Differentiating bacterial species is crucial for a variety of reasons, including detecting infection and ensuring food safety, as well as determining which species gives a cheese its unique flavour. Molecular techniques such as PCR, quantitative PCR, genome sequencing, and mass spectrometry can be used to distinguish bacterial species and even specific strains. Even without delving into the deep gritty of molecular biology, there are phenotypic variations between bacteria groups that can be utilised to distinguish them. This includes things like their morphology (bacilli vs. cocci, for example), their ability to grow in specific nutrients, and whether they prefer high or low oxygen settings. Bacterial species can be divided into broad groupings based on the trait being researched, but when combined, this information can substantially reduce the probable identities. The structure of bacterial cell walls is one such important classification, which determines whether a bacterium is Gram positive or Gram negative.

Difference in structure of Gram positive vs Gram negative bacteria

The thickness of the peptidoglycan layer and the presence or absence of the outer lipid membrane are the two essential factors that cause Gram positive and Gram negative organisms to have different visibility properties. This is due to the cell wall’s capacity to hold the crystal violet stain used in the Gram staining method, which can then be seen under a light microscope.

Gram positive bacteria have a thick peptidoglycan layer without an outer lipid membrane, whereas Gram negative bacteria have a thin peptidoglycan layer with an outer lipid membrane.

When referring to their structure rather than their staining properties, Gram positive bacteria are called monoderms because they lack an outer lipid membrane. Gram negative bacteria have an exterior lipid membrane, which is why they are called diderms when referring to their physical structure. Hans Christian Gram, a Danish bacteriologist, invented the Gram staining technique in 1884.

While a Gram stain will not tell you which species you’re looking at, it can help you narrow down your list of prospective choices and direct further tests if necessary.

At each stage of gramme staining, the colour of gramme positive and gramme negative bacteria

Preparing a sample for Gram staining

Your sample identification should be written on a clean glass microscope slide. Because the reagents used in the staining method destroy many inks, make sure you use a pencil.
If you’re using a liquid bacterial culture to make your slide, follow these steps:

Using a sterile loop, dab a little drop culture onto the slide. Smear the droplet in a circular motion over an area of about 1 cm in diameter. If your culture is particularly dense, you may need to dilute it first to guarantee that individual bacterial cells can be seen under a microscope after staining.

If the raw material comes from a bacterial plate, follow these steps:

To make a liquid culture, resuspend a loop of colony material in sterile phosphate buffered saline (PBS).

Pass the smeared slide through a flame two or three times once the spread has dried.

This kills the bacteria in the smear and adheres the sample to the slide; however, overheating the sample can damage cellular shape.

Gram stain procedure – Gram staining a sample

Apply a light coating of crystal violet to the smear and set aside for 1 minute. Slightly tilt the slide and gently rinse with tap or distilled water.

Crystal violet is a water-soluble pigment that penetrates the bacterial cell wall’s peptidoglycan layer.

Flood the smear gently with Gram’s iodine and let aside for 1 minute. Slightly tilt the slide and gently rinse with tap or distilled water. The smudge will now have a purple hue to it.

Gram’s iodine solution (iodine and potassium iodide) is used to create a complex with crystal violet, which is significantly bigger and water insoluble.

Use 95 percent ethyl alcohol or acetone to decolorize the stain. Apply the alcohol drop by drop until the alcohol runs practically clear, tilting the slide slightly (5-10 seconds). To avoid over-decolorization, immediately rinse with water.

Decolorizer shrinks and tightens the peptidoglycan layer by dehydrating it. The massive crystal violet-iodine complexes are unable to penetrate and escape the strong peptidoglycan layer in Gram positive bacteria, resulting in purple coloured cells. The outer membrane is destroyed in Gram negative bacteria, and the thin peptidoglycan layer is unable to hold the crystal violet-iodine complexes, resulting in colour loss.

Flood the area with safranin counterstain and wait 45 seconds. Slightly tilt the slide and gently rinse with tap or distilled water.

Safranin is a water-insoluble dye that will stain bacterial cells a light red, allowing Gram negative cells to be seen without interfering with the purple of Gram positive cells.

Blot the slide dry on filter paper, then examine the smear under oil immersion using a light microscope.

Gram positive vs Gram negative color

Gram positive bacteria

When Gram positive bacteria are examined under a light microscope after Gram staining, they display a unique purple colour. This is owing to the purple crystal violet stain remaining in the cell wall’s thick peptidoglycan layer. All staphylococci, all streptococci, and some listeria species are Gram positive bacteria.

All staphylococci, all streptococci, and some listeria species are Gram positive bacteria.

Gram negative bacteria

Following Gram staining, Gram negative bacteria appear pale reddish in colour under a light microscope. Because their cell walls are unable to hold the crystal violet stain, they are solely coloured by the safranin counterstain. Enterococci, Salmonella species, and Pseudomonas species are examples of Gram negative bacteria.

Conclusion

Gram staining is not a reliable method for determining bacterial phylogenetic relationships. The ancestral form of Gram-positive organisms is assumed to be a single membrane. Previously, it was considered that the second membrane present on Gram negative bacteria evolved only once, and that all Gram negative bacteria were therefore more closely related to one another than Gram positive bacteria. Genetic investigation has recently revealed that this is not the case, and it is more likely to have developed numerous times in separate lineages – a product of convergent evolution.