Staining

Types of Stains used in Microbiology

Different stains react or concentrate on different areas of a tissue or cell These properties can be utilized to highlight certain areas...

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This article writter by MN Editors on November 16, 2021

Microbiology Notes is an educational niche blog related to microbiology (bacteriology, virology, parasitology, mycology, immunology, molecular biology, biochemistry, etc.) and different branches of biology.

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Types of Stains used in Microbiology
Types of Stains used in Microbiology

There are present different types of stains which are used in microbiology laboratories to stain bacterial cells. All these stains are listed below;

Types of Stains

Different stains react or concentrate on different areas of a tissue or cell These properties can be utilized to highlight certain areas or regions. A few of the most well-known biological staining methods can be found below. If not otherwise indicated All of these dyes can be used on tissues and cells that are fixed as well as essential dyes (suitable for use in live organisms) are indicated.

1. Acridine orange

Acridine orange (AO) is an nucleic acid-specific fluorescent cationic dye that is useful to determine the cell cycle. It is a cell-permeable dye, and interacts with DNA as well as DNA through electrostatic or intercalation attraction. When bound to DNA it has spectrally very similar to fluorescein. Like fluorescein it is an excellent non-specific stain that can be used to backlight normally stained cells that are on the surface of the tissue (fluorescence backlit staining).

2. Bismarck brown

Bismarck brown (also Bismarck brown Y or Manchester brown) gives a yellow hue for acid mucins. and a deep brown colour on mast cells. The drawback of this stain’s properties is that it blocks out any other structure that is around it, which makes the quality of contrast poor. It is necessary to pair it with other stains to be effective. A few complementing stains that can be used with Bismark brown include Hematoxylin and Toluidine blue, which offer better contrast in an histology specimen.

3. Carmine

Carmine is a vibrant red dye that is used to stain glycogen. Carmine aluminum is an nuclear stain. Carmine staining is a result of the use of a mordant typically aluminum.

4. Coomassie blue

Coomassie blue (also brilliant blue) does not specifically stain proteins with and has a vivid blue color. It is frequently employed in electrophoresis in gels.

5. Cresyl violet

Cresyl violet is a dye that gives acids in the neurons’ cell cytoplasm with a violet hue, particularly the nissl body. It is often used in research on the brain.

6. Crystal violet

When coupled with a suitable mordant stain cell walls with a purple hue. It is the color employed for Gram staining.

7. DAPI

DAPI is a nuclear fluorescent stain, which is stimulated by ultraviolet light and displaying significant blue fluorescence when attached to DNA. It binds to A=T-rich repeats on the chromosomes. It is also invisible by regular transmission microscopy. It can be utilized in fixed or living cells. Cells stained with DAPI are particularly suitable to count cells.

8. Eosin

Eosin is used most commonly to counteract haematoxylin. It imparts the colour red or pink to membranes of cells, as well as some extracellular structures. It also provides a vibrant red color on red blood cell. Eosin is also used as a counterstain for certain varieties of Gram staining and in various other methods. 

There are actually two similar compounds, commonly known as Eosin. The most commonly used one is the compound eosin Y (also called eosin Y WS or eosin Yellowish) and has a slightly yellow cast. Another eosin compound is the eosin B (eosin imperial red) It has a faint blue cast. Both dyes can be used interchangeably and the choice of either one and the other one is an issue of preference and custom.

9. Ethidium bromide

Ethidium bromide intercalates and stain DNA, producing a stain that is red-orange. Although it cannot be able to stain cells that are healthy, it could be used to detect cells in the last stages of apoptosis . These cells are much better permeable cell membranes. This is why ethidium bromide is commonly employed as a marker to detect the apoptosis of cells as well as to identify DNA bands in electrophoresis in gels. 

The stain can also be utilized in conjunction with acridine (AO) for the process of counting viable cells. This combined stain triggers live cells to show green, whereas dying cells maintain the distinct red-orange fluorescence.

10. Acid fuchsin

Acid fuchsine can be used to color collagen, smooth muscles, or mitochondria. Acid fuchsin can be used as the cytoplasmic and nuclear stain in the Mallory’s trichrome method. Acid Fuchsin stain cytoplasm in a few variations of Masson’s Trichrome. In the case of Van Gieson’s picrofuchsine acid fuchsin gives its red hue to collagen fibers. Acid fuchsin can also be a well-known stain for mitochondria (Altmann’s method).

11. Haematoxylin

Haematoxylin (hematoxylin in North America) is a nuclear stain.Used in conjunction with a mordant the staining of nuclei is blue-violet or brown. It is usually utilized in conjunction with eosin for the stain H&E (haematoxylin as well as eosin) staining procedure, which is one of the most commonly used techniques in histology.

