Slides Stained with Bacterial Controls (circles on either end) and Unknown (circle in middle).  Slides Left to Right: Gram stained slide, Acid-fast stained slide, Endospore stained slide.

Differential Stains for Identifying Bacteria
 Gram, Acid-fast & Endospore 

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an important tool for distinguishing between two main types of bacteria—Gram-positive and Gram-negative.

Nearly all bacteria contain peptidoglycan, a molecule unique to the bacterial cell wall. Gram-positive bacteria have a cell wall composed almost entirely of peptidoglycan, present in many layers. Gram-negative bacteria have a cell wall with only a thin layer of peptidoglycan beneath an outer cell wall membranous layer composed of lipopolysaccharides (LPS), an endotoxin that can be harmful to organisms infected with Gram-negative bacteria.

During the Gram staining technique, a purple dye (crystal violet) is first applied to a prepared bacterial smear. After rinsing, iodine is then applied. The crystal violet and iodine bind to create a large molecule that is too big to exit the multiple layers of peptidoglycan in the Gram positive cells, so the purple color becomes trapped. 

Article Summary: The Gram, Ziehl Neelsen acid fast, and endospore stains are differential tests used to help identify bacteria. Here's how they work.
Gram, Acid-fast & Endospore Bacterial Stains
Mixed acid-fast stain  showing both Acid-fast (pink) and nonacid-fast  (purple) bacteria 1000xTM.
Gram stain of Gram+ Staphylococcus 1000xTM+
Gram stain of Gram+ Staphylococcus 1000xTM+.
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Mixed acid-fast stain  showing both Acid-fast (pink) and nonacid-fast  (purple) bacteria 1000xTM.
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Prokaryotic Cell, Mariana Ruiz
​Page last updated 4/2015

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Photographic guides to differential stains 
now available!
1. Gram
2. Acid-fast
3. Endopsore

Bacterial Smears of Gram, Acid-fast & Endospore Differential Stains
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How to Do a Gram Stain
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Differential stains work by using a series of dyes, and sometimes additional chemicals, to stain bacteria contrasting colors based on structural difference between bacterial cells. The Gram stain, acid-fast stain and endospore stain each reveal distinct information about the bacteria tested.


What the Gram Stain Reveals about Bacteria
The Gram stain, developed by Christian Gram in the 1800’s, was the first differential staining technique in use and is still 
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Acid-fast Stain
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After rinsing, a decolorizer is applied that removes the purple stain from Gram-negative cells, leaving them clear. After rinsing the decolorizer, a secondary stain (safrinin) is then applied to impart a pink color to the Gram-negative cells. After a final rinse, the slide can be viewed under oil immersion with a light microscope. Gram+ cells will appear purple and Gram- cells pink.

What the Acid-fast Stain Reveals about Bacteria
Acid-fast staining, also known as the Ziehl Neelsen stain, is used to identify specialized bacteria that have waxy mycolic acid in their cell wall. The presence of mycolic acid in bacteria is rare, only found in two genera—Mycobacteria and Nocardia. The bacteria that possess mycolic acid are considered “acid fast,” whereas the vast majority of bacteria, those that do not have cell walls containing mycolic acid, are considered “non-acid fast.”