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Block 1: Histology > Lecture 1 (Revised) > Flashcards

Lecture 1 (Revised) Flashcards

1
Q

Steps required in preparation of living tissues for microscopic examination include?

A

-Fixation
-Dehydration
-Clearing
-Embedding
-Sectioning
-Mounting
-Staining

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2
Q

Light Microscope is composed of?

A

A light source, condenser lens, objective lens, occular lens and a mechanical stage for supporting the specimen

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3
Q

Transmission election microscope is composed of?

A

Uses an electron beam as an “illuminating” beam, magnets for lens and a fluorescent screen to visualize structures of interest

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4
Q

Fixation?

A

To avoid autolysis or degradation by bacteria and to preserve the structure and molecular composition

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5
Q

What chemicals do you use for Fixation?

A

-Formaldehyde (LM)
-Glutaraldehyde (EM)

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6
Q

Formaldehyde?

A

(LM) Reacts with amine groups to stabilize same

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7
Q

Glutaraldehyde?

A

(EM) Cross-links proteins, followed by a second fixation in buffered Osmium Tetroxide that preserves membranes

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8
Q

Dehydration?

A

Water is extracted from fixed tissue fragments by bathing them successively in a graded series of ethanol/water mixtures

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9
Q

Ethanol?

A

-Used for Dehydration
-Fixed tissue is bathed in serious of ethanol and water mixtures
-Last solution is 100% ethanol

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10
Q

Clearing?

A

Making tissue clear or transparent so we can see through it

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11
Q

Xylene?

A

-Solution used in Clearing
-Xylene replaces ethanol is tissue to allow paraffin to penetrate tissue
-Xylene is miscible with embedding medium which makes tissue clear

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12
Q

Embedding?

A

Embedding tissue and making it a harder mold so we can cut it, if it is soft it’ll be hard to cut

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13
Q

Paraffin Wax?

A

-Step 1: Paraffin is melted to 58-60 degrees Celsius
-Step 2: Tissue is placed in melted wax –> heat causes xylene to evaporate
-Step 3: Paraffin cools and hardens

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14
Q

Epoxy Resin?

A

(EM) Used in cases where tissue has been cleared with plastic solvents

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15
Q

Chemicals used in Embedding?

A

-Paraffin Wax
-Epoxy Resin (EM)

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16
Q

Sectioning?

A

Cooled and hardened blocks of paraffin containing tissue are sectioned on a slicing machine called a microtome for LM or on an ultramicrotome for transmission EM

17
Q

Methods used in Sectioning?

A

-Microtome Machine
-Section Mounting
-Section Staining

18
Q

Microtome Machine?

A

Cuts 6u thick for LM and 0.06u thick for EM via ultramicrotome

19
Q

Section Mounting?

A

Glass slides for LM and copper wire grid for EM

20
Q

Section Staining?

A

-Step 1: Dissolve paraffin wax with xylene
-Step 2: Re-hydrate section through a series of alcohols stained with Hematoxylin (H-stain) in water
-Step 3: De-hydrate section through a series of alcohols stained with Eosin (E-stain)

21
Q

H & E Stain?

A

-Most Used
-Hematoxylin + Eosin

22
Q

H?

A

-Hematoxylin
-Stains the cell nuclei and RNA-rich organelles blue/purple
-Basic dye (+ charge) –> basophilia (reacts with negatively charged groups)

23
Q

E?

A

-Eosin
-Stains mitochondria, secretory granules and extracellular material such as collagen pink
-Acidic dye (- charge) –> acidophilic (reacts with positively charged groups)

24
Q

Artifacts of Preparation?

A

Artificial spaces, loss of molecules, shrinkage caused by tissue preparation

25
Q

Shrinkage causes?

A

-Fixation
-Dehydration (in ethanol)
-Heating (in melted paraffin during clearing)

26
Q

Artificial Spaces?

A

Holes between cells and extracellular components

27
Q

Artificial Spaces cause?

A

-Shrinkage
-Loss of molecules (due to chemicals used in tissue preparation)

28
Q

Artifacts of Prep?

A

Collection of distortions caused by prep procedures (shrinkage, artificial spaces, etc.)

29
Q

Artifacts of Prep?

A

Collection of distortions caused by prep procedures (shrinkage, artificial spaces, etc.)

30
Q

Resolution?

A

-Resolving Power
-Smallest distance seen between 2 particles
-Depends on quality of objective lens
-Quality of an image (clarity and detail) depends on resolving power

31
Q

Light Microscope?

A

(LM)
-Max resolving power= 0.2u
-Magnification= 1,000-1,500x

32
Q

Transmission Electron Microscope?

A

(EM)
-Max resolving power= 3nm
-Max magnification= 400,000x
-Magnification= 120,000x

33
Q

H&E preparations do not reveal much about the?

A

Chemical composition

Block 1: Histology (9 decks)

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