Gram stain is widely used in microbiology as it is one of the easiest ways to differentiate bacteria based on the composition of their cell wall. According to Gram, all bacteria can be divided into gram-positive (Gram (+)) and gram-negative (Gram (-)). The Gram stain method was developed in 1884 and has not lost popularity since then, although it has been modified several times.
Cell wall structure
Gram staining reveals whether a bacterium is Gram-positive or Gram-negative. The division of bacteria into Gram (+) and Gram (-) is carried out in accordance with the structure of their cell wall.
The cell wall contains the greatest amount of peptidoglycan (murein) - a complex substance, which includes peptapeptide and glycan. The glycan consists of alternating residues of N-acetylglucosamine and N-acetylmuramic acid linked to each other by β-1,4-glycosidic bonds. Peptidoglycan provides cell shape maintenance, osmotic protection, and antigenic functions.
Main differences between Gram-positive and Gram-negative bacteria
Different bacteria have different peptidoglycan layer thicknesses. In bacteria that are classified as gram-positive, it ranges from 15 to 80 nm, while in gram-negative it is from 2 to 8 nm. At the same time, Gram-negative bacteria have a special structure under the peptidoglycan layer, which Gram-positive bacteria do not have - the periplasmic space. This space is filled with hydrolytic enzymes - β-lactamase, ribonuclease 1, phosphatase. It is these enzymes that are responsible for the resistance of Gram-negative bacteria to many antibiotics.
The Gram(-) peptidoglycan layer of bacteria is bound to lipopolysaccharide, an antigenic structure containing endotoxin. In Gram(+) bacteria, teichoic acids perform similar functions.
Gram-negative bacteria have an additional structure - the outer membrane.
The essence of the staining method
Before you start staining, smears of the studied bacteria are prepared. To do this, water is dripped onto a glass slide and a culture of microorganisms is added there with a bacterial loop. Then, after the water has completely dried, the smear is fixed - the glass slide is carried several times over the burner flame. Gram stains are more effective than staining live bacteria - dye molecules bind better to dead cells.
Coloring is done in several stages:
- Small pieces of filter paper are placed on a fixed smear and the main dye is poured - gentian violet or methylene blue.
- After 3-5 minutes, remove the colored filter paper and fill the smear with Lugol's solution for 1 minute. In this case, the preparation darkens.
- The Lugol solution is drained and the smear is treated with pure ethyl alcohol: a few drops are dripped onto the preparation, drained after 20 seconds. The procedure is repeated 2-3 times.
- Rinse the test slide with distilled water.
- Produce additional staining - finish the preparation with fuchsin. After 1-2 minutes, the dye is washed off.
- After the water dries, examine the smear under a microscope. Gram-positive bacteria will be blue-violet, Gram-negative bacteria will be pink or red.
Causes of different staining patterns
As described above, Gram-staining of bacteria stains Gram-positive bacteria blue-violet, while Gram-negative bacteria stain red or pink. The reason for the differential staining of bacteria by this method is that after the soluble form of gentian violet enters the cell, the dye passes into the insoluble iodine form. During the treatment of bacteria with ethyl alcohol, lipids are extracted from the membrane under the action of this non-polar solvent. The membrane then becomes porous and is no longer a significant barrier to dye leaching. Howeverpeptidoglycan is more resistant to non-polar solvents, including alcohol. It is he who prevents the washing out of the dye, so bacteria with a thick murein layer turn blue-violet (gram-positive), and after treatment with alcohol they do not change their color.
Thin murein layer of gram-negative bacteria cannot hold the dye molecules in the cell, so after the action of alcohol they become colorless - they stain gram-negatively.
After exposure of a smear to fuchsin, Gram-stained bacteria remain blue-violet, while Gram-negative bacteria become pink-red.
Examples of Gram(+) and Gram(-) bacteria
Gram-negative bacteria include cyanobacteria, sulfur bacteria, iron bacteria, chlamydia, rickettsiae, acetic bacteria, many methylobacteria, thionic bacteria, arsenitobacteria, carboxybacteria.
Bifidobacteria, many aquatic bacteria, streptococci and staphylococci are Gram-positive.
