2.2 Gram stain

The Gram stain is the most important stain in microbiology. The first step is to heat-fix the bacteria to a microscope slide. Using a sterile loop, a few drops of water are transferred to the slide. The culture is then mixed with the water to give an emulsion and this is spread onto the slide. The secret of a good Gram stain is not to make the emulsion too thick. The emulsion is left to dry at room temperature.

Then the slide is passed through the Bunsen flame to fix the bacteria to the glass, so that they won’t be washed off by the staining. It’s important not to let the slide get too hot or the bacteria will burst. If it’s too hot for you, it will be too hot for the bacteria.

In Gram’s staining, the slide is flooded with crystal violet stain and left to stand for one minute. The crystal violet is washed off with slow-running tap water. The next step is to flood the slide with Gram’s iodine, which is also left for a minute. The iodine forms a complex with the crystal violet, giving a dark purple colour. The slide is rinsed again before the next step.

Next, 95% alcohol is used to wash the purple dye away. Some bacteria retain the dye; others don’t. After a rinse in water, the final step is to add a red dye, Safranin, which is left on for just 30 seconds.

After a final wash, the slide is blotted, dried, and then examined under the microscope. Some bacteria appear pink. Since they lost the original purple colour, they are called Gram negative. Other bacteria retain the purple colour, which masks the red dye, and these ones are called Gram positive. Round bacteria are called cocci. Rod-shaped bacteria are often known as bacilli.