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Histo 1 > Histology: Stains and Cell Ultrastructure > Flashcards

Histology: Stains and Cell Ultrastructure Flashcards

1
Q

Define: Histology

A

The study of functional tissue

aka Microanatomy

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2
Q

Nomarski (DIC)

A

pseudo 3D organelles

not used much with tissues

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3
Q

Giemsa Staining

A

common in research labs

uses eosin and methylene blue to stain cells

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4
Q

What is the most common stain?

A

Hematoxylin (blue-purple) and Eosin (orange-pink)

(H&E)

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5
Q

Basophilic structures

A
  • Stain with hematoxylin
  • ribosomes, nucleic acids, nucleus, rough er
  • Basophilic = Blue
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6
Q

Eosinophilic Structures

A

Acidophilic

stain with Eosin (orange/pink)

mitochondria, lysosomes, cytoplasm

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7
Q

What is normally lost during fixation?

A

Lipids/Carbohydrates

Ground substance (water, salt)

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8
Q

What are tissues normally processed in?

A

formaldehyde → it crosslinks proteins

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9
Q

How do you visualize cells in an H&E Stain?

A

Cytoplasm → pink

nucleus → purple

empty space → where glycogen and other carbohydrates were lost

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10
Q

Cresyl Violet (RNA)

A

Stains neurons, rough ER, and polysomes really well

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11
Q

Periodic Acid Schiff (PAS)

A

Stains carbohydrates (basement membrane, mucus) (magenta)

Schiffs reagent stains nuclei (DNA)

counterstain with Fast Green FCF (cytoplasm, collagen)

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12
Q

How does PAS work?

A

Periodic Acid or its sodium salt oxidizes glycols to aldehydes, which are made visible by exposure to Schiff’s Reagent

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13
Q

What is Schiff’ Reagent made from?

A

Basic Fuchsine and thionyl chloride

stains nuclei

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14
Q

What is the counterstain for PAS?

A

Fast Green FCF (stains cytoplasm and collagen)

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15
Q

What does PAS stain?

A

Carbohydrates (basement membrane, mucus)

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16
Q

Congo Red

A

Direct cotton dye

hydrogen binding to carbohydrates

elongated hydrophobic structure will bind amyloid fibrils under specific conditions (High pH, rinse with ethanol)

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17
Q

How does Congo Red stain work?

A

Hydrogen Binding to carbohydrates

Elongated hydrophobic structure will bind amyloid fibrils under specific conditions

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18
Q

What does Congo Red stain?

A

Amyloid deposits (shown in polarized light (green))

protein carbohydrates

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19
Q

What is a unique feature of Congo Red Stain?

A

unique feature of amyloid binding: dichroism

Reddish staining in bright-field

Bright Apple Green birefringence under polarized light (glows) → shows amyloid deposits

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20
Q

Where is Congo Red Staining mostly used?

A

Pathology

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21
Q

Alcian Blue

A

stains acid mucosubstances

does not usually stain nuclei or RNA

use pink or red counterstain (PAS is compatible)

different pHs stain dif things

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22
Q

What does Alcian Blue stain?

A

acid mucosubstances

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23
Q

What does Alcian Blue usually NOT stain?

A

Nuclei or RNA

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24
Q

What happens to a cell with Alcian Blue staining at pH = 2.5?`

A

all acid mucosubstances stain

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25
Q

What happens to a cell with Alcian Blue staining at pH = 1?`

A

only sulfated acid mucosubstances will stain

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26
Q

What is a counterstain for Alcian Blue?

A

pink or red counterstain (PAS is compatible)

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27
Q

What is a counterstain for PAS?

A

Fast Green FCF

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28
Q

Masson’s Trichrome

A

stain nuclei with iron-hematoxylin (brownish black)

stains cells and connective tissue different colors

Counterstain with Fast Green FCF (cytoplasm and CT) or aniline blue

and Biebrich Scarlet-Acid Fuchsine (Type I Collagen)

must be careful with fixative used

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29
Q

What does Masson’s Trichrome stain? with what?

