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INTSHP2 HISTOPATHOLOGY > CHAPTER 16 PRINCIPLES OF STAINING > Flashcards

CHAPTER 16 PRINCIPLES OF STAINING Flashcards

1
Q

is the process whereby tissue components are made visible in microscopic sections by direct interaction with a dye or staining solution.

A

Staining

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2
Q

Certain parts of cells and tissues that are acidic in character (e.g.?) have greater affinity for basic dyes, while basic constituents (e.g.?) take more of the acid stains.

A

nucleus

cytoplasm

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3
Q

Many dyes, however, require the use of a [?] - a chemical compound that reacts with the stain to form an insoluble, colored precipitate on the tissue and make the staining reaction possible.

A

mordant

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4
Q

The great majority of routine histology is done with [?], because it is quick, cheap and informative.

A

hematoxylin and eosin (H&E) staining

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5
Q

It involves the use of two contrasting stains, e.g., hematoxylin which stains the nuclear detail, and eosin which brings out the cytoplasmic detail of the cell and the tissue’s architecture.

A

hematoxylin and eosin (H&E) staining

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6
Q

is poorly permeable to most staining solutions and should therefore be removed from the section prior to staining.

A

Paraffin wax

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7
Q

This is usually done by immersing the paraffin section in a solvent (e.g. xylene) two times, at [?] duration each, for sections up to [?] thick.

A

1-2 minutes

10 micron

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8
Q

is not miscible with aqueous solutions and low graded alcohol, and should therefore be subsequently removed with absolute alcohol, followed by descending grades of alcohol to prevent damage and detachment of sections.

A

Xylene

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9
Q

The alcohol is then finally replaced with water before actual staining of section is performed. Such procedure is the exact reverse of impregnation and may be summed up by the phrase

A

“Sections to Water”.

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10
Q

If an alcoholic stain is to be used, there is no more need to replace the alcohol with water. After deparaffinization with xylene, the section is transferred to decreasing grades of alcohol, and in such instances, the term “?” is used, and the staining procedure is subsequently done unless the tissue has been fixed in mercuric chloride solution, in which case, the section is taken “to water”.

A

Sections to Alcohol

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11
Q

Sections must be left in the oven for a minimum of [?] before they are finally stained to avoid such problems.

A

30 minutes

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12
Q

is the process whereby the tissue constituents and general relationship between cell and tissue are demonstrated in sections by direct interaction with a dye or staining solution, producing coloration of the active tissue component.

A

Histological staining

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13
Q

Micro-anatomic stains, bacterial stains and specific tissue stains (e.g. muscles, connective tissue and neurologic stains) fall into this category.

A

Histological staining

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14
Q

is the process of giving color to the sections by using aqueous or alcoholic dye solutions.

A

Direct staining

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15
Q

Only one dye is used, which is washed away after 30–60 seconds, prior to drying and examination.

A

Direct staining

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16
Q

[?] is a classic example of a simple stain. This stain will color all cells blue, making them stand out against the bright background of the light microscope.

A

Methylene blue

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17
Q

Indirect staining is the process whereby the action of the dye is intensified by adding another agent or a

A

MORDANT

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18
Q

serves as a link or bridge between the tissue and the dye, to make the staining reaction possible.

A

MORDANT

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19
Q

combines with a dye to form a colored “lake”, which in turn combines with the tissue to form a “tissue- mordant-dye-complex” that is rendered insoluble in ordinary aqueous and alcoholic solvents.

A

MORDANT

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20
Q

This allows subsequent counterstaining and dehydration to be carried out easily.

A

MORDANT

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21
Q

It is an integral part of the staining reaction itself, without which no staining could possibly occur

A

MORDANT

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22
Q

may be applied to the tissue before the stain, or it may be included as part of the staining technique, or it may be added to the dye solution itself.

