How to Do a Gram Stain: Everything You Need to Know

Gram staining is one of the most widely used microbiological procedures. When used with a quality microscope, Gram stains are utilized for a variety of studies and even determining the early stages of treatment for patients in a hospital setting. 

How Gram Staining is Used in Microbiology

Developed in the late 1800s, this staining procedure has become crucial in medical laboratories and universities throughout the world as a means of quickly classifying and describing suspected bacterial pathogens in patients. Through the careful and precise application of staining mediums and other reagents, Gram staining allows microbiologists to promptly confirm if a bacterial organism breaks down into one of three large categories: Gram-Positive (purple staining), Gram-Negative (red staining) or Gram Variable/Indeterminate organisms. Another major benefit provided by Gram staining is that it allows for a microbiologist to determine the shape of an organism in question. 

Gram staining also enables doctors to quickly begin treating their patients. Bacteria are complex, microscopic organisms and while many of them are harmless, others, however, are capable of making people very sick and can even be fatal. Fortunately, a Gram stain allows physicians to quickly determine if bacteria are present in a sample–like a wound or a throat swab–and if they are something that the doctor should be truly concerned about.

How to Prepare a Gram Stain

Preparing a Gram stain is actually quite simple and straightforward. The microbiologist must first make a smear of the sample and then heat fix the smear to the slide. Heat fixing is important because it will prevent bacteria from being rinsed from the slide during the various rinsing steps. Once this step is completed, the microbiologist then applies the first stain. 

How to Identify Bacteria Using a Gram Stain

Using the stain Crystal Violet, the microbiologist floods the entire slide and allows the stain to sit for around 60 seconds. During this time, the bacteria on the slide will begin absorbing the Crystal Violet stain. Those bacteria we classify as Gram-Positive will absorb the stain into their cellular walls, where it will be bind with the protein, peptidoglycan.

Once the initial 60 seconds have passed, the microbiologist will then flood the slide a second time with iodine. Iodine acts as a binding agent, binding the Crystal Violet stain to the cells. The iodine should be allowed to sit for another 60 seconds, at which point the slide should be rinsed with running water. It is very important to take care when rinsing the slide, as even with all the steps taken to heat affix the slide, it is still possible to ruin the smear and wash off the bacteria.

Once the iodine has been completely rinsed from the slide, the next step to follow is the decolorization. Using either some variety of alcohol or acetone, a microbiologist will apply the decolorizer to the smear. Care must be taken here, as it is possible to over-decolorize the smear. Allow the alcohol to wash over the smear, and when the purple stain is no longer visible in the runoff, stop right away. What is happening in this step is that the alcohol is removing the Crystal Violet stain from cell envelopes and cell walls that do not have peptidoglycan. If watched closely, it is possible to observe the Crystal Violet from earlier in the process leaving the microscopic cells. This then allows those cells without peptidoglycan to uptake the next and final stain.

The final stain is then applied to the smear and left to sit for another 60 seconds. This allows the bacteria that had the Crystal Violet stain removed during the decolorization phase to be stained a bright red color, which is key in classifying bacteria as either Gram-Positive or Gram-Negative. Many disease-causing bacteria, like E. coli, are Gram-Negative and need to be treated by a medical professional as soon as possible.

Final Thoughts

Gram stains are incredibly important in modern medicine. Even though preparing a Gram stain is a relatively quick process, it is all too easy to perform poorly or incorrectly. Through a well-performed Gram stain, a physician can quickly establish if bacteria are present in a given sample and how to begin proceeding with the treatment. Purchase a stain kit and learn how to properly prepare a Gram stain today!