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Histology M1 > Histology Overview of Methods (M1) > Flashcards

Histology Overview of Methods (M1) Flashcards

1
Q

What is Histology

A

The scientific study of microscopic structures of tissues and organs of the body

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2
Q

Histology is also called…

A

Microscopic Anatomy

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3
Q

What is Histopathology?

A

Microscopic study of diseased tissue

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4
Q

What is Histotechnology?

A

Processing of tissues in such a manner as to enable microscopy / study of tissue

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5
Q

Modern histology includes many aspects of…

A

molecular and cellular biology, which help describe cell organization and function

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6
Q

What are the 7 main steps to prepare a slide?

A
  1. Fixation 2. Dehydration 3. Embedding 4. Sectioning 5. Mounting 6. Staining 7. Observation
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7
Q

What is the primary purpose of Fixation ?

A

Purpose: Maintenance of tissue architecture by cross linking proteins and inhibiting autolysis.

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8
Q

What is the procedure in Fixation?

A
  1. A large specimen is received 2. Cut into smaller pieces 3a. For Light Microscope, specimen is dipped in formalin 3b. Electron Microscope, specimen is dipped in Glutraldehyde and then Osmium tetroxide. 4. Wait 24 hours
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9
Q

What is the primary purpose of Dehydration and Clearing?

A

To remove all the water because the Paraffin is immiscible in water.

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10
Q

What are the steps in Dehydration and Clearing?

A
  1. Dehydration- solution is placed in increasing concentrations of alcohol beginning at 50% and increasing to 100%. Each step takes 2-3 hours. 2. Clearing- Removing the alcohol and replacing it with a chemical that is miscible in both alcohol and paraffin. Xylene solution is used to infiltrate the tissues. Smaller tissues take up to an hour while larger ones take 2-4 hours.
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11
Q

What is Embedding?

A

Tissues are placed in an oven containing liquid paraffin that infiltrates it.

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12
Q

What is occurs during the Embedding process?

A

The high temperatures of the oven (52-60 C) evaporates the Xylene in order to obtain a block of Paraffin. A Light Microscope uses Paraffin and plastic resins while an Electron Microscope uses resins for the medium.

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13
Q

What are the advantages/disadvantages to using Paraffin as the embedding medium?

A

Advantage- it stains reliably and is easy to work with Disadvantage- slices cannot be cut very thin

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14
Q

What is involved in Sectioning and what step is done to allow sectioning to take place?

A
  1. Trimming- a process in which excess paraffin is removed from the block to expose tissue to be cut by the microtome. 2. Sectioning- involves using a microtome to cut sections very thin in the form of ribbons. Ribbons are cut and mounted on glass slides. *Light Microscope- sections are 5-10 micrometers thick *Electron Microscope- sections are 0.02-0.1 micrometers thick
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15
Q

What is a disadvantage of cutting thick sections?

A

Overlapping, which causes a decrease in resolution

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16
Q

In Mounting and Staining of the sample, what are the most common dyes used and give a description of when we use each.

A

Most Common Stains: Hematoxylin and Eosin Hematoxylin- it is a BASIC dye and it colors the Acidic components of cells by giving a bluish-purple tint. It reacts with negatively charged ionized phosphate groups in nucleic acids (basophilic structures) Eosin- An ACIDIC dye and it colors the Basic components a pinkish tint. It reacts with positively charged cationic groups in cells and tissues, particularly amino groups of proteins (eosinophilic structures)

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17
Q

Give examples of when we would use Hematoxylin

A

Use for protein rich areas like Nuclei, Rough Endoplasmic Reticulum, collagen matrix

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18
Q

Give examples of when we would use Eosin

A

Use for basic components of cells such as cytoplasm, collagen fibres, mitochondria, lysosomes, muscle, connective tissue, colloid, red blood cells.

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19
Q

If reagent Masson’s trichrome were used, what would result?

A

Black: Nuclei

Red: Muscle, keratin, cytoplasm

Light Blue: mucinogen, collagen

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20
Q

If reagent Orcein’s elastic stain were used, what would result?

A

Brown: elastic fibers

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21
Q

If reagent Weigert’s elastin stain were used, what would result?

A

Blue: elastic fibers

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22
Q

If reagent Silver stain were used, what would result?

A

Black: reticular fibers

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23
Q

If Iron hematoxylin were used, what would result?

