Gram Stain

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

  • At the end of the gram staining procedure, gram-negative bacteria are pink.

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.

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.

Fig.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).

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.

Fig. 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).

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.

Fig. 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!