Gram Stain

How do you identify bacteria in a sample if you have no prior information? There are millions of bacterial strains! Yes, you can look at their shape, the way they aggregate to form colonies... but that won't give you all the information. You have to use a variety of observations. One of these that is widely used is Gram staining. By using the Gram staining technique we can understand if a bacteria strain has thick or thin cell walls, which combined with other observations gives us a better picture of what bacteria strain we might be staring at.

Gram Stain Gram Stain

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Table of contents

    What is Gram staining?

    Gram staining is a staining technique which uses crystal violet to dye bacteria. Crystal violet binds to the peptidoglycans on the bacterial cell wall and can help identify if a bacterium has a thick or thin cell wall.

    Gram staining is a technique that was named after its creator, Danish scientist Hans Christian Gram. He developed this technique in the late 1800s. Gram realized that bacteria can be stained in predictable, orderly ways when exposed to certain substances. These stains can then be used to group bacteria into categories based on which stain the bacteria pick up. You may have done Gram staining in chemistry or biology lab, and scientists and doctors still use this method when analyzing things like mucous samples from a patient.

    Gram staining results

    There are four possible outcomes of gram staining, which help us to categorize bacteria. Two are common, two are uncommon. We will briefly describe how each staining outcome looks.

    Common gram stain outcomes

    • Gram-positive

      • At the end of the gram staining procedure, gram-positive bacteria are purple.

    • Gram-negative

    Uncommon gram stain outcomes

    • Gram variable

      • Gram-variable bacteria have a mixed purple and pink appearance

    • Gram indeterminate

      • These are the bacteria that do not pick up any stain well. They're neither pink nor purple. They are colourless at the end of the gram staining process.

    Gram stain method

    Gram staining steps:

    1. A culture with unknown substances is brought out to be examined.

    2. A smear or sample of bacteria is taken from this culture.

    3. This sample gets heat-fixed.

    4. Substance #1: A dye, crystal violet is added to the sample.

    5. Substance #2: Iodine is added to the sample to help the crystal violet stick.

    6. Substance #3: Ethanol (or acetone or some other alcohol) is added as a decolorizing agent, which means to wash other colours out.

    7. Substance #4: A new dye, Safranin is added. This step is called counterstaining.

    Heat fixation is a process of passing heat around or through a sample of a bacterial culture. This procedure is typically done by researchers manually, in a laboratory, with the source of heat being something like a Bunsen burner. A slide of bacteria may be passed several times over a Bunsen burner, such that the bacteria on the slide are fixed, or immobile.

    Some of the bacteria in the sample may be killed during this process, but enough of them will still be viable to do whatever test is needed (i.e. to perform a gram stain).

    We already know that overall, gram-positive bacteria stain purple ultimately, and gram-negative bacteria stain pink. But what do they look like following each of the steps we outlined above? Are they ever the same and at what point can we observe their colour distinction?

    How does Gram staining work?

    Gram staining has a scientific logic behind it: crystal violet can bind to the peptidoglycans present in the cell wall of bacteria. Depending on the thickness of this cell wall, the dye will remain after adding the decolourizer or not, conditioning the colour of the bacteria: purple if it remained (crystal violet), and pink if it didn't, due to the cell turning the colour of safranin.

    Gram-positive stain

    Gram-positive bacteria have a thick cell wall (Fig. 3). This cell wall is outside of their cell membrane and is made up of a material called peptidoglycan (macromolecule formed by peptides and sugars).

    After crystal violet has been added, and iodine is used as a mordant to fix the crystal violet to the bacteria, the thick peptidoglycan cell wall complexes solidly with the crystal violet-iodine duo. Then, the decolourizer ethanol is added. However, because the peptidoglycan wall is so thick, instead of removing the crystal violet-iodine complex, what happens is that ethanol causes the pores present in the cell wall to close, and the bacterium itself to dehydrate and shrink. Because the pores are closed, the crystal violet dye cannot exit the cell wall and be washed out. Thus, the purple, primary stain remains, even after the decolourizer is added. Then, when the secondary counterstain safranin is added, it cannot take hold in the bacterial cell wall because crystal violet is already there and was not washed out.

    Gram-negative stain

    Gram-negative bacteria, on the other hand, have a very thin peptidoglycan cell wall, and outside of this cell wall, they have an outer membrane layer full of lipids and lipoproteins. When gram-negative bacteria encounter crystal violet dye, they do take it up and become purple, and iodine does act as a mordant to help them fix this purple colour. However, once the ethanol decolourizer is added, the alcohol disturbs and dissolves the lipids in the outer membrane. With the outer membrane weakened and only a thin layer of peptidoglycan, the crystal violet can escape the cell and the bacteria becomes colourless at this stage. Then, the counterstain safranin stains the Gram-negative cell wall pink, because crystal violet has already been washed out.

    Bacillus subtilis Gram stain

    Bacillus subtilis is our example organism to demonstrate what happens during gram staining of gram-positive organisms. Its genus: Bacillus, tells us that it is a rod-shaped bacteria. But what colour would these rods appear during the gram staining process (Fig. 1)?

    • First, of course, we'd take a culture containing B. subtilis.

      • When examining these bacteria under the microscope, they are naturally colourless.

    • Then, we'd heat-fix a smear of B. subtilis.

      • No colour changes are observed.

    • Now, we add the first substance: crystal violet.

      • Crystal violet is a dye that causes the bacteria in the sample to take on a purple colour.

    • Next, we add the second substance: iodine

      • Iodine naturally has a dark brown or dark blue colour. It is not a dye, so the bacteria are still purple.

