Chapter 2, Problem 1CHI

Compare, Hypothesize, Invent

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.

Summary Introduction

Danish physician, Christian Gram in 1884, developed a gram staining procedure. In bacteriology, this method is employed widely. This technique will be carried out in clinical laboratories during the diagnosis of infectious disease. This staining method is an example of the differential staining technique. Using this technique, most of the bacteria are classified into two groups.

Explanation of Solution

In Gram staining, sample (bacterial cells) will be stained with crystal violet. They all will appear in purple colour. Next step is the addition of mordant-iodine, and then the stain will be fixed in the sample due to the formation of crystal violet (CV) and iodine complex. The third step is de-staining with water plus alcohol. If the stain remains then the bacteria are Gram-positive (purple) and if colour come out, they are Gram-negative group. These will appear in red colour.

Students who are performing the Gram-staining procedure should carry out all the steps carefully. This because an error in each might result in false positive result or many issues. Some of the issues that might happen when the procedure is performed well are given below:

Insufficient heat fixation: Sufficient heat is required so that the sample will be fixed over the slide.

Over-vigorous washing: If the sample is washed vigorously, the cells might be removed from the slide

Improper staining or destaining: De-staining procedure or staining must be carried out for required time otherwise the bacterial cells will not take the stain.

Over decolourization and focussing: Over decolourization will result in the removal of CV⁺ iodine complex, making the Gram-negative positive cells appear like Gram-negative cells. This will affect the result.