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Histology > Microscope Lab > Flashcards

Microscope Lab Flashcards

1
Q

Compound Light Microscope: Ocular Lens definition

A

The eye lens

Additional 10X mag

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2
Q

Compound Light Microscope: Objective Lens definition

A

Magnifies sample

4X, 10X, 20X, 40X

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3
Q

Compound Light Microscope: Objective Turret definition

A

Hold objectives

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4
Q

Compound Light Microscope: Stage definition

A

Location of slide placement

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5
Q

Compound Light Microscope: Slide holder definition

A

Holds slide snugly

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6
Q

Compound Light Microscope: Mechanical Stage Controls definition

A

X and Y movement

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7
Q

Compound Light Microscope: Course Focus

A

This can drive objective through slide

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8
Q

Compound Light Microscope: Fine Focus

A

Mostly use this one

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9
Q

Compound Light Microscope: Condenser

A

Focuses light on specimen

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10
Q

Compound Light Microscope: Iris diaphragm level

A

Controls light through condenser

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11
Q

Compound Light Microscope: Rheostat

A

Controls light intensity (on side of base)

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12
Q

Compound Microscope

A
  • Has more than 2 sets of lens
  • Considered binocular because it has 2 eyepieces rather than 1
  • Allows you to alter the distance of the 2 eyepieces to accommodate your interpupillary distance to view single image
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13
Q

Resolution

A

Defined as the ability to distinguish 2 items that are closely spaced as separate entities

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14
Q

Resolving Power

A
  • 200microm or 0.2mm

- Items closer than 200 microm from each other cannot be distinguished without optical assistance

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15
Q

Electron microscope resolving power

A

2-3 nm

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16
Q

Fixation

A
  • Formaldehyde for LM
  • Glutaraldehyde for TEM
  • poor fixation = poor slide quality
  • During fixation, cell water is replaced with a buffered (physiological pH) chemical that binds (fixes) proteins
  • Most fixatives are aldehydes
  • Critical parameter of time spent in fixture: 18-24 hrs
  • Critical parameter (fixative volume to tissue volume ratio): 10X more fixative
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17
Q

Problems from Bad Fixation

A
  1. Excessive fixation time can cause the tissue to be brittle
  2. Too little time won’t let the fixative diffuse throughout the entire tissue sample
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18
Q

Processing

A
  • Paraffin for LM
  • Epoxy/resin for TEM
  • Replacing the aqueous fixative solution within the cells with an embedding medium
  • Tissue is dehydrated through an increasing graded series of alcohol (removes all the water from the cells and replaces it with ethanol)
  • Add solvent that is miscible with the embedding medium (xylene for paraffin), (propylene oxide for eposy/resin)
  • Sample is placed in pure solvent, then 50% solvent/50% EM & then 100% EM
19
Q

Embedding

A
  • Paraffin for LM
  • Epoxy/resin for TEM
  • Requires proper orientation of the sample within the embedding mold (cassette for LM; capsule for TEM) followed by polymerization/ curing of EM
  • once sample is oriented properly within the EM, it is filled with the embedding media
  • Paraffin embedding requires cooling @ room temp to solidify
  • Epoxy/resin embedded requires polymerization with heat
  • Final product = block
  • **Once embedded, the sample is protected from degradation and blocks are stored @ room temp
20
Q

Sectioning

A
  • LM: 4-8 microm
  • TEM: 60-90nm
  • act of slicing the tissue block thinly and placing it on a glass slide (LM) or a copper grid (TEM)
  • LM: microtome, stainless steel knife
  • TEM: ultramicrotome, precision glass knives/diamond knives
21
Q

Staining

A
  • LM: hematoxylin & eosin
  • TEM: uranyl acetate/lead citrate
  • adding dyes/stains, antibodies, fluorescent probes to sections to make them viewable
  • For LM, paraffin must be removed from the tissue (needs to happen or it will inhibit the cells fro taking up/reacting with stains & dyes)
  • xylene is used to remove paraffin from the tissue section followed by rehydration through a decreasing graded series of alcohol from 100% alcohol/water
22
Q

Intensity of Staining is due to:

A
  1. abundance of a macromolecule, structure, or component
  2. tissue section thickness
  3. staining method
  4. age of slide
23
Q

Hematoxylin

A
  • chemistry of dye: basic stain (acidophilic)
  • chemistry of structures: acidic structures
  • results on slides: nucleic acids (DNA & RNA)
  • color: blue, purple
  • **
  • stains the nucleus (DNA), nucleolus, and cytoplasm in areas of abundant RNA (rER)
  • Makes them visible through LM
  • color of the nucleic acids stained with hematoxylin will range in the intensity from blue to purple to almost black
24
Q

Pattern of Staining for Hematoxylin informs what:

A

1) cell metabolic activity
2) cell cycle & mitotic phase of cell
3) cell viability (apoptosis)

25
Q

Eosin

A
  • chemistry of dye: acidic stain (basophilic)
  • chemistry of structures: basic structures
  • results on slides: proteins
  • color: pink, red
26
Q

Pattern of Staining for Eosin informs what:

A
  1. Presence of connective tissue
  2. Muscle types
  3. Abundance of mitochondria
27
Q

What are some other facts of H&E staining?