A

Black: striations of muscle, nuclei, erythrocytes

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24
Q

If Periodic acid-Schiff were used, what would result?

A

Magenta: glycogen, carbohydrate-rich molecules

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25
Q

If Wright’s and Giemsa stains were used, what would result?

A

Pink: erythrocytes, eosinophil granules Blue: cytoplasm of monocytes and lymphocytes

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26
Q

What is Wright’s and Giemsa stain used for?

A

Differential staining of blood cells

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27
Q

What are histochemical and cytochemical procedures based on?

A

Specific binding of a dye with a particular cell component exhibiting inherent enzymatic activity of a cell component.

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28
Q

When the extracellular matrix displays basophilia, what structures is stained?

A
  1. Heterochromatin and nucleoli of the nucleus (mainly because of ionized phosphate groups in nucleic acids of both) 2. Cytoplasmic Components such as the ergastoplasm (also because of ionized phosphate groups in ribosomal RNA) 3. Extracellular Materials such as the complex carbohydrates of the matrix of cartilage (because of ionized sulfate groups)
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29
Q

When the extracellular matrix displays acidophilia, what structures is stained?

A
  1. Most cytoplasmic filaments, especially those of muscle cells 2. Most intracellular membranous components and much of the otherwise unspecialized cytoplasm 3. most extracellular fibers (primarily because of ionized amino groups)
30
Q

What is Metachromasia?

A

Absorption of certain wavelengths of light and emit a different wavelength.

31
Q

Why Metachromasia?

A

Polyanions of tissues bind with dye molecules result in polymer or dimers of dye molecules (appear as different color than expected- methylene blue gives red or purple color)

32
Q

What are metachromatic substances?

A

Ionized SO4, PO4 of cartilage

33
Q

Where can you find ionized SO4, PO4 of cartilage?

A

Mast cell granules (heparin) and rough Endoplasmic Reticulum of plasma cells.

34
Q

What is PAS?

A

Periodic Acid Schiff. It is a special stain

35
Q

PAS positive substances?

A

Carbohydrate (glycogen) or carbohydrate rich molecules, basement membrane, reticular fibers.

36
Q

Aldehyde binds with Schiff to produce what color?

A

Magenta or Pink * Hexose rings of Carbohydrates contain adjacent carbons, each bears a hydroxyl group * Hexosamines of glycosaminoglycans contain adjacent carbons, one of which bears a hydroxyl group, but the others bear an amino group

37
Q

What stain is used for nuclear proteins?

A

Feulgen Stain

38
Q

Why can’t RNA be stained by Feulgen?

A

Because it targets a specific sugar-deoxyribose (RNA has ribose)

39
Q

Explain what occurs in a Feulgen stain for nuclear proteins.

A
  1. Acid hydrolysis or cleaves proteins from deoxyribose of DNA, which leads to opening of sugar group and formation of aldehyde 2. Schiff binds and gives magenta color to aldehyde 3. It is useful to quantify the amount of DNA (by usingh spectrophotometry of Feulgen stained tissu)
40
Q

If DNA is stained by Feulgen what color would the DNA be and what color would the Cytoplasm be?

A

DNA: Red - Purple Cytoplasm: green

41
Q

Why are special stains used?

A

Used to identify certain normal and abnormal substance present in the cells and tissue, which cannot be identified on routine

42
Q

Haematoxylene and Eosin staining are better appreciated on?

A

a special stain

43
Q

What is the basement membrane largely composed of?

A

Type IV collagen and laminin

44
Q

What is a Reticular fiber consist of?

A

Consists of a very thin fiber of type III collagen which are widespread in connective tissue throughout the body.

45
Q

What does Reticulin Stain-Gomori’s method help see?

A

Reticular fibers and basement membrane material

46
Q

When is Trichrome stain used ?

A

To evaluate the type and amount of extracellular material like collagen, muscle and elastic fiber

47
Q

What techniques are used for Trichrome stain?

A
  1. Masson trichrome stain 2. Van Gieson Stain 3. Verhoeff-Van Gieson (VVG) stain
48
Q

When is Masson trichome stain used?

A

for detection of collagen fibers in the tissues such as skin, stomach, intestine and lung.

49
Q

Masson trichome stain is used to differentiate between what?

A

Differentiate between collagen and smooth muscle in tumour.

