Background:
âBelinda!â called Kyle, running to catch up to her. Belindaturned around and waited for him. âAre you heading to the lab rightnow?â asked Kyle. âYes.â she said. âI want to look over the Gramstain protocol one more time before we go in.â âYeah, good idea.Iâve been trying to memorize the steps. But itâs hard.â Belinda andKyle spent the twenty minutes remaining before the start of labgoing over Chapters 3 and 4 of their textbook, which listed thesteps of the Gram stain and how the staining results relate to thestructure of the bacterial cell wall.
Kyle and Belinda took a sample of the bacteria provided by theirinstructor and each performed the Gram stain. They focused on theirslides with the oil immersion lens. âGot it!â they said almostsimultaneously, grinning proudly. âSo, what do you have?â askedBelinda. âI have Gram-positive bacilliâ announced Kyle with an airof triumph. âHuh? I have Gram-negative bacilli.â âThatâs not right,we used the same sample! Let me see yours.â Kyle peered throughBelindaâs microscope. âYour bacteria look like they have the sameshape as mine, but the color is different. Mine are obviouslypurple, but yours look much lighter. They are definitely pink.Maybe you did something wrong.â âWhy me? You could have donesomething wrong.â Kyle looked up from Belindaâs microscope. âFairenough.â Kyle continued âI prepared a bacterial smear like welearned last week, then I added safranin, then Gramâs iodine, thendecolorizer, then I added crystal violet.â âYou switched up thecrystal violet and the safranin,â said Belinda. âI did? Let me seethat protocol again. Hmm⦠I guess youâre right. Let me try itagain.â
Questions to address:
1. Explain how the difference in the structure of the cell wallresults in the different colors after performing the Gramstain.
2. Why did Kyleâs Gram stain give the wrong result?
3. Are there any other mistakes that can result in gram-negativebacteria staining purple? Which mistakes can result ingram-positive bacteria staining pink?