When carrying out the Gram stain procedure, adding too little or too much alcohol to the sample is a common mistake. Suppose a student added too little alcohol to a sample of Gram-negative bacteria. What would she observe? Why?

In the decolorization step alcohol is used which destain the gram-negative bacteria but not gram-positive because alcohol degrades the LPS cell wall of gram-negative bacteria which allow the crystal violet to moves out of the cell.

So if we use too little alcohol then the alcohol will not able to decolorize all the gram-negative bacteria and most of the cell in the culture will appear violet or purple because crystal violet retained in the cell wall of bacteria.

So the sample of gram-negative bacteria will appear as gram-positive bacteria under the microscope.

this protocol describes a detailed procedure for intracellular flow cytometry staining.