Gram staining technique

Introduction

Staining techniques are of different types, such as simple staining, negative staining and differential staining. Gram staining is an example of differential staining techniques.

Differential staining differentiates bacteria into different groups or which can differentiate different structure cell. In differential staining, specimen is subjected to series of stains in which different organism or different parts of cell stained differently. So that they can be distinguished from each other.

For differential staining, at least four chemical reagents are required, primary stain, mordant, decolorizing agent and counter stain.

Primary stain is the first stain. Its function is to impart its color to all cells. Mordant is second stain which intensify the color of the primary stain. In order to establish a color contrast, the third reagent used is the decolorizing agent. Based on the chemical composition of cellular components, the decolorizing agent may or may not remove the primary stain from the entire cell or only from certain cell structures. The final reagent, the counterstain, has a contrasting color to that of the primary stain.

 

Principle:

Gram staining is the most important differential stain used in bacteriology. It was introduced by Dr. Hans Christian Gram (1884). It divides bacterial cells into two major groups, gram positive and gram negative, which makes it an essential tool for classification and differentiation of microorganisms.

The Gram stain reaction is based on the difference in the chemical composition of bacterial cell walls. Gram-positive cells have a thick peptidoglycan layer, whereas the peptidoglycan layer in gram-negative cells is much thinner and surrounded by outer lipid containing layers.

Peptidoglycan is mainly a polysaccharide composed of two chemical subunits found only in the bacterial cell wall. These subunits are N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM). With some organisms, as the adjacent layers of peptidoglycan are formed, they are cross-linked by short chains of peptides by means of a trans-peptidase enzyme, resulting in the shape and rigidity of the cell wall.

In the case of gram-negative bacteria and several of the gram-positive such as the Bacillus, the cross-linking of the peptidoglycan layer is direct because the bacteria do not have short peptide tails. Early experiments have shown that a gram-positive cell denuded of its cell wall by the action of lysozyme or penicillin will stain gram-negative.

The Gram stain uses four different reagents.

1. Primary Stain (Crystal Violet): This violet stain is used first and stains all cells purple.
2. Mordant (Gram’s iodine): This reagent serves not only as a killing agent but also as a mordant, a substance that increases the cells’ affinity for a stain. The reagent does this by binding to the primary stain, thus forming an insoluble complex. The resultant crystal-violet–iodine (CV-I) complex serves to intensify the color of the stain. At this point, all cells will appear purple-black.
3. Decolorizing Agent (Ethyl Alcohol; 95%): This reagent serves a dual function as a protein-dehydrating agent and as a lipid solvent. Its action is determined by two factors, the concentration of lipids and the thickness of the peptidoglycan layer in bacterial cell walls. In gram-negative cells, the alcohol increases the porosity of the cell wall by dissolving the lipids in the outer layers. Thus, the CV-I complex can be more easily removed from the thinner and less highly cross-linked peptidoglycan layer. Therefore, the washing-out effect of the alcohol facilitates the release of the unbound CV-I complex, leaving the cells colorless or unstained. The much thicker peptidoglycan layer in gram-positive cells is responsible for the more stringent retention of the CV-I complex, as the pores are made smaller due to the dehydrating effect of the alcohol. Thus, the tightly bound primary stain complex is difficult to remove, and the cells remain purple. Note: Be careful not to over-decolorize the smear with alcohol.
4. Counter stain (Safranin): This is the final reagent, used to stain pink those cells that have been previously decolorized. Since only gram-negative cells undergo decolorization, they may now absorb the counterstain.

Requirements:

1. Fresh culture sample: 24-hour agar culture of Staphylococcus epidermidis/ 24-hour agar culture of Bacillus subtilis/ 24-hour agar culture of Escherichia coli
2. Primary stain: crystal violet
3. Mordant: gram’s iodine
4. Decolorizing agent: alcohol (95% ethanol)
5. Counter stain: safranin
6. Bunsen burner
7. Inoculating loop
8. Microscope
9. Distilled water
10. Soft cotton or tissue paper
11. Microscopic Slides
12. China-marking pencil or permanent marking pen

 

Gram staining procedure.

1. Take a clean glass slide.
2. Using aseptic technique, prepare a smear of given sample organisms. Do this by placing a drop of water on the slide, and then transferring sample organism to the drop of water with a sterile, cooled loop. (Note: If bacteria are taken from a broth culture, the drop of water is not required. Place a loop of bacterial suspension directly on the glass slide.)
3. Allow smears to air-dry and then heat fix in the smear
4. Gently flood smears with crystal violet and let stand for 1 minute.
5. Gently wash with distilled water.
6. Gently flood smears with the Gram’s iodine mordant and let stand for 1 minute.
7. Gently wash with distilled water.
8. Decolorize with 95% ethyl alcohol. Note: Do not over-decolorize. Add reagent drop by drop until the alcohol runs almost clear, showing only a blue tinge.
9. Gently wash with distilled water.
10. Counterstain with safranin for 45 seconds.
11. Gently wash with distilled water.
12. Let the slide for air dry
13. Examine under 10X, 40X and under oil immersion.
14. As you observe each slide under oil immersion, record your observation in record file.
15. Draw a representative microscopic field.
16. Describe the cells according to their morphology and arrangement.
17. Describe the color of the stained cells.
18. Classify the organism as to the Gram reaction: gram positive or gram negative.

 

References

1. Cappuccino, J.G and Welsh C. Microbiology: a laboratory manual (2018). Pearson education limited, England. 11 edition.
2. Manandhar S, Sharma S (2006): practical approach to microbiology, Graphic plus printers, Kathmandu
3. Shah P.K, Dahal P.R, Amatya J (2009): Practical microbiology, Delta offset press, Thapathali, Kathmandu

Gram staining technique