50
Q

Masson trichome is used to help identify?

A

To identify increased collagen deposition in condition like cirrhosis, keloid, benign prostatic hyperplasia, prostatic hyperplasia, membranoproliferative glomerulonephritis etc.

51
Q

What color is resulted from using Masson trichome stain in nuclei, collagen, cytoplasm?

A

Nuclei: black Collagen: blue Cytoplasm, RBCs- Red

52
Q

Van Gieson Stain is used for the detection of?

A

Collagen

53
Q

What is the principle behind Von Kossa Method for Calcium?

A

Principle: The tissue sections are treated with silver nitrate solution, silver is deposited by replacing the calcium and then it is reduced by the strong light and visualized as metallic silver.

54
Q

When is Von Kossa Method used?

A

For use in abnormal deposits of calcium. H&E stain causes calcium to appear deep blue-purple. Von Kossa method will have it appear black.

55
Q

What colors result from utilizing the Von Kossa Method for Calcium salts, nuclei and cytoplasm?

A

Calcium Salts- black Nuclei- red Cytoplasm- pink

56
Q

What are the various uses for PERL’S-Prussian Blue Reaction for Iron?

A
  1. To demonstrate ferric iron in tissue sections 2. Small amounts of iron are found normally in spleen and bone marrow 3. Excessive amounts are present in hemochromatosis- with deposits found int he liver and pancreas and hemosiderosis- with deposits in the liver, spleen, lymph nodes 4. To access the bone marrow iron content
57
Q

What colors result from using PERL’S-Prussian Blue for Iron, Nclei, and the background?

A

Iron- blue Nuclei- red Background- pink

58
Q

What is Immunocyto/histochemical staining?

A

a technique to identify cellular or tissue constituents (antigen), by means of antigen-antibody interactions

59
Q

How is the site of antibody binding identified in Immunocyto/histochemical staining?

A

Is identified by direct labeling of antibody or by using a secondary labeling method

60
Q

Immunocyto/histochemical staining name if it is in tissue/cell?

A

Tissue: Immunohistochemistry Cell: Immunocytochemistry

61
Q

What is an antigen?

A

a protein or carbohydrate or lipid molecule. It has one ore more antibody binding sites. A highly specific topographical region composed of a small number of amino acids or monosaccharide unit known as antigenic determinant group or epitopes.

62
Q

What is an antibody and what are the two types?

A

Antibody is a molecule that can recognize and bind to the antigen. Polyclonal Antibody and Monoclonal Antibody

63
Q

What are characteristics of polyclonal antibody?

A
  1. An antibody that can bind to several similar epitopes 2. It may cross react with other molecules 3.Has low sensitivity.
64
Q

What are the characteristics of Monoclonal antibody?

A

An antibody which is sensitive to only one epitope. Does not cross react with other molecules and High sensitivity.

65
Q

What happens in direct immunofluorescence?

A

A fluorochrome-labeled primary antibody reacts with a specific antigen within the tissue sample.

66
Q

What does the indirect method involve?

A

Involves 2 processes. First, the specific primary antibodies react with the antigen of interest. Second, the secondary antibodies, which are fluorochrome labeled, react with the primary antibodies.

67
Q

What are artifacts?

A

Found in the specimen slides and are wrinkles or folds, which are well-defined dense staining regions in the section where detail is obscured.

68
Q

What happens during a fold?

A

It depends on the type of tissue but sometimes it is tissue that has doubled up on itself.

69
Q

What are three other types of artifacts that may present itself in a specimen slide?

A
  1. Foreign body introduction- occurs when the slide had its coverslip applied. 2. Irregular distribution of staining- a clump of stain precipitate that most likely occurred during the staining process. 3. A torn slide- specimen is torn or separated and is introduced when processing and/or sectioning.
70
Q

What is a knife mark?

A

Scratches present on the microtombe blade’s surface that then scored the section as it was cut.

71
Q
  1. What type of tissue is this?
  2. What staining was used
  3. Identify what structure each of the three lines are identifying.
A
  1. Skeletal Muscle Tissue
  2. Hematoxylin was used to give the acidic components of the cell a bluish-purple color. Eosin was used to give the basic comonents of the cell a pinkish color.
  3. Top Arrow- Nuclei

Middle Arrow- Muscle Fibers

Bottom Arrow- Muscle Fibers

Histology M1 (2 decks)

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