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Staining is a technique used to enhance and contrast biological specimens under a microscope. Stains and dyes are used to highlight specimens for study at higher magnifications. Common staining techniques include Gram staining to identify bacteria, endospore staining to detect endospores, and hematoxylin and eosin staining for histopathology. Specimens must first be prepared through fixation, use of mordants, and permeabilization to allow effective staining. Common biological stains include crystal violet, iodine, safranin, and methylene blue.

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25 views

What Is Moreree

Staining is a technique used to enhance and contrast biological specimens under a microscope. Stains and dyes are used to highlight specimens for study at higher magnifications. Common staining techniques include Gram staining to identify bacteria, endospore staining to detect endospores, and hematoxylin and eosin staining for histopathology. Specimens must first be prepared through fixation, use of mordants, and permeabilization to allow effective staining. Common biological stains include crystal violet, iodine, safranin, and methylene blue.

Uploaded by

alia
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© © All Rights Reserved
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Download as PDF, TXT or read online on Scribd
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What is Staining?

Staining, in microbiology, can be defined as a technique which is used to


enhance and contrast a biological specimen at the microscopic level.
Stains and dyes are used to highlight the specimen at the microscopic
level to study it at higher magnification for histopathological studies and
diagnostic purposes.

However, staining is not just limited to biological specimens, it can also be


used to study the structure of crystalline polymers.

Preparation of the Biological Specimen


The preparation of the biological specimen to be analysed under
a microscope depends on the type of staining. Given below are some
procedures that are carried out.

• Wet Mounting: Living biological specimens are mounted on a glass slide with
water and specific stains.
• Fixation: It is a multi-step process which is done to preserve the shape of cells
and tissues. Heat fixation is done to kill and adhere the specimens. Chemical
fixation is done to generate strong bonds and increase the rigidity of the
samples. Common chemical fixatives used are formaldehyde, picric acid,
methanol and ethanol.
• Mordant: Mordants are chemical agents that are used along with dye to make
the specimen stainable, which otherwise is unstainable. Mordants are of two
types:
• Basic Mordants: They react with acidic dyes.
• Acidic Mordants: They react with basic dyes.

When staining is done with the help of mordants, it is known as indirect


staining. On the other hand, when staining is done without the help of
mordants, it is known as direct staining.

• Permeabilisation: This procedure involves treating the specimen with a


surfactant that dissolves the cell membrane allowing easy staining with the
dye.

Types of Staining Techniques


1. Gram’s staining: This staining procedure is used to identify bacteria based on
their cell wall composition. There are two types of Gram’s staining, and the
bacteria can be divided into gram positive or gram negative bacteria. It uses
crystal violet for the staining of cell walls, iodine as the mordant and safranin or
fuchsin as the counterstain.
2. Endospore staining: This technique is used to find out the presence
of endospores in bacteria. Endospores are tough, dormant structures that are
less permeable to dyes and stains. Their presence makes the bacteria difficult
to kill. The process involves heat fixing the specimen and dying it with a blend
of safranin and 5% diluted malachite green.
3. Ziehl-Neelsen staining: This technique is used to stain acid-fast bacteria such
as Mycobacterium tuberculosis that do not stain with Gram’s staining. Carbol
fuchsin is used as a stain, and methylene blue is used as a counterstain.
4. Haematoxylin and Eosin staining: This staining procedure is used in
histopathological studies to observe thin tissue sections. The hematoxylin stain
colours the nuclei in blue and the cytoplasm and its components in red or pink.
5. Periodic Acid-Schiff (PAS) staining: PAS staining is used to stain carbohydrate
molecules. It is used on liver tissues, kidney, pancreas and ovaries to detect the
presence of glycogen and help in the diagnosis of diseases.
6. Masson staining: Masson’s trichrome is a three-colour staining procedure
which is used for differentiating cells from connective tissues. It colours the
muscle fibres and keratin to red, collagen to blue-green, cytoplasm to red-pink
and nuclei into black colour.
Read more: Differentiate between Simple and Differential Staining

Biological Stains
Some of the most commonly used biological stains are listed below:

• Acridine orange: It is a fluorescent cationic dye that is selective to nucleic


acids. It is used during the cell cycle to analyse DNA molecules.
• Coomassie blue: It is used in gel electrophoresis to stain the proteins blue.
• Crystal violet: It is used in Gram’s staining along with iodine to stain the
bacterial cell wall in purple colour.
• Eosin: It is used as a counterstain to hematoxylin that imparts a red colour to
the cytoplasm and its components.
• Ethidium bromide: It provides a red-orange fluorescent stain to the DNA after
intercalating with the molecule.
• Iodine: It is used as a mordant in Gram’s staining.
• Malachite green: It gives a blue-green colour when used as a counterstain
against safranin that is used in endospore staining.
• Methylene blue: It is used to stain animal cells because it enhances the nuclei.
• Safranin: It is a red cationic dye that is used as a counterstain in both Gram’s
staining and

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