Digital Flashcard Assignment

20 stains!

Centers for Disease Control and Prevention, V. McPheeters. (1965). Ziehl–Neelsen-stained specimen showing Mycobacterium tuberculosis [Photomicrograph]. Public Health Image Library, Image ID 23000.

Stain Name

Ziehl–Neelsen, Kinyoun, and Fite Acid-Fast Bacteria Stains

Purpose

Demonstrates acid-fast bacteria.

• Ziehl–Neelsen/Kinyoun: Mainly Mycobacterium tuberculosis

• Fite: Mainly Mycobacterium leprae

Color Results

• Acid-fast bacteria: Red/pink

• Background tissue: Blue

Reagents Responsible for Color

• Carbol fuchsin: Stains bacteria red

• Acid-alcohol: Removes excess stain

• Methylene blue: Stains background blue

• Heat: Helps stain penetrate in Ziehl–Neelsen

• Concentrated carbol fuchsin: Replaces heat in Kinyoun

• Xylene–peanut oil: Protects weakly acid-fast bacteria in Fite

ZEISS. (n.d.). Detection of acid-fast bacilli [Photomicrograph].

Stain Name:
Auramine–Rhodamine Fluorochrome Stain

Purpose:
Demonstrates acid-fast bacteria, especially Mycobacterium tuberculosis.

Color Results:

• Acid-fast bacteria: Bright yellow-orange or reddish-yellow

• Background: Black/dark

Reagents Responsible for Color:

• Auramine O and Rhodamine B: Produce fluorescent yellow-orange bacteria

• Potassium permanganate: Quenches background fluorescence, making it dark

Image citation:
Newcomer Supply. (n.d.). Gram, Brown-Hopps stain kit [Photomicrograph]. Fisher Scientific.

Stain Name:
Brown and Hopps Modified Gram Stain

Purpose:
Demonstrates and differentiates gram-positive and gram-negative bacteria in tissue.

Color Results:

• Gram-positive bacteria: Blue/violet

• Gram-negative bacteria: Red

• Nuclei: Red

• Background: Yellow

Reagents Responsible for Color:

• Crystal violet + Gram iodine: Blue/violet gram-positive bacteria

• Basic fuchsin: Red gram-negative bacteria and nuclei

• Tartrazine or picric acid: Yellow background

• Gallego solution/Cellosolve: Differentiates the bacteria

National Society for Histotechnology. (2021). Crystal violet-stained amyloid tissue [Photomicrograph].

Stain name:
Crystal Violet Stain

Purpose:
Rapid screening method used to demonstrate amyloid deposits in tissue.

Color results:

• Amyloid: reddish-purple or purple/violet

• Background tissue: blue to blue-violet

Reagents responsible for color:

• Crystal violet: produces the reddish-purple metachromatic color in amyloid and the blue-violet background staining.

Uthman, E. (2011). Helicobacter pylori, stomach biopsy, Giemsa stain [Photomicrograph]. Wikimedia Commons.

Stain name:
Giemsa Stain for Microorganisms

Purpose:
Demonstrates microorganisms, especially Helicobacter pylori, certain parasites, and some fungi in tissue.

Color results:

• Microorganisms: dark blue to purple

• Nuclei: blue to purple

• Cytoplasm/background: pale blue to pink

Reagents responsible for color:

• Methylene blue and azure dyes: stain microorganisms and nuclei blue-purple

• Eosin: stains cytoplasm and background pink

Newcomer Supply. (n.d.). Fungus, Grocott methenamine silver stain [Photomicrograph].

Stain name:
Grocott–Gomori Methenamine Silver (GMS) Stain

Purpose:
Demonstrates fungi and Pneumocystis organisms in tissue.

Color results:

• Fungal organisms: black to dark brown

• Background tissue: light green

Reagents responsible for color:

• Chromic acid: oxidizes fungal cell-wall carbohydrates to aldehydes

• Methenamine–silver nitrate: deposits metallic silver, producing black organisms

• Gold chloride: tones the silver deposits black

• Light Green SF Yellowish: produces the green background

Silver-stain extra credit:

• Reaction: argentaffin

• Developer/reducer used: no external developer

• Reducing agent: aldehyde groups in the fungal cell wall reduce the silver

• Silver solution: alkaline methenamine–silver nitrate

University of Utah WebPath. (n.d.). Spirochetes with Warthin–Starry silver stain [Photomicrograph].

Stain name:
Warthin–Starry Silver Stain

Purpose:
Demonstrates spirochetes, Helicobacter pylori, Legionella, and Bartonella organisms in tissue.

Color results:

• Microorganisms: black to dark brown

• Nuclei: brown

• Background tissue: yellow to golden brown

Reagents responsible for color:

• Acidified silver nitrate: impregnates the microorganisms with silver

• Hydroquinone: reduces silver ions to black metallic silver

• Gelatin: stabilizes the reducing solution

Silver-stain extra credit:

• Reaction: argyrophilic

• Developer/reducer used: yes

• Developer/reducing agent: hydroquinone

• Silver solution: acidified silver nitrate

iHisto. (n.d.). Luxol Fast Blue-stained central nervous system tissue [Photomicrograph].