A

MORDANT

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23
Q

Examples of mordants are

A

potassium alum with hematoxylin in Ehrlich’s hematoxylin, and iron in Weigert’s hematoxylin.

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24
Q

is not essential to the chemical union of the tissue and the dye.

A

ACCENTUATOR

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25
Q

It does not participate in the staining reaction, but merely accelerates the reaction.

A

ACCENTUATOR

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26
Q

Examples of ACCENTUATOR are

A

potassium hydroxide in Loeffler’s methylene blue and phenol in carbol thionine and carbol fuchsin.

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27
Q

is the process whereby tissue elements are stained in a
definite sequence, and the staining solution is applied for specific periods of time or until the desired intensity of coloring of the different tissue elements is attained.

A

PROGRESSIVE STAINING

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28
Q

Once the dye is taken up by the tissue, it is not washed or decolorized. The differentiation or distinction of tissue detail relies solely on the selective affinity of the dye for different cellular elements.

A

PROGRESSIVE STAINING

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29
Q

With this technique, the tissue is first overstained to obliterate the cellular details, and the excess stain is removed or decolorized from unwanted parts of the tissue, until the desired intensity of color is obtained.

A

REGRESSIVE STAINING

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30
Q

is the most common method utilized for microanatomical studies of tissues, using the regressive staining which consists of overstaining the nuclei, followed by removal of superfluous and excessive color of the tissue constituent by acid differentiation.

A

Routine Hematoxylin and Eosin (H&E) staining

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31
Q

is the selective removal of excess stain from the tissue during regressive staining in order that a specific substance may be stained distinctly from the surrounding tissues.

A

DIFFERENTIATION (DECOLORIZATION)

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32
Q

uses more than one chemical stain to better differentiate between various microorganisms or structures/cellular components of a single organism.

A

Differential Staining

33
Q

This is usually done by washing the section in simple solution (e.g. water or alcohol), or by the use of acids and oxidizing agents.

A

Differential Staining

34
Q

One commonly recognizable use of differential staining is the

A

Gram stain.

35
Q

Gram staining uses two dyes: [?] (the counterstain) to differentiate between Gram-positive bacteria (large Peptidoglycan layer on outer surface of cell) and Gram-negative bacteria.

A

Crystal violet and Fuchsin or Safranin

36
Q

entails the use of specific dyes which differentiate particular substances by staining them with a color that is different from that of the stain itself (metachromasia).

A

METACHROMATIC STAINING

37
Q

Tissue components combine with these dyes to form a different color from the surrounding tissue. This is particularly employed for staining cartilage, connective tissues, epithelial mucins, mast cell granules, and amyloid.

A

METACHROMATIC STAINING

38
Q

is a process where specific tissue elements are
demonstrated, not by stains, but by colorless solutions of metallic salts which are thereby reduced by the tissue, producing an opaque, usually black deposit on the surface of the tissue or bacteria.

A

METALLIC IMPREGNATION

39
Q

is the selective staining of living cell constituents,
demonstrating cytoplasmic structures by phagocytosis of the dye particle (cytoplasmic phagocytosis), or by staining of pre-existing cellular components (true vital staining), as in the staining of mitochondria by Janus green.

A

VITAL STAINING

40
Q

is done by injecting the dye into any part of the animal body (either intravenous, intraperitoneal or subcutaneous), producing specific coloration of certain cells, particularly those of the reticulo-endothelial system.

A

INTRAVITAL STAINING

41
Q

Common dyes used are lithium, carmine and India ink.

A

INTRAVITAL STAINING

42
Q

Although methyl violets, of which crystal violet is one, do give metachromatic staining, they are not considered to be the most effective for the purpose. The azures or toluidine blue are more effective usually.

A

METACHROMATIC STAINING

43
Q

is a method of staining used in microscopy to examine
living cells that have been removed from an organism.

A

SUPRAVITAL STAINING

44
Q

It differs from intravital staining, which is done by injecting or otherwise introducing the stain into the body.

