B.Sc. Biotech Biochem II BM Unit-4.2 Staining

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Staining Technique

Course : B.Sc. Biotechnology, BiochemistrySem IISub: Basic MicrobiologyUnit 4.2

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Aims of Staining in microbiology

• To increase visibility & contrast• To study the morphology• To detect extracellular and intracellular

components of microbes• To help in the primary level of diagnosis

Why Stain Cells?

• The most basic reason that:– Enhance visualization of the cell or certain cellular

components under a microscope. – Cells may also be stained to highlight metabolic

processes or to differentiate between live and dead cells in a sample.

– Cells may also be enumerated by staining cells to determine biomass in an environment of interest.

How Are Cells Stained and Slides Prepared?

• Cell staining techniques and preparation depend on the type of stain and analysis used. One or more of the following procedures may be required to prepare a sample:– Smear preparation– Permeabilization– Fixation– Mounting– Staining

Smears and Staining

• Bacteria must be stained (dyed) so they can be seen with the microscope

• Before staining a smear must be made• A smear is just a film of bacteria on a glass

slide• After the smear dries it is heat fixed, this– Kills the bacteria– Helps adhere the cells to the slide– Makes the cells more receptive to the dye

Stains

• Stains are dyes• Stains carry either a positive charge (basic dyes) or a

negative charge (acidic dyes)• Bacteria typically carry a slight negative charge on

the cell surface so they attract a basic dye• Most of the stains used in the lab are basic dyes• A negative stain uses acidic dyes that do not stain the

cell but rather the background

1. Basic dyes—methylene blue, basic fuchsin, crystal violet, safranin, malachite

green—have positively charged groups (usually some form of pentavalent nitrogen) and are generally sold as chloride salts. Basic dyes bind to negatively charged molecules like nucleic acids and many proteins. Because the surfaces of bacterial cells also are negatively charged, basic dyes are most often used in bacteriology.

2. Acid dyes— eosin, rose bengal, and acid fuchsin—possess negatively charged groups such as carboxyls (— COOH) and phenolic hydroxyls (— OH). Acid dyes, because of their negative charge, bind to positively charged cell structures.

Staining Techniques

• Simple Stain– Uses only one basic dye– Provides basic information

about cell shape and arrangement

• Differential Stain– Uses more than one dye– These procedures react

differently with different kinds of bacteria

– Helps distinguish between different kinds of bacteria

– Most common and important differential stain is the GRAM STAIN

Gram Stain

• Most important differential staining technique• Differentiates all bacteria based on cell wall

composition• Bacteria are either Gram + and stain blue or

Gram- and stain red• Gram stain is usually the first step in

identifying an unknown bacteria

Gram Stain

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Gram stain

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Acid-fast Stain

• Differential stain• Identifies bacteria with MYCOLIC ACID in their

cell walls• Very important human pathogens that can be

identified with this stain is Mycobacterium tuberculosis

• All members of genus Mycobacterium are acid-fast

Acid-fast Stain

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Special stains

• Negative stain• Acidic dye stains background, not cell• Used to determine cell shape and size• Spore stain• Used to identify bacteria that can form spores

Special Stains

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References

Images:1. https://www.withfriendship.com/images/d/17558/basis-for-the-gram-stain.jpg 2. http://2.bp.blogspot.com/-cLbZ0C-O_so/TjwJa2f7mxI/AAAAAAAAAGc/

2vvVrfVg1ds/s1600/Gram-Staining-Procedure.jpg 3. http://www.dermpedia.org/files/images/histioid_leprosy_Fite_oil.jpg4. https://lh6.ggpht.com/PZTVqr-v7Set_4U8p5il7UU-yrRPMENYClH9-

jc1MZYbw0miu6xFdoVo6RvfE-hrdjsEhA=s117

Books:1. Microbiology by pelczar

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