A

Nuclei → Iron-hematoxylin

Cytoplasm and CT → Fast Green FCF or aniline Blue

Type 1 Collagen (Muscle, Cytoplasm, Keratin) → Biebrich Scarlet-Acid Fuchsine

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30
Q

What fixatives work well with Masson’s Trichrome?

A

Mercury-containing like SUSA

or Zinc-formaline (Kiernan)

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31
Q

What is used as a counterstain for Masson’s Trichrome?

A

Fast Green FCF (cytoplasm and CT) or aniline Blue

and

Biebrich Scarlet Acid Fuchsine (Type I Collagen)

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32
Q

Which counterstain is the differentiation step in Masson’s Trichrome?

A

Biebrich Scarlet-Acid Fuchsine

it stains muscle, cytoplasm, and keratin red

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33
Q

Prussian Blue

A

Reacts chemically to iron present in the tissue and produces an insoluble blue pigment (ferric ferrocyanide)

detects cysteine residues, serotonin, catecholamines, and melanin precursors

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34
Q

What is Prussian Blue made of?

A

Ferric Ion and ferricyanide

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35
Q

What does Prussian Blue Detect?

A

detects cysteine residues, serotonin, catecholamines, and melanin precursors

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36
Q

What is Prussian Blue used for pathologically?

A

to detect iron deposits in tissue

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37
Q

Mucicarmine

A

Stains Epithelial mucins deep red

Carmine stains nuclei

adds aluminum, changing chemistry

will also stain cryptococcus neoformans (fungus)

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38
Q

What does Mucicarmine stain? what color?

A

epithelial mucins → deep red

carmine stains nuclei

fungus (Cryptococcus Neoformans)

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39
Q

What is Carmine from Mucicarmine made from?

A

ground insects

or synthetic alternatives

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40
Q

Verhoeff-van Gieson (VVG)

A
  • Stains elastic fibers
  • Two parts:
    • Verhoeff’s stain →stains elastic fibers nuclei, and myelin sheaths black
    • Van Gieson’s reagent → stains collagen red (Acid fuchsine) and cytoplasm yellow (Picric Acid)
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41
Q

What does VVG stain? what color?

A

elastic fibers, nuclei, myelin sheaths → black

collagen → red

cytoplasm → yellow

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42
Q

What is VVG made of?

A
  • Verhoeff’s stain → iron hematoxylin + iodine, potassium Iodide
  • Van Gieson’s reagent
    • Acid Fuchsine (red)
    • Picric acid (yellow)
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43
Q

Acid Fast-Bacilli (AFB)

A

Stains red for mycobacteria (includes causative agents for TB and leprosy)

Ziehl-Neelson method

Uses heat (and sometimes phenol) to permeabilize bacterial cell walls: dye retained after cooling

uses basic fuchsine or pararosaniline

counterstain with methylene blue

44
Q

What does AFB stain? what color?

A

Mycobacterium (including causative agents for TB Leprosy) → Red

45
Q

How does AFB work?

A

Uses heat (and sometimes phenol) to permeabilize bacterial cell walls

dye retained after cooling

Uses Basic Fuchsine or Pararosaniline

46
Q

What do you counterstain AFB with?

A

methylene blue

47
Q

Gram Stain

A
  • Stain with Crystal Violet (checks cell proliferation)
  • precipitate dye with iodine-potassium iodine solution
  • extract insoluble dye with organic solvent (etOH, Acetone, aniline)
    • gram negative → don’t retain dye
    • gram positive → retain dye, it cannot be extracted
  • Stain with safranin
    • stains gram-negative red
48
Q

Gram-negative

A

Bacteria that lose dye

stained red with safranin

49
Q

Gram positive

A

bacteria that retain dye in a gram stain

fungal hyphae and spores are gram positive

50
Q

Gomori Methenamine Silver Nitrate (GMS)

A
  • Detects bacteria known as spirochetes
    • carbohydrates in fungal wall
  • stains fungi, cellulose, starch, and chitin black
    • also stains melanin granules, glycogen, or mucus
  • counterstain: Fast Green FCF
51
Q

What does GMS use?

