Question

1. Please describe the gram staining procedure. Be sure to include: crystal violet, iodine, alcohol, safranin, and any other terms you think are important to the technique. You do not need to re- write the lab procedure here please describe what is occurring conceptually to both gram + and gram-cells during the procedure. What would a gram positive cell look like under oil immersion if a researcher forgot to use crystal violet? Please provide a rationale for your response. 3. What is the shape and gram stain of the bacteria in this photo? 4. What might happen to any bacterial cells on the slide if 95% ethanol was dispersed directly onto the smear during the staining procedure for twice the recommended time? What could a researcher do help ensure this does not happen (where should they aim the ethanol)?

Answer 1

1. Gram staining is a staining technique used to differentiate two groups of bacteria, i.e. Gram-Positive Bacteria, 2. Gram-Negative, based on their different cell wall constituents. The staining procedure distinguishes between them by colouring these cells with safranin and crystal violet.
Gram-positive bacteria take up crystal violet due to the presence of a thick layer of peptidoglycan in their cell walls, which retains the crystal violet. Whereas, Gram-negative bacteria stains red, due to a thinner peptidoglycan wall, which does not retain the crystal violet during the decolouring process.

2. A gram-positive bacteria, when stained without crystal violet, will look the same as a gram-negative bacteria are the removal of the crystal violet stain help to distinguish gram-positive bacteria from gram-negative bacteria.

3. The cells in the photo represent gram-positive cocci due to its pink colour and dotted shape.

4. Adding 95% ethanol directly to the cells causes both the gram-positive as well as the gram-negative cells to get decolourized due to the high concentration of alcohol along with a higher flow rate of alcohol
A Researcher should gently pour the alcohol at the edges of the slide and let the alcohol flow should the sample, this helps to keep the flow rate low and does not decolorize more than required.