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Gram Stain

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Synapsis

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I understand that Gram-positive bacteria absorb the stain well because of their thick peptidoglycan cell wall and Gram-negative don't because of their thin cell wall. EK Bio says that the Gram-positive cells appear purple and the Gram-negative cells appear pink. However, I just did a passage in TBR Bio (Ch. 6, passage 11) that outlines the gram-staining procedure. One of the questions asks which color each type of cell will be at the end. According to TBR, Gram-positive cells will be blue, and Gram-negative cells will be red. Now, even if you had read EK before doing this passage, you could still figure out the correct answer from the procedure they give you. But which of these is correct? Or are they supposed to be the same thing?
 

sakabato93

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I understand that Gram-positive bacteria absorb the stain well because of their thick peptidoglycan cell wall and Gram-negative don't because of their thin cell wall. EK Bio says that the Gram-positive cells appear purple and the Gram-negative cells appear pink. However, I just did a passage in TBR Bio (Ch. 6, passage 11) that outlines the gram-staining procedure. One of the questions asks which color each type of cell will be at the end. According to TBR, Gram-positive cells will be blue, and Gram-negative cells will be red. Now, even if you had read EK before doing this passage, you could still figure out the correct answer from the procedure they give you. But which of these is correct? Or are they supposed to be the same thing?

Positive Gram-staining is purplish, and negative Gram-staining is pinkish. In reality the colors are fairly flexible in their ranges, sort of like pH indicators. So a blue and red staining wouldn't be uncommon for positive and negative stains, respectively.

However, your evaluation on the nature of the staining seems a little flawed. Gram-staining is an evaluation of how well the peptidoglycan cell is exposed to the environment. Gram-positive bacteria do have a thick cell wall, but Gram-negative bacteria have a peptidoglycan cell wall that is a little thinner and is situated between two phospholipid membranes (in what's called the periplasmic space). The existence of the outer lipid layer coupled with the relative thinness of the cell wall accounts for the negative staining. Generally Gram-negative bacteria are harder to eradicate as well, due to the presence of the outer membrane.
 

Synapsis

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Positive Gram-staining is purplish, and negative Gram-staining is pinkish. In reality the colors are fairly flexible in their ranges, sort of like pH indicators. So a blue and red staining wouldn't be uncommon for positive and negative stains, respectively.

However, your evaluation on the nature of the staining seems a little flawed. Gram-staining is an evaluation of how well the peptidoglycan cell is exposed to the environment. Gram-positive bacteria do have a thick cell wall, but Gram-negative bacteria have a peptidoglycan cell wall that is a little thinner and is situated between two phospholipid membranes (in what's called the periplasmic space). The existence of the outer lipid layer coupled with the relative thinness of the cell wall accounts for the negative staining. Generally Gram-negative bacteria are harder to eradicate as well, due to the presence of the outer membrane.

Thanks for the explanation!
 

MedSprinter

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Positive Gram-staining is purplish, and negative Gram-staining is pinkish. In reality the colors are fairly flexible in their ranges, sort of like pH indicators. So a blue and red staining wouldn't be uncommon for positive and negative stains, respectively.

However, your evaluation on the nature of the staining seems a little flawed. Gram-staining is an evaluation of how well the peptidoglycan cell is exposed to the environment. Gram-positive bacteria do have a thick cell wall, but Gram-negative bacteria have a peptidoglycan cell wall that is a little thinner and is situated between two phospholipid membranes (in what's called the periplasmic space). The existence of the outer lipid layer coupled with the relative thinness of the cell wall accounts for the negative staining. Generally Gram-negative bacteria are harder to eradicate as well, due to the presence of the outer membrane.

The explanation of why the color of Gram+ and Gram- bacteria may vary is spot on as well as Gram- bacteria being more difficult to eradicate.

However, I believe your explanation of the Gram-staining procedure is incorrect. Whether the bacteria are stained purplish or pinkish is based on the thickness of the peptidoglycan layer of their cell walls. The reason for this is because the acetone-alcohol destaining step will solubilize the outer membrane of the Gram- bacteria and expose this layer to the destaining solution. Crystal violet (primary stain) diffuses through the outer membrane and stains the thin layer of peptidoglycan in Gram- bacteria, but the destaining solution will remove this stain. If you don't wash the destaining solution off after a few seconds both Gram+ and Gram- bacteria will lose their crystal violet stain and retain the secondary safranin stain and appear pinkish. Gram+ bacteria do not lose their crystal violet stain during the destaining step because their peptidoglycan layers are much thicker. Gram+ bacteria still take up the safranin stain, but the crystal violet is so much darker so that is all you see.

I have a Bachelor's in microbiology if that helps convince you and if I am incorrect, someone please correct me!
 

sakabato93

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The explanation of why the color of Gram+ and Gram- bacteria may vary is spot on as well as Gram- bacteria being more difficult to eradicate.

However, I believe your explanation of the Gram-staining procedure is incorrect. Whether the bacteria are stained purplish or pinkish is based on the thickness of the peptidoglycan layer of their cell walls. The reason for this is because the acetone-alcohol destaining step will solubilize the outer membrane of the Gram- bacteria and expose this layer to the destaining solution. Crystal violet (primary stain) diffuses through the outer membrane and stains the thin layer of peptidoglycan in Gram- bacteria, but the destaining solution will remove this stain. If you don't wash the destaining solution off after a few seconds both Gram+ and Gram- bacteria will lose their crystal violet stain and retain the secondary safranin stain and appear pinkish. Gram+ bacteria do not lose their crystal violet stain during the destaining step because their peptidoglycan layers are much thicker. Gram+ bacteria still take up the safranin stain, but the crystal violet is so much darker so that is all you see.

I have a Bachelor's in microbiology if that helps convince you and if I am incorrect, someone please correct me!

Thanks!
 
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