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M2C > Histology & Its Methods of Study: Preparation of Tissues for Study > Flashcards

Histology & Its Methods of Study: Preparation of Tissues for Study Flashcards

1
Q

Define Histology

A

the study of the tissues of the body & how these tissues are arranged to constitute organs
- involves tissue biology, focusing on how cells’ structures & arrangement optimize functions specific to each organ

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2
Q

What is the ECM and what does it do?

A

extracellular matrix
- supports the cells

  • contains fluid transporting nutrients to the cells
  • carries away wastes & secretory products
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3
Q

What happens to cells during development?

A
  • they become functionally specialized with their associated ECM
  • create distinct tissues with characteristic structural features
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4
Q

Define microtome

A

instrument used for sectioning paraffin-embedded tissues for light microscopy
- sections placed on glass slides,
allowed to adhere,
deparaffinized, and stained for
light microscopy

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5
Q

Define cryostat

A
  • type of microtome in a cabinet at subfreezing temperature that is used to section the block with tissue
  • frozen sections are placed on slides for rapid staining
  • freezing of tissues used for very sensitive enzymes
  • freezing, unlike fixation, does not inactivate most enzymes
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6
Q

Define Paraffin

A
  • white/colorless soft solid hydrocarbon mixture derived from petroleum
  • commonly used as embedding medium in histology to support and preserve tissue samples for microscopic examination
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7
Q

What is fixation?

A
  • 1st step in tissue preparation
  • small pieces of tissue placed in solutions
  • chemicals that cross-link proteins and inactivate degradative enzymes for preservation of tissue and structure
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8
Q

Dehydration

A
  • 2nd step in tissue preparation
  • tissue transferred through a series of increasingly concentrated alcohol solutions
  • ends with 100% ethanol to remove all water
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9
Q

Clearing

A
  • 3rd step in tissue preparation
  • alcohol is removed in organic solvents in which both alcohol + paraffin are miscible (mixable)
  • replaces dehydrating agent (ethanol) with a clearing agent to make the tissue transparent
  • preserves structure for accurate microscopic examination.
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10
Q

Infiltration

A
  • 4th step in tissue preparation
  • tissue placed in melted paraffin until tissue is properly infiltrated with the embedding medium (paraffin)
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11
Q

Embedding

A
  • 5th step in tissue preparation
  • paraffin-infiltrated tissue is placed in small mold with melted paraffin and left to harden at room temperature
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12
Q

Trimming

A

-6th (mostly unmentioned) step in tissue preparation
- paraffin block is trimmed to expose the tissue for sectioning (slicing) on a microtome

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13
Q

Section Mounting

A
  • trimmed tissue sections are mounted on glass slides for staining required to reveal specific cellular and tissue components with microscope.
  • mounted on paraffin block holder
  • each turn of drive wheel advances generally 1 - 10 um
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14
Q

Describe the H&E staining method

A
  • Hematoxylin & Eosin
  • most common staining method
  • H stains DNA in nucleus, RNA-rich regions of cytoplasm, & matrix of cartilage dark blue/purple
  • E stains collagen & other cytoplasmic structures pink
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15
Q

Define Anionic & Identify properties/associations

A
  • describes cell components such as nucleic acids with a negative charge and an affinity for basic dyes
  • basophilic
  • DNA
  • RNA
  • glycosaminoglycans
  • Hematoxylin
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16
Q

Define Cationic & Identify properties/associations

A
  • describes cell components such as proteins with many ionized amino groups
  • stain more readily with acidic dyes
  • acidophilic
  • mitochondria
  • secretory granules
  • collagen
  • Eosin
17
Q

Enzyme Histochemistry

A

lightly fixed or unfixed tissue sections to produce visible products in the specific enzyme locations.

  • Fixation and paraffin embedding denatures most enzymes, so histochemistry usually uses frozen tissue sectioned with a cryostat.phosphatases, dehydrogenases, and peroxidases, with peroxidase often conjugated to antibodies used in immunohistochemistry.
18
Q

Periodic Acid-Schiff (PAS) Reaction

A
  • utilizes hexose rings of polysaccharides and other carbohydrate-rich tissue structures and stains such macromolecules purple or magenta
19
Q

How can the DNA of cell nuclei be specifically stained?

A

using a modification of the PAS procedure called the Feulgen reaction

20
Q

How can basophilic or PAS-positive material be further identified?

A

via enzyme digestion, pretreatment of a tissue section with an enzyme that specifically digests one substrate
- ex: pretreatment with ribonuclease will greatly reduce cytoplasmic basophilia with little overall effect on nucleus, making RNA important for this process

21
Q

What is a less common method of staining and what does it entail?

A
  • metal impregnation
  • uses solutions of silver salts to visual certain ECM fibers & specific nervous tissue cellular elements
22
Q

How are lipid-rich structures revealed?

A
  • by avoiding processing steps that remove lipids (heat, organic solvents)
  • staining with lipid-soluble dyes such as Sudan black and Oil Red O
23
Q

What are lipid-soluble dyes like Sudan black useful for?

A

diagnosis of metabolic diseases involving intracellular accumulations of cholesterol, phospholipids, or glycolipids

24
Q

How long does slide preparation, from tissue fixation to observation with a light microscope, take depending on the size of the tissue, embedding medium, and the method of staining?

A

12 hours to 2 1/2 days

25
Q

What is the final step before microscopic observation?

A

mounting a protective glass coverslip on the slides with a clear adhesive

26
Q

Interpretation of Structures in Tissue Sections

A

certain steps distort tissues slightly, producing structural abnormalities called artifacts not present in the living tissue
- shrinkage caused by fixative, ethanol, or heat
- spaces caused by loss of lipids not preserved
- small wrinkles or precipitates (clumps) from stain
- TEM allows observation of cells in ECM but only a few can be conveniently studied in small samples
- Sections of cells or tissues are essentially 2D planes through 3D structures, and understanding this fact is important for their correct interpretation and study

M2C (11 decks)

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