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.
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
Gram-negative
Uncommon gram stain outcomes
Gram variable
Gram indeterminate
Gram stain method
Gram staining steps:
A culture with unknown substances is brought out to be examined.
A smear or sample of bacteria is taken from this culture.
This sample gets heat-fixed.
Substance #1: A dye, crystal violet is added to the sample.
Substance #2: Iodine is added to the sample to help the crystal violet stick.
Substance #3: Ethanol (or acetone or some other alcohol) is added as a decolorizing agent, which means to wash other colours out.
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)?
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).
| Substance added | Colour change it leads to in gram-positive bacteria |
| Crystal violet | Purple |
| Iodine | No change - still purple |
| Ethanol | No change - still purple |
| Safranin | No 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:
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).
| Substance added | Colour change it leads to in gram-negative bacteria |
| Crystal violet | Purple |
| Iodine | No change, still purple |
| Ethanol | Colourless |
| Safranin | Pink |
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!
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.
Explanations 
Exams 
Magazine 
