Question

5. You use the Gram stain to try to differentiate bacteria in a sample. You find that all of the cells are Gram negative, but the teaching assistant insists there are both Gram positive and Gram negative cells present in the sample you are given. You definitely smeared a loop of the sample onto the slide and your fingers are stained a little purple and also a little red from handling the sides of the slide. Give a reason why your effort at carrying out the Gram stain failed 6. On another day in the lab you use simple staining to check that there are cocci present in your culture. Unfortunately, after washing away the stain, you see nothing on the slide. What are possible reasons for this result? 2

Answer 1

1. under the microspoce, the gram + cells appear purple while the gram - cells appear red. If however we see that all cells look gram - (red) this might be because of improper handling of the reagents.

• The reagents used may be old and have lost some of their properties.
• The incubation time provided for each step might not be enough.
• Overuse of the Red stain.
• Also, chosing a sample that contained non-viable gram + cells may also result in this kind of a situation.

2. This may so happen in case of excessive washing of the sample. While we destain or wash any sample, it is very important to be gentle on the sample and also to keep in mind the incubation time required for washing. However, improper handling at any stage might result in loss of the sample or the stain from the cells and cause this kind of problems.

All the Best :)