Prescott's Microbiology 8th Edition by Joanne Willey, Linda Sherwood, Christopher J. Woolverton, Lansing Prescott, John Harley, Donald Klein

Exercise 1

If you prepared a sample of a specimen for light microscopy, stained it with the Gram stain, and failed to see anything when you looked through your light microscope, list the things that you may have done incorrectly.

Step 1 of 3

In order to perform a Gram stain, one must first fix cells onto a slide called a smear. The smear is then stained with a dye called crystal violet. This stains all of the cells purple and is the primary stain. Iodine is then added, which acts as a mordant to help the dye stick to the cell. Next, ethanol or acetone is added to de-stain the cells.

If the cells are gram-positive, they do not lose the stain easily. If the cells are gram-negative, they lose the stain and appear clear. In order to better visualize gram-negative cells, the smear is counterstained with safranin, which stains the gram-negative cells a pink or red color. The counter stain stains all of the cells. The purple color is much darker than the pink and overpowers the color.