A
  1. With H&E staining, the hematoxylin will fade before the eosin so the old slide looks light pink in color
  2. An abundance of hematoxylin & eosin doesn’t stain tissue
28
Q

Amphophilic

A
  • having the same affinity for basic and acidic dyes
  • example: neutrophil
  • neutral in chemistry as the tissue and cell chemistry has acidic and basic structures
29
Q

Trichrome

A

-stain collagen
-common recipes in this course: Masson’s trichrome & Mallory’s trichrome
-importance: provides contrasting color for collagen vs. other cells/tissues carrying various proteins
-stains collagen as blue/green
***
Masson’s trichrome staining:
-nuclei: black
-cytoplasm: pink, red, brown
-keratin, erythrocytes, myelin & muscle: red
-collagen: blue, green

30
Q

Why is trichrome staining different from H&E?

A
  • In H&E, all proteins stain at a different intensity of red as it’s hard to distinguish pink cardiac muscle (healthy) from pink collagen (scar tissue)
  • With trichrome, college is specifically blue/green
31
Q

Periodic Acid Schiff’s (PAS) Stain

A
  • oxidizes hexose ring of monosaccharides to determine the location and abundance of carbohydrates
  • macromolecules of note: glycogen, glycoprotein, proteoglycans
  • Can be found in: (1) connective tissue, (2) glycocalyx of cells, (3) basal laminae of epithelial cells, (4) muscins of mucus-secreting cells and tissues
  • demonstrates fungi and yeast cells
  • reaction produces a HOT PINK/MAGENTA color
  • hematoxylin is used as a counterstain to reveal the nuclei
32
Q

Elastic stains

A
  • stains elastin and elastic fibers
  • Verhoff’s stains elastin as black
  • Aldehyde-fuchsin stains elastin dark purple
  • Orcein stains elastin bright pink/magenta
33
Q

Silver stains

A

-reduce metal salts causing them to precipitate on certain structures and components within cells and tissues
-Example: silver salts
-Helps visualize the fine structure of the nervous system and connective tissues in delicate organs
-demonstrate fungi and spirochees
***
Golgi Stain
-originally used the gold salt, replaced with silver salt
-stained with silver salt is going to be black
**Was usually used in the nervous system pathways

34
Q

Reticulin Stain

A
  • reticular fibers are an extracellular matrix component formed by the Type 3 collagen
  • Example of a silver stain used to demonstrate the complexity of the reticular network in various organs (esp. smooth muscle and lymphoid organs)
  • this is a type of a silver staining method so the result would be black precipitate on the reticular fluids
35
Q

Total Magnification

A

Objective magnification X ocular lens magnification (always 10X)

36
Q

Field of View

A

-circle you look through the eyepiece of your LM
-field numbers (located on side of ocular lens) = 16-22
-always in mm
*******
diameter of FOV = field number/objective we have in place

37
Q

Lumen

A

middle/opening of the hollow, tubular organ (blood vessel, intestines) that substances pass through

38
Q

Artifacts

A
  • aberrations on slide
  • some of these could be due to processing (different rates of shrinkage can cause some tissues to separate from another to create an artificial space
  • sectioning errors: knife marks
  • section placements: wrinkles are common, especially in large slides
  • staining: dye can fall out of solution and collect on tissue
  • cover slipping: air bubbles from dirty coverslip
39
Q

Interphase nucleus

A
  • pale white/gray areas contain euchromatin (open DNA that can be actively used in the cell)
  • Under H&E, these same areas would stain very little and appear clear to very pale blue
  • the dark black areas are heterochromatin (DNA not actively transcribed)
  • Under H&E, these same areas would stain as dark blue and purple
  • Some of the heterochromatin represents the redundant DNA abundant in eukaryotes
  • The pattern of euchromatin and heterochromatin, its ratio can change for a cell type based on its metabolic activity
40
Q

Endothelial cell nucleus

A
  • marginated heterochromatin pattern
  • the heterochromatin is packaged against the nuclear envelope
  • the heterochromatin outlines the borders of the nucleus within the cytoplasm of this cell
  • without the packaging, the nuclear edges within the cytoplasm of a cell will be vague due to the inability to resolve the nuclear envelope itself under LM
41
Q

Metabolically active cell

A
  • typical nucleus of a cell producing abundant protein

- Has euchromatic nucleus, prominent nucleolus, and some marginated heterochromatin

42
Q

Nucleolus

A
  • Sphere will stain blue and purple as it contains both RNA and DNA
  • the ribosomal subunits exit the nucleus through nuclear pores within the nuclear envelope
43
Q

Rough Endoplasmic Reticulum

A
  • Ribosomes are the location where RNA is translated into protein
  • All cells make proteins that either remain within the cytoplasm for the cell’s day to day operations or is secreted for other purposes
  • the cytoplasmic proteins are translated on polyribosomes that are free within the cell’s cytoplasm
  • protein is packaged and secreted is translated on rER
44
Q

Mitochondria

A
  • Under TEM, they look like striped tigers

- In H&E staining, an abundance of mitochondria will result in a deeply red cytoplasm

Histology (7 decks)

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