A

Chromium trioxide, sodium metabisulfite, methenamine-silver stock (with borax for working solution)

52
Q

What was Gomori looking for when using GMS?

A

glycogens and mucus

53
Q

What does Gomori Methenamine Silver Nitrate (GMS) stain? what color?

A

Fungi, cellulose, starch, and chitin

stains black

also stains melanin granules, glycogen, or mucus

54
Q

What is the counterstain for Gomori Methenamine Silver Nitrate (GMS) stain?

A

Fast Green FCF

55
Q

Reticulin

A
  • Used for reticular fibers and basement membrane
    • collagen fibers with hexose sugars bound
  • similar to PAS but uses silver
  • can use trichrome (Lillie’s allochrome)
  • Fibers are birefringent using picro-sirius red
    • but basement membranes will not show as well
56
Q

What does Reticulin stain for?

A

Reticular fibers and basement membranes

57
Q

What can be used to counterstain Reticulin?

A
  • trichrome (Lillie’s Allochrome)
  • Picro-sirius Red (stains fibers red)
    • basement membranes won’t show well
58
Q

What are the acidic (acidophilic) dyes?

A
  • Eosin
  • Picric Acid (Verhoeff-Van Giesen VVG)
  • Acid Fuchsine (collagen)
  • Fast Green FCF (CT)
  • Biebrich Scarlet
  • Picric-Sirius red
59
Q

What are the basic (Basophilic) dyes?

A
  • Hematoxylin
  • Methylene Blue (Giemsa)
  • Basic Fuchsine (pararosaniline)
  • Crystal Violet (Gram Stain)
  • Cresyl Violet (Nissl Stain)
  • Orcein (mixture; elastin)
  • Safranine
60
Q

What is osmium stain used for?

A

lipids

myelin sheath around nerves

fixes lipids in place

61
Q

What is Periodic Acid Schiff (Pas) used for?

A

Carbohydrates (glycogen in hepatocytes)

62
Q

What is silver stain used for?

A

proteins

nerve fibers, reticular fibers

63
Q

What are trichrome stains used for?

A

will stain connective tissue components different colors

64
Q

What happens when lipids/carbs are lost during fixation?

A

Dark purple areas show protein only

lighter color areas are heavily glycoylated

65
Q

Why is osmium better than H&E for staining the myelin sheath?

A
  • H&E
    • can’t really see myelin sheath
  • Osmium
    • stains myelin sheath black
    • can be used to see damage to myelin sheath
66
Q

What stain is better for Nerve fibers?

H&E or Silver?

A

Silver; makes it easier to see nerve fibers

67
Q

What do you do if you cannot distinguish between cells morphologically?

A
  • Look at the proteins they express
    • B cells vs. T cells
    • NK cells vs. T Helper cells
    • Glial Cells in the CNS
  • they all looks similar but express different proteins
68
Q

How do you histologically look at protein expression?

A
  • Immunohistochemistry (IHC)
    • on cultured cells → immunocytochemistry (ICC)
69
Q

How does immunohistochemistry work?

A

It uses antibodies to recognize specific proteins

it detects antibodies through bound moieties (direct or indirect)

70
Q

What is a direct moiety in IHC?

A

moiety bound to a primary antibody

71
Q

What is an indirect moiety in IHC?

A

moiety bound to secondary antibody that recognized primary antibody

72
Q

How does IHC work?

A
  1. Fluorescein isothicynate (FITC)
  2. Antibody that recognizes antibody (ed. Goat anti-rabbit)
  3. Antibody that recognizes protein of interest (e.g. Akt)
  4. Cell growing on glass surface (or cell in tissue slice on glass slide)
    1. fixed and permeabilized
73
Q

How is IHC visualized?

A

Colorimetric → Visible on light microscope

Fluorescent → more sensitive

74
Q

What is an issue with Non-IHC H&E Staining of the Epidermis?

A

Can see Keratinocytes but not Langerhans cells

75
Q

What is the pro of colorimetric IHC staining?