A

SUPRAVITAL STAINING

45
Q

New Methylene Blue and Brilliant Cresyl Blue for reticulocyte staining

A

SUPRAVITAL STAINING

46
Q

To achieve desired effects, the stains are used in very dilute solutions ranging from

A

1:5,000 to 1:50,000.

47
Q

Common dyes used are:

A

1 Neutral red
2. Janus green
3. Trypan blue
4. Nile blue
5. Thionine
6. Toluidine blue

48
Q

-probably the best vital dye.

A

1 Neutral red

49
Q

-especially recommended for mitochondria.

A
  1. Janus green
50
Q

-one gram of dye is dissolved in 100 ml. of sterile distilled water to be used immediately;

A
  1. Trypan blue
51
Q

it is dangerous to allow the suspension to stand for more than one hour, because it is likely to become toxic to the cell.

A
  1. Trypan blue
52
Q

is the corner stone of tissue-based diagnosis.

A

Hematoxylin and Eosin (H&E) staining

53
Q

The process stains thin tissue sections so that pathologists can visualize tissue morphology.

A

Hematoxylin and Eosin (H&E) staining

54
Q

plays a significant role in tissue-based diagnosis by coloring otherwise transparent tissue sections, and allowing cell structures including the cytoplasm, nucleus, and organelles and extra-cellular components to be clearly visible under the microscope.

A

Routine H&E staining

55
Q

Fixation: Most fixatives can be used except osmic acid solutions which inhibit hematoxylin.

A

ROUTINE H&E STAINING in Paraffin Embedded Section (Regressive Staining)

56
Q

For tissues fixed with mercuric chloride, the staining time in hematoxylin should be increased slightly while duration of eosin staining should be reduced. The mercury should be removed using a [?] in [?] and rinsed in water. The iodine is then removed by placing the slide in [?] solution for [?] and washing it well in running water for [?].

A

0.5% solution of iodine

80 to 95% alcohol

3% sodium thiosulfate

1 to 5 minutes

3 to 5 minutes

57
Q

Alternatively, mercury deposits may be removed after sections are hydrated, by immersing the sections in [?] for 5 minutes, followed by [?] and subsequently washing the section in water prior to staining.

A

Gram’s or Lugol’s iodine

sodium thiosulfate

58
Q

Staining may be prolonged for [?]-fixed tissues (e.g. Flemming’s fluid), for tissues subjected to long acid decalcification, and after prolonged storage in acid formalin or 70% alcohol.

A

chromium and osmium

59
Q

The following staining methods are commonly employed for frozen sections, the choice depending upon the personal preference of the pathologist and the type of tissue section to be stained.

A
  1. Hematoxylin-Eosin method
  2. Thionine method
  3. Polychrome Methylene Blue method
  4. Alcoholic Pinacyanol method (used also for supravital staining of
    mitochondria and primarily for color sensitization in photography)
60
Q

It is somewhat less favored than regressive staining due to the difficulty of producing sufficiently intense progressive staining of cell structures without staining other parts, thereby resulting in diffused color and obscured details.

A

H & E staining of Frozen Sections for Rapid Diagnosis (Progressive Staining)

61
Q

For convenience, reagents for this rapid H&E stain are generally arranged in sequence using a series of Coplin jars.

A

H & E staining of Frozen Sections for Rapid Diagnosis (Progressive Staining)

62
Q

This method takes only 5-10 minutes and produces well-differentiated sections that are semi-permanent and can be stored.

A

H & E staining of Frozen Sections for Rapid Diagnosis (Progressive Staining)

63
Q

The remaining portion of tissue must be kept for routine processing and are made for comparison with frozen sections.

A

H & E staining of Frozen Sections for Rapid Diagnosis (Progressive Staining)

64
Q

Stains may be effectively removed from the skin by prompt topical application of [?], followed by rinsing with tap water.

