How to Gram Stain


Want to learn a cool microbiology technique? Well in this blog I will teach you a simple way on how to perform a gram stain, a technique used often to classify different types of bacteria as either gram positive or negative. The ability to perform a gram stain is a technique one can put on their resume or CV, especially those wanting to go to graduate school for some sort of clinical research. Gram staining is done in research facilities, doctor’s offices, vet’s offices, etc– so it is a pretty universal technique.

Today in my microbiology lab we used a gram stain in order to determine whether an unknown bacteria was gram positive or negative. First off we had to create controls using two known bacteria– Escherichia coli (E. coli) which is gram negative and Staphylococcus aureus or Staph which is gram positive. We placed these two known bacteria and the unknown bacteria on a slide using a sterile inoculating ring and then used a heat fixation technique (placed the slide through a Bunsen burner flame) in order to do three things– kill the bacteria, make the bacteria more permeable to the stain, and to hold the bacteria in place on the slide which prevents it from possibly being washed off. Once the bacteria are properly heat fixed the first stain can be applied. The first stain is the crystal violet stain and it is to stay on the slide for approximately 60 seconds. After about 60 seconds the crystal violet can be washed off with deionized water. The crystal violet makes the bacteria look a purplish blue color. Next comes the mordant. The mordant is used to help lock in the stain, and the particular mordant we use in our lab is an iodine solution. You then add the iodine to the stain and allow it sit for another 60 seconds. The bacteria still remain a purple/ blue color. Wash the iodine solution from the slide with deionized water and then add the decolorizing agent (ethanol) to the slides. Allow the decolorizing agent to sit for only 5 seconds and then rinse with deionized water. After this step a gram positive bacteria will remain purple/ blue and the gram negative will appear colorless. Lastly you apply the safranin which is a red stain. The safranin should sit for approximately 60 seconds and then can be washed afterward with deionized water. Gram-positive bacteria remains purple/ blue and the safranin stain turns gram negative bacteria into a reddish pink color. Once the staining process is complete and the slides have dried they can be viewed under the microscope using the oil immersion lens. The controlled gram positive and negative stains are important because they serve as a viewing key when trying to determine what our unknown is. According to the gram stain the unknown we worked with today is gram negative because it appeared more red/ pink under the microscope and looked most similar with the known gram negative bacteria, E. coli.

I hope you all learned something today and that this peaked your interest in microbiology or general biology. Research facilities and clinical offices are always looking for individuals that have experience with specific lab techniques, which is why is looks so great on a resume.

Until next time… take care!



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One Response to How to Gram Stain

  1. Godfrey A.K-Medical student says:

    Thanx,i’v learnt it gud.

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