A

You can see Keratinocytes (purple) and Langerhans Cells (Gold)

76
Q

What is the pro oof Fluorescent IHC Staining?

A

Good for showing the morphology of Langerhan Cells but Cannot see Keratinocytes

77
Q

How are fluorescent dyes classified?

A
  • based on excitation/emission spectra
    • light absorbed at one wavelength (excitation
    • light emitted at lower energy (higher wavelength)
78
Q

What are commonly used fluorophores? What color do they show?

A

DAPI → blue

FITC → Green

Cy3 → Red

79
Q

How do you determine which stain to use?

A

Based on what structure you want to look at and the chemistry of the stain

80
Q

What are the major organelles and their general functions in mammalian cells?

A
  • Nucleus → information storage
  • Cytoskeleton → structure
  • Endoplasmic Reticulum → protein/lipid production
  • Mitochondria → Energy production
  • Golgi Apparatus → Protein production
  • Cytoplasm → Ionic balance, protein aggregates
81
Q

Define: DNA

A

higher order organization by histones

82
Q

Define: Nucleolus

A

ribosome production

proteins imported from cytoplasm

83
Q

Define: Euchromatin (EC)

A

unraveled chromatid

Doesn’t stain as intensely b/c it is unraveled

actively transcribed

84
Q

Define: Heterochromatin (HC)

A

Condensed chromatid

not actively transcribed

The more HC the more inactive

85
Q

What does a flat/compacted nucleus mean?

A

it is inactive

86
Q

What does a round nucleus mean?

A

it is active

87
Q

Define: Microfilaments

A

Structural (red)

88
Q

Define: Microtubules

A

Organization (green)

89
Q

Define: Intermediate Filaments

A

signal transduction

90
Q

Define: Endoplasmic Reticulum

A

Protein (rough) and lipid (smooth) production

Specific conditions required for normal protein folding

calcium storage

91
Q

Define: Mitochondria

A

Energy production by protein complexes across inner and outer membranes

Matrix has specific chemistry (highly eosinophilic-pink areas)

Calcium Storage

Closely associated with microtubules and ER

92
Q

Which star has more mitochondria?

A
  • Red star
    • more eosinic
    • more energy
    • more active transport
    • more mitochondria (pinker)
  • alot of mitochondria = stain heavily with Eosin
93
Q

Define: Golgi Apparatus

A

Found to one side of the. nucleus

Vesicle production

Protein processing and sorting

Associated with microtubule organizing center (MTOC)

doesn’t typically stain

94
Q

What is an artifact?

A

Debris or damage from preparation of the tissue

95
Q

What are the types of artifacts?

A

Separation

Tears

Folds

Dust

Bubbles

Chatter

Loss of delicate structures

96
Q

What is a Separation artifact?

A

Most common between two types of tissues

tissue separates

organelles pull apart

97
Q

What is a Tear artifact?

A

Found in the middle of tissue

tissue rips during preparation

nothing lining it (lumen is usually lined with epithelia)

98
Q

What is a fold artifact?

A

Tissue folds during preparation

Common in cartilage

99
Q

What is a Dust artifact?

A

Dust particles get trapped in slide during preparation

100
Q

What is a chatter artifact?

A

Blade used to cut tissue doesn’t cut through tissue smoothly

changing blade helps avoid

causes lines in tissue

101
Q

What is a loss of delicate tissues?

A

Area cibrosa in the renal pyramid

tissue gets removed during preparation

102
Q

What is photomicrography?

A

Taking pictures of cells

103
Q

What are the two types of cameras?

A

Non-digital (rare)

Digital

104
Q

What is a non-digital camera?

A

Best quality on color reversal film (slides)

requires a projector

images must be scanned for digital

(What Neary Used for Thesis)

105
Q

What is a digital camera?

A
  • Basic will capture color
  • More advanced will capture pixels
    • pseudo-coloring used for fluorescent channels
  • Best files are high resolution TIFF (can use JPEG)

Histo 1 (13 decks)

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