Question

Background:

“Belinda!” called Kyle, running to catch up to her. Belinda turned around and waited for him. “Are you heading to the lab right now?” asked Kyle. “Yes.” she said. “I want to look over the Gram stain protocol one more time before we go in.” “Yeah, good idea. I’ve been trying to memorize the steps. But it’s hard.” Belinda and Kyle spent the twenty minutes remaining before the start of lab going over Chapters 3 and 4 of their textbook, which listed the steps of the Gram stain and how the staining results relate to the structure of the bacterial cell wall.

Kyle and Belinda took a sample of the bacteria provided by their instructor and each performed the Gram stain. They focused on their slides with the oil immersion lens. “Got it!” they said almost simultaneously, grinning proudly. “So, what do you have?” asked Belinda. “I have Gram-positive bacilli” announced Kyle with an air of triumph. “Huh? I have Gram-negative bacilli.” “That’s not right, we used the same sample! Let me see yours.” Kyle peered through Belinda’s microscope. “Your bacteria look like they have the same shape as mine, but the color is different. Mine are obviously purple, but yours look much lighter. They are definitely pink. Maybe you did something wrong.” “Why me? You could have done something wrong.” Kyle looked up from Belinda’s microscope. “Fair enough.” Kyle continued “I prepared a bacterial smear like we learned last week, then I added safranin, then Gram’s iodine, then decolorizer, then I added crystal violet.” “You switched up the crystal violet and the safranin,” said Belinda. “I did? Let me see that protocol again. Hmm… I guess you’re right. Let me try it again.”

Questions to address:

1. Explain how the difference in the structure of the cell wall results in the different colors after performing the Gram stain.

2. Why did Kyle’s Gram stain give the wrong result?

3. Are there any other mistakes that can result in gram-negative bacteria staining purple? Which mistakes can result in gram-positive bacteria staining pink?

Answer 1

!. Gram staining is the basic staining procedure of staining bacteria and it distinguishes them on the basis of the composition of their cell wall. There are two main dyes used in this- The crystal violet which is the primary stain and the safranin that is the secondary stain. Alcohol is used as decolourizer and iodine is used as mordant.

Now the difference comes in the results of the stain colours produced. Gram positive bacteria have a thick peptidoglycan mesh like cell wall which binds a lot of crystal violet dye and are thus stained purple. Gram negative bacteria on the other hand have a thin peptidoglycan layer that dissolves when alcohol is added. Thus it gets the pink colour of the safranin and as a result are stained pink.

At first, both gram negative and gram positive bacteria are coloured purple by the crystal violet dye. The difference comes from the decolourization by adding alcohol. Thicker cell wall of gram positive bacteria does not decolourize and doesnot take up the colour of safranin and thus appear purple.

2. kyle's gram stain gave her the wrong result because of the interchanging of dyes. She added safranin as the primary stain and crystal violet as counter stain.

3. One of the other mistake that can result in gram negative bacteria staining purple is that the smear was not properly washed with alcohol which did not decolourized it.

Gram positive bacteria could stained pink because of the following reasons:

a. The sample of bacteria could be taken from an older culture that would have been incubated for more than 18 hours of incubation time.

b. Other reason could be that it had been over-decolourized by using excess amount of alcohol.

c. The bacteria which is grown on certain differential media such as EMB can also show variable staining results.

d. The smear could have been thick which cause the dye to be unevenly distributed throughout the smear,