giving color to the given sections by using aqueous or alcoholic dyes
Direct staining
A staining method wherein the action of dye is intensified by using a mordant which serves as a link between tissue and dye
Indirect staining
T/F, nucleus is basic in nature hence stains blue by Hematoxylin
False, acidic
T/F, Cytoplasm is basic in nature hence in stained by Eosin pink
True
A dye of combination of an acid and basic dye
Neutral dye
Example where Neutral dye is used?
Leishman staining
It gives a protective cover over the smear and make a permanent bond between the coverslip and the slides
Mounting media
Requirement for a mounting media
a. same refractive index as the coverslip and glass slide (1.52 -1.54)
b. should be colorless
c. quick drying and stick to the slide
d. resist contamination particularly the growth of microbes
e. miscible with clearing agent
What structures can PAS stains?
a. glycoprotein
b. mucin
c. mucoprotein
d. glycoprotein
e. basement membranes
f. capsules
g. blood vessels
Color stain of PAS for positive
Purple/ Red/Magenta
Color stain of PAS for nuclei
Blue
Modified PAS which can stain DNA of cell nuclei
Feulgen reaction
Lipid soluble dyes that lipid rich structure (phospholipids and neutral fats)
Sudan black
Color stain of Sudan black if positive
blue black
Color stain of Sudan black for nuclei
red
Uses silver salts to stain certain ECM fibers and specific cellular elements in nervous tissues
Metal impregnation
What is produced in Metal impregnation?
opaque black deposit on bacteria or cell surface
T/F, Light is either passed through or Reflected in Brightfield microscopy
True
Other term for eyepiece
Ocular lens
T/F, eyepiece or ocular lenses improves the resolution of the object
False, it does not improve but only enlarges the image
Objective used for locating objects and scanning slides quickly
Scanning objective
Color and magnification of line in scanning obj
red line- 4x
color and magnification of line in Low power obj
yellow line- 10x
color and magnification of line in high power obj
blue line- 40x
Lens that allows you to find and center object in a slide
Low power object
Lens used to zoom in for closer viewing
High power objective
It determines the quality of the image, clarity, and richness of detail
Resolving power
What is the fluorescent stain used?
Acridine orange
T/F, acridine orange binds only to the DNA
False, both DNA and RNA
Light emitted by DNA and RNA under fluorescence microscopy
DNA- Yellow
RNA- orange
Specimen for Phase contrast microscopy
unstained cells and tissue sections
uses lens that produces visible images from transparent objects
PCM
Used to study cell cycle in live cell
PCM
Digitally reconstructs the specimen into a 3D image; records ion movements; determines enzymatic activities in tissues
Confocal microscopy
Used in detecting cardiac amyloidosis (apple green birefringence)
cardiac amyloidosis
It can visualize individual viral particles, diagnosis of glumerulopathies
Electron microscopy
Fixative for electron microscopy
Glutaraldehyde
Cut object in TEM
40-90 nm
T/F, in TEM, electrons pass directly through the specimen
true
T/F. in SEM, electrons bounce off the surface of the specimen at an angle and 3D image is produced
true
Complex solutions that is used to sustain cells and tissue culture
Salts, Amino acid, vitamins, serum and specific growth factor
Refers to as low passage or finite cell lines, aka diploid
Established cell line
T/F, established cell lines can be maintained longer than primary cell line
True
Altered cells that can be maintained indefinitely, the cells are heteroploid
Continuous cell lines
W1-38, MRC-5, IMR-90 are examples of what cell line
Established cell line
Hela, Hep-2, A549 and vero cells are example of what cell lines
Continuous cell line
What is used in autoradiography as microdetector
Silver bromide crystals
In autoradiography, they indicate the cells or regions of cells where macromolecules are synthesized
Silver grains
Technique used to identify diagnostically useful enzyme or other tissues
Enzyme histo/Cytochemistry
T/F, histochemistry and cytochemistry serves to detect early metabolic changes in biopsies and autopsy tissues
true
T/F, immunohistochemistry, enables us to visualize the distribution and localization of specific cellular components within cell or tissue
true
The most important requirement of Immunohistochemistry is the availability of an antibody against the protein to be detected
true
A procedure in IHC wherein it uses heat , couple with specific buffered solutions to recover antigen reactivity
Antigen retrieval
Procedure for IHC
(insert photo)
Clinical application for IHC
useful in cancer diagnosis
Allows a specific identification of sequences in genes or RNA
hybridization technique
