Download Distinguishing Bacteria Using Differential Stains

Whitney Brown
October 29, 2014
English 202C
Distinguishing Bacteria Using Differential Stains
Differential staining is a procedure used to distinguish different kinds of bacteria. These differential
stains work by using a series of dyes and additional chemicals to stain the bacteria in order to reveal
physical and chemical properties of different groups of bacteria. The Gram Stain and Acid-Fast Stain
are amongst the most commonly used differential staining methods and help to distinguish these
different types of bacteria into groups.
The Gram Stain
The gram stain is a scientific staining procedure used to classify bacteria into two large groups:
gram-positive and gram-negative. The gram stain classifies these bacteria by detecting
peptidoglycan1 using four different liquid applications. Some bacteria retain these liquid
applications while some do not, therefore differentiating the bacteria into a gram-negative or grampositive group. The overall process can be broken down into the following essential steps:
Source: Microbiology: An Introduction, 2013 <www.lifescienceacademy.net/gramstaining.html>.
1. Crystal Violet Application: A heat fixed smear slide containing cells is covered with a crystalviolet dye. This dye transfers its purple color to all the cells present, therefore it is referred
to as the primary stain. The crystal violet dye is eventually washed off of the slide using
water, but cells still remain colored.
1
The structural molecule of bacterial cell walls consisting of the molecules N-acetylglucosamine, N-acetylmuramic
acid, tetrapeptide side chain, and peptide side chain.
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Whitney Brown
October 29, 2014
English 202C
2. Iodine Application: An iodine solution2, used as a mordant3, is added to the slide of the
colored cells. When the iodine is washed off, gram-positive and gram-negative bacteria
appear dark violet or purple.
3. Alcohol Wash: In this step, the slide is washed with alcohol or an alcohol-acetone solution
that is used as a decolorizing agent. This decolorizing agent removes the purple from some
species but not from others.
4. Counterstain Application: In this step of the gram staining process, the alcohol is rinsed off
and then stained with a red counterstain dye, safranin. After the safranin has been added,
the slide is washed again, dried, and examined microscopically.
After this process, some of the bacteria retain the primary stain (crystal violet) and others are
decolorized by the alcohol. The bacteria which retain the crystal violet color are identified as grampositive; the other bacteria are gram- negative. Gram-negative bacteria remain colorless after the
alcohol wash and are essentially invisible. As a result, the safranin is applied to turn gram-negative
bacteria pink, because the gram-positive bacteria retain the original primary stain and therefore are
not affected by the safranin counterstain.
The Acid-Fast Stain
The Acid-Fast Stain is another important differential stain that divides bacteria into distinctive
groups. This stain is used to identify bacteria that contain mycolic-acid4 in their cell wall which is
only found in the genera of Mycobacteria and Norcardia. The stain uses a primary stain, an alcoholic
decolorizer, and a counterstain to distinguish the physical and chemical characteristics that group
these different types of bacteria into acid-fast or non-acid-fast bacteria. The overall process can be
broken down into the following essential steps:
Source: Microbiology: An Introduction, 2013 <www.lifescienceacademy.net/acidfast.html>.
2
A solution made up of iodine and potassium iodine.
A substance added to a staining solution to make it stain more intensely.
4
Long chained, branched fatty-acids characteristics of members of the genus Mycobacterium and Norcadia.
3
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Whitney Brown
October 29, 2014
English 202C
1. Carbolfuchsin Application: Carbolfuchsin, a red dye, is applied to a heat fixed smear and is
used as the primary stain, because it transfers its purple color to all the cells present. The
carbolfuchsin dye is eventually washed off of the slide using water, but cells still remain
colored.
2. Alcohol Wash: In this step, the slide is washed with an acid-alcohol solution that is used as a
decolorizing agent. This decolorizing agent removes the red stain left from the carbolfuchsin
stain from bacteria that are not acid-fast while the acid-fast bacteria still remain pink.
3. Methylene Blue Application: In this step of acid-fast staining, the alcohol is rinsed off and
then stained with a counterstain dye, methylene blue. After the counterstain is added, the
slide is washed again, dried, and examined microscopically.
After this process, some of the bacteria retain the primary stain (carbolfuchsin red dye) and others
are decolorized by the alcohol. The bacteria which retain the red color are identified as acid-fast;
the other bacteria are non-acid-fast. In the non-acid-fast bacteria, the cell walls lack mycolic acid,
leaving the cells colorless and essentially invisible. As a result, the bacteria was stained with the
methylene blue counterstain, so non-acid-fast bacteria appear blue; the acid-fast bacteria are not
affected by the counterstain, because they still retain the primary stain.
Conclusion
Gram staining and Acid-Fast staining are two differential test used to distinguish bacteria into
certain groups. Gram staining divides bacteria into gram-negative and gram-positive bacteria,
whereas acid-fast divides them into acid-fast or non-acid fast bacteria. It is important to note that
gram staining and acid-fast staining techniques are not interchangeable (this means a gram stain
test cannot determine if certain bacteria are acid-fast and vice versa). These two differential test
are important, because they provide valuable information for the treatment of disease. Knowing
these characteristics of different bacteria help to link these bacteria to their diseases, and they help
to identify what medicines would work best with the bacteria causing the problem.
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Whitney Brown
October 29, 2014
English 202C
References
Tortora, Gerard J., Berdell R. Funke, and Christine L. Case. Microbiology : An introduction.
Boston: Pearson, 2013. Print.
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