      • Iodine is considered a mordant, which means it's a fixing agent. It helps fix the purple colour to the bacteria.

    • Now, we add the third substance: ethanol

      • Ethanol, or whichever alcohol is used in this step, is a decolourizer

      • However, gram-positive bacteria like B. subtilis are NOT decolourized by it. They retain their purple colour.

    • Lastly, we add the fourth substance: safranin

      • Safranin is the counterdye, and it has a pink colour

      • However, safranin can only affect bacteria that have been properly decolourized. Since Gram-positive bacteria are NOT decolourized by ethanol, they are not affected by safranin and end this entire process with a purple colour.

    Gram staining Bacillus subtilis Gram positive StudySmarterFig.1. Bacillus subtilis Gram stain. You can see the rods are purple, which means they are Gram-positive.

    A chart summarizing the effects of each substance on gram-positive bugs like Bacillus subtilis is below (Table 1).

    Substance added Colour change it leads to in gram-positive bacteria
    Crystal violetPurple
    IodineNo change - still purple
    EthanolNo change - still purple
    SafraninNo change - still purple

    Table 1: Gram stain steps for gram-positive bacteria.

    E coli Gram stain

    For our step-by-step example of a gram-negative organism, we shall use another rod-shaped bacteria: Escherichia coli (Fig. 2). E. coli is generally harmless, but it can cause a host of different illnesses, ranging from UTIs to traveller's diarrhoea to meningitis in newborns. The steps for E coliI Gram staining are the same as for any bacteria:

    • First, we'd take our colourless culture of E. coli and heat-fix it.

    • Then we'd apply crystal violet.

      • The bacteria now appear purple.

    • Next, we'd apply our mordant, iodine.

      • No colour changes will be observed, the E. coli will still be purple.

    • Then we'd use our ethanol wash as a decolourizer.

      • The E. coli lose their colour and become colourless.

    • Lastly, we apply safranin, the counterstain.

      • The E. coli take on the colour of the counterstain and become pink.

    E coli gram stain gram negative StudySmarterFig. 2. E. coli Gram staining. The rod bacteria are pink, which means they are Gram-negative.

    A chart summarizing the effects of each substrate on gram-negative bugs is below (Table 2).

    Substance addedColour change it leads to in gram-negative bacteria
    Crystal violetPurple
    IodineNo change, still purple

    Table 2: Gram stain steps for gram-negative bacteria

    Gram stain of Staphylococcus aureus

    As another example of a Gram-positive bacteria, we have Staphylococcus aureus, which as the name indicates is a cocci bacteria. In this case, the staining process is the same (crystal violet, then iodine, then alcohol and finally safranin). However, the final image under the microscope will show purple spheres rather than purple rods like with Bacillus subtilis.

    Staphylococcus aureus Gram positive gram stain cocci StudySmarterFig. 3. Staphylococcus aureus Gram stain. You can see that the cocci are purple, thus they are Gram-positive.

    After this article, we hope you have a better understanding of Gram staining: how it works, what it indicates, and how colours are a key part of microscopy!

    Gram Stain - Key takeaways

    • There are two main possible outcomes with gram stain: gram-negative and gram-positive.
    • The four steps of gram staining are: primary staining, fixing, decolorizing, and counterstaining.
    • Gram-negative bacteria stain pink because of their thin cell walls and lipid outer membrane.
    • Gram-positive bacteria stain purple because of their thick peptidoglycan cell walls.
    Frequently Asked Questions about Gram Stain

    What is Gram staining?

    Gram staining is a staining technique used to stain and classify bacteria. 

    How does Gram staining work?

    Gram staining works by exposing a bacteria culture to a dye containing crystal violet (the purple dye that gives Gram-positive bacteria their characteristic colour) and alcohol (which takes away the purple colour from bacteria that are not Gram-positive). Cells with a thick cell wall retain the dye (crystal violet) even after alcohol exposure, whilst bacteria with a thin cell wall will lose the dye when exposed to alcohol.

    What is the purpose of Gram staining?

    The purpose of Gram staining is to colour bacteria in a culture and detect if they are Gram-positive or Gram-negative. This gives information about which strain of bacteria they could be.

    What are the 4 steps of Gram staining?

    The steps of Gram staining are:

    1. A dye, crystal violet is added to the bacteria sample.

    2. Iodine is added to the sample to help the crystal violet stick.

    3. Ethanol (or acetone or some other alcohol) is added as a decolorizing agent, which is intended to wash other colours out.

    4. A new dye, Safranin is added. This step is called counterstaining and is meant to stain the cells that didn't retain the crystal violet stain.

    Why is Gram stain important in microbiology?

    Gram staining is one of the most important staining methods in microbiology and serves to identify if the bacteria in a culture are Gram-positive or Gram-negative.

    How is Gram staining used to identify bacteria?

    Gram staining is used to understand if bacteria in a culture are Gram-positive or Gram-negative. Each bacteria strain is Gram-positive or negative, so this dye reduces the types of bacteria to take into account when trying to identify the types of bacteria present in a sample.

    Why do we do gram staining?

    Gram staining is done to determine if bacteria in a sample are Gram-positive or Gram-negative.

    What is gram staining used for?

    Gram staining is used to determine if bacteria in a sample are Gram-positive or Gram-negative.

    Test your knowledge with multiple choice flashcards

    What color does safranin turn gram negative bacteria to?

    What kind of substance can be used as a decolorizer?

    Which dye is used first, crystal violet or safranin


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