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  • Is endospore staining an example of positive, negative, or differential staining?

Flagella staining

Flagella (singular: flagellum) are tail-like cellular structures used for locomotion by some bacteria, archaea, and eukaryotes. Because they are so thin, flagella typically cannot be seen under a light microscope without a specialized flagella staining technique. Flagella staining thickens the flagella by first applying mordant (generally tannic acid, but sometimes potassium alum), which coats the flagella; then the specimen is stained with pararosaniline (most commonly) or basic fuchsin ( [link] ).

Three red rectangles on a clear background are shown. Each rectangle has many thin, wiggly lines projecting from it.
A flagella stain of Bacillus cereus , a common cause of foodborne illness, reveals that the cells have numerous flagella, used for locomotion. (credit: modification of work by Centers for Disease Control and Prevention)

Though flagella staining is uncommon in clinical settings, the technique is commonly used by microbiologists, since the location and number of flagella can be useful in classifying and identifying bacteria in a sample. When using this technique, it is important to handle the specimen with great care; flagella are delicate structures that can easily be damaged or pulled off, compromising attempts to accurately locate and count the number of flagella.

A table of simple stains is shown. Basic stains include: methylene blue, crystal violet, malachite green, basic fuschsin, carbolfuschsin, and safranin. Basic stains stain negatively charged molecules and structures, such as nucleic acids and proteins. The outcome of this positive stain is dark cells on a light background. Acidic stains include eosine, acid fuchsin, rose Bengal, and Congo red. Acid stains stain positively charged molecules and structures such as proteins. Acidic stains can either be positive or negative stains depending on the cell’s chemistry. Negative stains include india in k and nigrosine. Negative stains stain the background, not the specimen and produce a dark background with a light specimen.
(credit “basic stains”: modification of work by Centers for Disease Control and Prevention; credit “Acidic stains”: modification of work by Roberto Danovaro, Antonio Dell’Anno, Antonio Pusceddu, Cristina Gambi, Iben Heiner, Reinhardt Mobjerg Kristensen; credit “Negative stains”: modification of work by Anh-Hue Tu)
A table of differential stains is shown. The Gram stain uses crystal violet, Gram’s iodine, ethanol (decolorizer), and safranin. The purpose of the Gram stain is to distinguish cells by cell-wall type (Gram-positive, Gram-negative). Gram-positive cells stain purple/violet. Gram-negative cells stain pink. The acid fast stain: after staining with basic fuchsin, acid-fast bacteria resist decolonization by acid-alcohol. Non-acid-fast bacteria are counterstained with methylene blue. The acid-fast stain is used to distinguish acid-fast bacteria such as M. tuberculosis, from non-acid-fast cells. Acid-fast bacteria are red; non-acid-fast cells are blue. The Endospore stain uses heat to stain endospores with malachite green (Schaeffer-Fulton procedure), then cell is washed and counterstained with safranin. The endospore stain is used to distinguish organisms with endospores from those without; used to study the endospore. Endospores appear bluish-green; other structures appear pink to red. Flagella stain: flagella are coated with a tannic acid or potassium alum mordant, then stained using either pararosaline or basic fuchsin. The flagella stain is used to view and study flagella in bacteria that have them. Flagella are visible as thin strands if present. Capsule stain: negative staining with india ink or nigrosine is used to stain the background, leaving a clear area of the cell and the capsule Counterstaining can be used to stain the cell while leaving the capsule clear. The capsule stain is used to distinguish cells with capsules from those without. Capsules appear clear or as halos if present.
(credit “Gram stain”: modification of work by Nina Parker; credit “Acid-fast stain”: modification of work by American Society for Microbiology; credit “Endospore stain”: modification of work by American Society for Microbiology; credit “Capsule stain” : modification of work by American Society for Microbiology; credit “Flagella stain”: modification of work by Centers for Disease Control and Prevention)

Preparing specimens for electron microscopy

Samples to be analyzed using a TEM must have very thin sections. But cells are too soft to cut thinly, even with diamond knives. To cut cells without damage, the cells must be embedded in plastic resin and then dehydrated through a series of soaks in ethanol solutions (50%, 60%, 70%, and so on). The ethanol replaces the water in the cells, and the resin dissolves in ethanol and enters the cell, where it solidifies. Next, thin sections are cut using a specialized device called an ultramicrotome ( [link] ). Finally, samples are fixed to fine copper wire or carbon-fiber grids and stained—not with colored dyes, but with substances like uranyl acetate or osmium tetroxide, which contain electron-dense heavy metal atoms.

Photograph a shows a blue solid specimen sits below a mechanical dial. Photograph b shows a person holding a dial on a machine.
(a) An ultramicrotome used to prepare specimens for a TEM. (b) A technician uses an ultramicrotome to slice a specimen into thin sections. (credit a: modification of work by “Frost Museum”/Flickr; credit b: modification of work by U.S. Fish and Wildlife Service Northeast Region)

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Source:  OpenStax, Microbiology. OpenStax CNX. Nov 01, 2016 Download for free at http://cnx.org/content/col12087/1.4
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