Lab Practical 1 Stains

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Simple Staining

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the use of a single stain to color a bacterial cell useful in determining basic morphology and the presence or absence of certain kinds of granules

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Commonly used dyes

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Methylene blue, basic fuchsin and crystal violet. All of these work because they are colo bearing ions ( chromophores that are positively charged cationic.

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Basic Dyes

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Methylene blue- methylene + chloride -

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Acidic Dyes

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Eosin- Sodium + eosinate -

Anionic chromophore- eosinate - will not stain bacteria because of the electrostatic repelling forces.

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Pleomorphism

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pertains to irregularity of form. Different shapes

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Metachromatic granules

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distine reddish purple granules within cells that show up when the organisms are stain with methylene blue these granules are masses of volutin a polymetaphosphate

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Palisade arrangement

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pertains to parallel arrangement of rod shaped cells.

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Methylene blue

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Stain for one minute,wash briefly, and dry

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Negative stains

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-Useful in studying the morphology of bacterial cells.
-Characterizing external structures such as capsules.
-stains are acidic, have negative charge chromophore doesn't penetrate the cell because of the simular charge.
-India ink nigrosine
- good for observing spirochetes

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Negative stain procedure

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-disperse organism into a small drop of nigrosine
-use spreader slide to drag the suspension over the slide
-No heat fix
-Air Dry

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Capsule

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Bacterial cells surrounded by a slime layer capsule
Streptococcus pneumoniae causes pneumoniae
Prevent phagocytic white blood cells from engulfing this bacterial pathogen.
Means for bacteria to attach to solid surfaces (streptococcus mutans and teeth)

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Capsular Staining

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preform a negative stain head fix then stain with crystal violet for one minute wash off with water and blot dry.
Capsule will appear as a halo like structure surrounding purple cells against a dark background

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Gram stain

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Differential stain. since cells or structures within cells display dissimilar staining reactions that can be distinguished.
Gram positive bacteria retain crystal violet iodine complex through discoloration with alcohol or acetone
Positive bacteria = Purple
Negative- =Pink to Red alcohol or acetone removes the crystal violet iodine complex and counterstains with safranin red.

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Gram Staining Procedure

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Primary stain- crystal violet ---> Gram iodine is a mordant that complexex with the crystal violes and forma an insoluble complex in gram positive cell. ---> Dye mordant complex is not removed from gram positive bacteria but is leached from gram negative by acetone or alcohol. ---> final step safranin counterstain is applied which stains the colorless g negative cells.

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Contributing factor to gram positive stain

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G positive cells have a thick layer of peptidoglycan that comprises cell wall of these organisms.

G negative cell wall in gram negative consists of an outer membrane that covers a much thinner layer.

This determines whether gram mordant is removed or not.

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Endospores

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When bacteria belonging to the genera Bacillus and Clostridia exhaust essential nutrients they undergo developmental cycle that produces resting stages.
Allows bacteria to survive environmental conditions that are not favorable for growth
Dehydrated structures
Heat resistant, radiation, acids and many chemicals

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Exosporium-

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that forms a protective barrier around the spore.
Heat resistance associated with water content of endospores.

Higher water content lower heat resistance