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Transcript
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. Page 1 of 4 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 Page 2 of 4 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. Page 3 of 4 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. Page 4 of 4