Road Stop (Techniques of light microscopy) While the Gram stain is more likely t
ID: 276712 • Letter: R
Question
Road Stop (Techniques of light microscopy) While the Gram stain is more likely to be extensively covered in a laboratory, the principles underlying why a Gram stain differentiates between different groups of bacteria is intimately linked to the nature of the cell envelope (1) For each of the following steps, indicate what the anticipated color of a Gram-positive or a Gram-negative bacterium would be: Gram-positive bacteria Gram-negative bacteria Gram stain step After crystal violet After Gram's iodine After alcohol After counter-stain (safranin) (2) What information can be gained from a Gram stain? (3) Which of the solutions added acts as a mordant? What is a mordant? (4) If you forgot to add the alcohol, what color would you expect the Gram-positive bacteria to be? What about the Gram negative bacteria?Explanation / Answer
1) Step.............................Gram positive bacteria ......................................Gram negative bacteria
After crystal violet...................... Purple ..................................................................Purple
After gram's iodine..................... Purple ..................................................................Purple
After alcohol ...............................Purple ..................................................................Colourless
After counter stain ......................Purple................................................................... Pink
2) Gram staining separates bacteria into two groups depending on the cell wall composition. One group is Gram positive bacteria which stains purple due to thick layer of peptidoglycan in cell wall and the other group is Gram negative bacteria which stains pink/red because of thin layer of peptidoglycan in cell wall and high lipid content.
3) Iodine is added as a mordant. It forms the crystal violet-iodine complex by changing the shape of the dye and trapping it in cell wall so that it cannot be removed easily.
4) Alcohol is used as a decolouriser which dissolves lipids found in the outer cell membrane of gram-negative bacteria. This allows the crystal violet-iodine complex to leak out of the thinner peptidoglycan layer. After adding alcohol, gram negative bacteria become colourless while gram negative one continues to look purple. If alcohol is not added, both types of bacteria will show purple stain at the end of the staining process.
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