Stain name:
Luxol Fast Blue (LFB) Myelin Stain

Purpose:
Demonstrates myelin and identifies areas of demyelination in central nervous system tissue.

Color results:

• Myelin: blue

• Demyelinated areas and background: pale or colorless

Reagents responsible for color:

• Luxol Fast Blue: stains myelin blue

• Lithium carbonate and 70% alcohol: remove excess dye from the background and differentiate gray matter from white matter

Van Orden, S. (1966). Treponema pallidum spirochetes in tissue stained with Steiner silver stain [Photomicrograph]. Centers for Disease Control and Prevention.

Stain name:
Steiner–Steiner Modified Silver Stain

Purpose:
Demonstrates spirochetes and other microorganisms, including Helicobacter pylori, Legionella, nonfilamentous bacteria, and some fungi.

Color results:

• Microorganisms: dark brown to black

• Background: golden brown

Reagents responsible for color:

• Silver nitrate: impregnates the microorganisms with silver

• Hydroquinone: reduces the silver to dark metallic silver

• Uranyl nitrate: sensitizes the microorganisms

• Gum mastic: stabilizes and controls silver development

Silver-stain extra credit:

• Reaction: argyrophilic

• Developer/reducer used: yes

• Developer/reducing agent: hydroquinone

• Silver solution: 1% aqueous silver nitrate

Newcomer Supply. (n.d.). Luxol Fast Blue–Cresyl Violet stained nervous tissue [Photomicrograph].

Stain name:
Luxol Fast Blue–Cresyl Echt Violet Stain

Purpose:
Demonstrates myelin, neuronal cell bodies, Nissl substance, and areas of demyelination in nervous tissue.

Color results:

• Myelin: blue

• Nissl substance and nuclei: violet

• Neurons: pink to violet

Reagents responsible for color:

• Luxol Fast Blue: stains myelin blue

• Cresyl Echt Violet: stains Nissl substance, nuclei, and neurons violet

• Lithium carbonate and 70% alcohol: differentiate the blue stain and remove excess dye

iHisto. (n.d.). Cresyl Echt Violet-stained neural tissue showing Nissl substance [Photomicrograph].

Stain name:
Cresyl Echt Violet (Nissl) Stain

Purpose:
Demonstrates Nissl substance in neurons and evaluates neuronal structure or loss.

Color results:

• Nissl substance and nuclei: dark blue to purple

• Background: pale or colorless

Reagents responsible for color:

• Cresyl Echt Violet acetate: stains RNA-rich Nissl substance and nuclei blue-purple

• 70% alcohol: removes excess dye and produces a pale background

Polysciences, Inc. (n.d.). Bielschowsky-stained brain tissue [Photomicrograph].

Stain name:
Modified Bielschowsky Silver Stain

Purpose:
Demonstrates axons, nerve fibers, neurofibrillary tangles, and senile plaques in nervous tissue.

Color results:

• Neurofibrillary tangles and plaques: black

• Axons: black

• Nerve fibers: brown to black

• Background: yellow to brown

Reagents responsible for color:

• Ammoniacal silver nitrate: impregnates neural structures with silver

• Formaldehyde developer: reduces silver ions to black metallic silver

• Gold chloride: tones silver deposits black

• Sodium thiosulfate: removes unreduced silver and fixes the stain

Silver-stain extra credit:

• Reaction: argyrophilic

• Developer/reducer used: yes

• Developer/reducing agent: formaldehyde

• Silver solution: ammoniacal silver nitrate

Woerly, S., Doan, V. D., Sosa, N., et al. (2001). Holmes-stained horizontal sections of spinal cord [Photomicrograph].

Stain name:
Holmes Silver Nitrate Method for Nerve Cells and Fibers

Purpose:
Demonstrates nerve cells, axons, neurofibrils, nerve fibers, and nerve endings.

Color results:

• Axis cylinders: blue to black

• Nerves and nerve endings: black

• Background: gray to rose

Reagents responsible for color:

• Silver nitrate: impregnates neural structures

• Hydroquinone and sodium sulfite: reduce silver to visible metallic silver

• Gold chloride and oxalic acid: tone and develop structures blue-black

• Sodium thiosulfate: removes unreduced silver and fixes the stain

Silver-stain extra credit:

• Reaction: argyrophilic

• Developer/reducer used: yes

• Developer/reducing agent: hydroquinone and sodium sulfite

• Silver solution: aqueous silver nitrate followed by an alkaline silver nitrate impregnating solution

Namazi, H., Emami, M. J., Dehghani Nazhvani, F., & Dehghani Nazhvani, A. (2019). Differentiated nerve bundle showing myelinated neurons, Weil’s stain, 400× [Photomicrograph].