A

0.5% acid alcohol

65
Q

Paraffin ribbons containing air bubbles, torn or inadequately infiltrated sections are likely to float from the slide when deparaffinized and stained.

A

COLLODIONIZATION

66
Q

also recommended for sections that will be subjected to strong alkaline or acid solutions and for tissues that contain glycogen for demonstration.

A

COLLODIONIZATION

67
Q

is soluble in absolute alcohol, and will be removed if absolute alcohol is used in the final dehydration prior to clearing of stained sections. Instead, sections treated with 95% alcohol may be transferred to a mixture of equal parts of chloroform, absolute alcohol and xylene (C.A.X,) then treated with xylene and mounted in Xam.

A

Cellulose nitrate (celloidin)

68
Q

Old, bleached or faded sections may be re-stained: the slide is usually immersed in xylene for [?], or gently heated until the mounting medium begins to bubble.

A

24 hours

69
Q

It is placed in a [?] solution for [?], rinsed in tap water and subsequently immersed in [?]for [?] or until the section is decolorized. After washing it again in running tap water for another 5 minutes, the section may then be re-stained with the appropriate staining technique.

A

0.5 potassium permanganate

5-10 minutes

5% oxalic acid

5 minutes

70
Q

is the process whereby various constituents of tissues are studied thru chemical reactions that will permit microscopic localization of a specific tissue substance.

A
71
Q

Chemical ions such as calcium, molecules such as bile pigments, and biopolymers such as cellulose, DNA and specific enzymes are among the tissue components that can be identified using

A

HISTOCHEMICAL STAINING (HISTOCHEMISTRY)

72
Q

The staining techniques employed for histochemistry are also usually applied for staining of histologic structures. Examples of such type of stains are

A

Perl’s Prussian blue reaction for hemoglobin, and Periodic Acid Schiff staining for carbohydrates.

73
Q

is a combination of immunologic and histochemical techniques using a wide range of polyclonal or monoclonal, fluorescent labeled or enzyme-labeled antibodies to detect and demonstrate tissue antigens (e.g., proteins) and phenotypic markers under the microscope.

A

IMMUNOHISTOCHEMICAL (IHC) STAINING

74
Q

is widely used in the diagnosis of abnormal cells such as those found in cancerous tumors, in the localization of biomarkers and differentially expressed proteins in different parts of a biological tissue, and in the detection of specific molecular markers that are characteristic of particular cellular events such as proliferation or cell death (apoptosis)

A

IMMUNOHISTOCHEMICAL (IHC) STAINING

75
Q

Visualizing an antibody-antigen interaction can be accomplished in a number of ways. In most cases, an antibody is conjugated to an enzyme, such as peroxidase, that can catalyze a color-producing reaction. Alternatively, the antibody can also be tagged with a fluorophore, such as fluorescein or rhodamine.

A

IMMUNOHISTOCHEMICAL (IHC) STAINING

76
Q

Commercially produced antibodies most frequently originate from mice, and less frequently from rabbits. The degree of autolysis or putrefaction, the selection of fixation medium, fixation duration, incubation period, and concentration of the selected antibodies can be crucial factors that can affect the results of immunohistochemical staining protocols.

A

IMMUNOHISTOCHEMICAL (IHC) STAINING

77
Q

are used to label defined antigens with monoclonal and polyclonal antibodies.

A

IMMUNOHISTOCHEMICAL (IHC) STAINING

78
Q

Unlike conventional histological staining methods, immunohistochemical techniques are based on antigen–antibody bindings, which can be affected by inappropriate fixative selection and duration.

A

IMMUNOHISTOCHEMICAL (IHC) STAINING

79
Q

Unlike conventional histological staining methods, immunohistochemical techniques are based on antigen–antibody bindings, which can be affected by inappropriate fixative selection and duration.

A

IMMUNOHISTOCHEMICAL (IHC) STAINING

INTSHP2 HISTOPATHOLOGY (14 decks)

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