12. Hoechst stains

Hoechst is an bis-benzimidazole derivative chemical that is able to bind to the minor grooves of DNA. Most often, it is used in fluorescence microscopes to aid in DNA staining Hoechst staining is yellow when they are dissolved in aqueous solutions. They also emit blue light when exposed to ultraviolet excitation. There are two main varieties of Hoechst: Hoechst 33258 and Hoechst 33342. Both are functionally identical, however with slight differences in structure. Hoechst 33258 has an hydroxyl group at the terminal end and, consequently, is more soluble in water but this also decreases its capability for penetration into the membrane of plasma. Hoechst 3342 has an ethyl-based substitution for the terminal group of hydroxyl (i.e. the ethylether groups) which makes it more hydrophobic, facilitating plasma membrane flow

13. Iodine

Iodine is utilized in chemistry to indicate for starch. When starch is combined with iodine in a solution it produces a deep dark blue hue develops which is a sign of a starch/iodine combination. Starch is a component that is common to all plant cells, and therefore, a weak solution of iodine can stain the starch in cells. Iodine is a component of the staining process called Gram staining employed in microbiology. As a mordant used in Gram’s staining, it enhances the flow of stain through pores in the cell’s membrane or cell wall.

Lugol’s solution, also known as Lugol’s iodine (IKI) is brown solution that changes to black when exposed to starches. It is also used as a stain for cells which makes the nuclei of the cell more obvious.

Utilizing common vinegar (acetic acid) Lugol’s solution can be utilized to determine pre-cancerous or cancerous changes to vaginal and cervical tissues in “Pap smear” exams that follow-up to prepare for biopsy. The acetic acid causes abnormal cells to become white, while normal tissues color a mahogany brown because of the Iodine.

14. Malachite green

Malachite green (also called diamond green B, or victoria green B) is utilized for a counterstain of blue-green for safranin within the Gimenez staining method for bacteria. It is also used to stain spores directly.

15. Methyl green

Methyl green is commonly used when using bright-fields as well as with fluorescence microscopes to color the cells’ chromatin so that they can be more easily observed.

16. Methylene blue

Methylene blue can be used to color animals’ cells, like human cheek cells to enhance the visibility of their nuclei. Also used to color blood film in the field of cytology.

17. Neutral red

The neutral red (or the toluylene color) The color is neutral red (also known as toluylene red). Nissl substance to red. It is often employed as a counterstain conjunction along with various dyes.

18. Nile blue

Nile blue (or Nile blue A) stains nuclei blue. It can be used in conjunction with living cells.

19. Nile red

Nile red (also called Nile blue Oxazone) is created through the boiling of Nile blue in sulfuric acid. It results in a mixture with Nile Red as well as Nile blue. Nile red can be described as a lipophilic stain, it is able to be absorbed into lipid globules in cells, and stain the cells with red. Nile red is a stain that can be utilized in living cells. It has a strong fluorescence when divided into lipids, but never in an water solutions.

20. Osmium tetroxide (formal name: osmium tetraoxide)

Osmium Tetraoxide is employed in optical microscopy for staining the lipids. It dissolves in fats and is then reduced by organic substances to the elemental osmium. It is a visible black substance that is easily visible.

21. Propidium iodide

Propidium iodide is a light-emitting intercalating agent that is used to color cells. Propidium Iodide is utilized as an DNA stain for flow cytometry to assess the viability of cells or DNA content during cell cycle analysis, as well as using microscopy for the purpose of observing the nucleus as well as other organelles that contain DNA. Propidium Iodide cannot cross the cell membrane and is therefore able for separating necrotic, apoptotic, and healthy cells. Also, PI is a RNA-binding protein, requiring treatment with nucleases to differentiate between DNA staining and RNA.

22. Rhodamine

Rhodamine is a specific protein stain that is used for fluorescence microscopy.

23. Safranine

Safranine (or Safranine O) is an ionic red dye. It is a binding agent for nuclei (DNA) and various other tissues, like glycosaminoglycans that are found in cartilage as well as mast cells, as well as components of plastids and lignin in plants’ tissues. Safranine is not to be confused with the saffron. which is a costly natural color utilized in certain methods to give a yellow hue to collagen, in contrast with red and blue colors that other dyes impart to cytoplasm and nuclei of the cytoplasm of animal (including human) tissues.

An incorrect name “safranin” has become widespread usage. The -ine suffix is suitable for safranine O because the dye comes from an amine.

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Microbiology Notes is an educational niche blog related to microbiology (bacteriology, virology, parasitology, mycology, immunology, molecular biology, biochemistry, etc.) and different branches of biology.

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