Stain name:
Weil Iron Hematoxylin Stain

Purpose:
Demonstrates myelin sheaths and identifies areas of demyelination in nervous tissue.

Color results:

• Myelin: blue-black to black

• Nuclei: blue

• Red blood cells: black

• Background: clear to yellow

Reagents responsible for color:

• Potassium dichromate: mordants the myelin

• Hematoxylin and ferric ammonium sulfate (iron alum): stain myelin blue-black and nuclei blue

• Iron alum and potassium ferricyanide-borax: differentiate the tissue and clear the background

Centers for Disease Control and Prevention. (n.d.). Papanicolaou test showing cervical adenocarcinoma [Photomicrograph]. Public Health Image Library, Image ID 19471.

Stain name:
Papanicolaou Stain (Pap Stain)

Purpose:
Demonstrates cellular and nuclear detail in cytology specimens and helps detect abnormal, precancerous, and cancerous cells.

Color results:

• Nuclei: blue to blue-black

• Keratinized cells: orange

• Superficial cell cytoplasm: pink

• Intermediate, parabasal, and glandular cell cytoplasm: blue-green

Reagents responsible for color:

• Hematoxylin: stains nuclei blue to blue-black

• Orange G-6: stains keratinized cells orange

• Eosin Y in EA-50 or EA-65: stains cytoplasm pink

• Light Green SF in EA-50 or EA-65: stains cytoplasm blue-green

iHisto. (n.d.). Prussian Blue-stained tissue demonstrating ferric iron deposits [Photomicrograph].

Stain name:
Prussian Blue Stain for Ferric Iron

Purpose:
Demonstrates ferric iron, especially hemosiderin, in tissue and bone marrow.

Color results:

• Ferric iron and hemosiderin: bright blue

• Nuclei: red

• Background tissue: pink

Reagents responsible for color:

• Hydrochloric acid: releases ferric iron from tissue proteins

• Potassium ferrocyanide: reacts with ferric iron to form blue ferric ferrocyanide

• Nuclear Fast Red: stains nuclei red and background tissue pink

IMAGE B

Tan, C., Shichinohe, H., Abumiya, T., et al. (2014). Turnbull blue-stained tissue in a rat model of lacunar infarction [Photomicrograph]. ResearchGate.

Stain name:
Turnbull’s Blue Stain for Ferrous Iron

Purpose:
Demonstrates ferrous iron (Fe²⁺) in tissue.

Color results:

• Ferrous iron: bright blue

• Background tissue and nuclei: pink-red

Reagents responsible for color:

• Hydrochloric acid and potassium ferricyanide: react with ferrous iron to produce the blue Turnbull pigment

• Nuclear Fast Red: stains the background and nuclei pink-red

Abcam. (n.d.). Fontana–Masson-stained human skin melanoma demonstrating melanin [Photomicrograph].

Stain name:
Fontana–Masson Silver Stain

Purpose:
Demonstrates melanin and argentaffin granules in tissue.

Color results:

• Melanin and argentaffin granules: black

• Nuclei: red

• Background tissue: pale pink

Reagents responsible for color:

• Ammoniacal silver nitrate: produces black metallic silver deposits

• Gold chloride: tones the deposits black

• Sodium thiosulfate: removes unreduced silver

• Nuclear Fast Red: stains nuclei red and background pink

Silver-stain extra credit:

• Reaction: argentaffin

• Developer/reducer used: no external developer

• Reducing agent: melanin or argentaffin granules

• Silver solution: ammoniacal silver nitrate

El Chediak, A., Janom, K., & Koubar, S. H. (2020). Bile casts visualized by Hall stain [Photomicrograph]. Renal Replacement Therapy, 6, Article 15.

Stain name:
Hall’s Bilirubin (Bile) Stain

Purpose:
Demonstrates bile pigments, especially bilirubin, and distinguishes bile from other pigments.

Color results:

• Bile and bilirubin: emerald green to olive green

• Collagen: red

• Muscle and cytoplasm: yellow

Reagents responsible for color:

• Fouchet’s reagent: oxidizes bilirubin to green biliverdin

• Acid fuchsin in Van Gieson solution: stains collagen red

• Picric acid in Van Gieson solution: stains muscle and cytoplasm yellow

Abcam. (n.d.). Von Kossa-stained bone demonstrating calcium deposits [Photomicrograph].

Stain name:
Von Kossa Stain for Calcium Salts

Purpose:
Demonstrates calcium salts and areas of mineralization in tissue.

Color results:

• Calcium salts: black to dark brown

• Nuclei: red

• Background tissue: pink

Reagents responsible for color:

• Silver nitrate and bright light: produce black metallic silver at sites containing calcium phosphate or carbonate

• Sodium thiosulfate: removes unreduced silver and fixes the reaction

• Nuclear Fast Red: stains nuclei red and background tissue pink