Question

QUESTIONS Predict the effect on Gram-positive and Gram-negative cells of the following mistakes made when performing a Grum stain. Consider cac mistake independently a. Failure to add the iodine. b. Failure to apply the decoloriser. 3 c. Failure to apply the safranin. d. Reversal of crystal violet and safranin stains. Both crystal violet and safranin are basic stains and may be used to do simple stairs on Gram-positive and Gram-negative cells. This being the case, explain how they end up stainting Gram-positive and Gram-negative cells differently in the Gran stan. 6 If you saw large, eukaryotic cells in the preparation made from your gumline, they were most likely your own epithelial cells. Are you Gram-positive or Gram-negative (You can make a good guess about this even if you didn't see your cells.) One of your lab partners has followed the recommended procedure of noring Gram-positive and Gram-negative control organisms on her Gram stain of an sonknown species. Her choices of controls were Escherichia coli and Bacillus subtilis. She tries several times and each time concludes she is decolorizing too long because both controls have pink cells (one more than the other). What might you suggest she try and why?

Answer 1

​​​​​​A. Iodine is used as a mordant in Gram staining technique. Mordant is a trapping reagent which helps in the trapping of stain molecules in a specific organelle. So iodine forms big sized complexes with Crystal Violet. These Crystal Violet iodine complexes get entrapped into the network of peptidoglycan of bacteria. If we forget to add iodine, than these complexes will not be formed and the bacteria will not be able to retain Crystal Violet in the cell wall. Hence the bacteria will not appear purple to us.

B. Decolorizer is used for making Gram Negative bacteria colourless. If we forget to add decolorizer, then Gram Negative bacteria will also appear purple to us. Decolorizer removes all the Crystal Violet iodine complex is present in the peptidoglycan of Gram Negative bacteria. This complexes are not easily lost from Gram Positive Bacteria because Gram Positive Bacteria has a thick layer of peptidoglycan but Gram Negative has a very thin layer of it.

​​​​​​C. If we fail to apply Saffranin, then gram negative bacteria will remain colourless and will not be visible to us. Safranin stains Gram Negative bacteria red.

D. If we reverse the use of Crystal Violet and safranin stain, then Gram Positive Bacteria will appear red and Gram Negative bacteria will appear purple. Saffranin will form complexes with iodine and will get trapped into the peptidoglycan of Gram Positive Bacteria.

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