The Gram stain, developed in 1884 by Danish pathologist Christian Gram, is a crucial differential staining technique in bacteriology. By applying this method, bacteria can be classified into two major groups: Gram-positive (G+) and Gram-negative (G−).

  I. Purpose:

Understand the principle of Gram staining and become familiar with the procedural steps; also, be aware of the precautions associated with bacterial Gram staining.

II. Experimental Principle:

Microbial cells are small and transparent, making them difficult to distinguish under a conventional light microscope. Therefore, they must be stained to produce a distinct color contrast between the stained cells and the background, enabling clearer observation of their morphology and structure. Consequently, microbial staining techniques are an essential method for examining the morphology and architecture of microorganisms.

The Gram stain reaction is an important characteristic for bacterial classification and identification. It was developed in 1884 by the Danish physician Hans Christian Gram. The Gram staining method not only allows observation of bacterial morphology but also categorizes all bacteria into two major groups: those that stain blue‑purple are designated Gram‑positive, abbreviated as G+; those that stain red (the counterstain color) are designated Gram‑negative, abbreviated as G−. The differential response of bacteria to the Gram stain arises from differences in the composition and structure of their cell walls. In Gram‑positive bacteria, the cell wall consists primarily of a mesh‑like structure of peptidoglycan; during staining, ethanol treatment causes dehydration, which reduces the pore size of this mesh and decreases permeability, thereby retaining the crystal violet–iodine complex within the cell and preventing decolorization, resulting in a blue‑purple appearance. In contrast, Gram‑negative bacteria have a cell wall with lower peptidoglycan content and higher lipid content; ethanol treatment solubilizes these lipids, increasing cell‑wall permeability, which allows the crystal violet–iodine complex to be extracted and the cell to lose its initial color, followed by uptake of the counterstain (safranin), yielding a red color.

Gram staining requires four distinct solutions: a primary stain (basic dye), a mordant, a decolorizing agent, and a counterstain. As described in the basic principles of bacterial single‑staining methods, the primary stain is typically crystal violet. The mordant enhances the affinity or adhesion between the dye and the cell, helping the dye to remain firmly bound and preventing it from being easily washed away; iodine is a commonly used mordant. The decolorizing agent removes the stain from the cells; different cell types respond differently—some can be decolorized, while others cannot. A 95% ethanol solution is often employed as the decolorizing agent. The counterstain is also a basic dye, but its color differs from that of the primary stain. Its purpose is to impart a color to cells that have been decolorized, while leaving unstained cells retaining the original primary‑stain color, thereby distinguishing bacteria into two major groups: Gram‑positive and Gram‑negative. Safranin is a frequently used counterstain.

III. Materials Required for the Gram Staining Experiment

1. Strains: Escherichia coli, Bacillus subtilis

2. Equipment: microscope, slides, staining jar, etc.

3. Staining solutions: ammonium oxalate–crystal violet stain, Lugol’s iodine solution, 95% ethanol, and 0.5% safranin stain.

IV. Experimental Procedure

1. Smear Preparation: Prepare smears of Bacillus subtilis cultured for 14–16 hours and Escherichia coli cultured for 24 hours, respectively (note that the smears should not be too thick). Allow the smears to dry, then fix them. During fixation, pass the slide over a flame 1–2 times; avoid overheating—stop when the slide is no longer hot to the touch.

2. Dyeing:

(1) Primary staining: Add one drop of ammonium oxalate–crystal violet, allow to stand for about one minute, then rinse with water.

(2) Mordanting: Add iodine solution, rinse off any residual water, and allow it to sit for about one minute, then wash with water.

(3) Destaining: Carefully blot off the water from the slide, place it against a white background, and rinse with 95% ethanol until the effluent no longer exhibits a purplish hue, which typically takes about 20–30 seconds. Immediately rinse away the ethanol with distilled water.

(4) Counterstaining: Stain with safranin for 1–2 minutes, then rinse with water.

(5) Microscopic examination: After drying, observe under the oil immersion objective. Gram-negative bacteria appear red, while Gram-positive bacteria appear purple. The Gram stain reaction should be interpreted based on well‑dispersed bacteria; densely packed cells often yield false‑positive results.

(6) In the same experiment, a mixed smear was prepared on a single microscope slide using Escherichia coli and Bacillus subtilis, and Gram staining was performed for comparative analysis.

The key to Gram staining lies in strictly controlling the degree of alcohol decolorization: excessive decolorization can cause gram-positive bacteria to be misstained as gram-negative, while insufficient decolorization may lead to gram-negative bacteria being misstained as gram-positive. In addition, bacterial age also affects the staining outcome; for instance, prolonged incubation of gram-positive bacteria, or the presence of dead cells and partial autolysis, often results in a negative reaction.

V. Precautions

1. The key to the success or failure of Gram staining lies in the decolorization time: if decolorization is excessive, Gram-positive bacteria may be decolorized and mistakenly identified as Gram-negative; if decolorization is too brief, Gram-negative bacteria may be misidentified as Gram-positive. Therefore, it is essential to strictly control the decolorization time.

2. It is advisable to use bacteria cultured for 18–24 hours; if the cells are too old, cell death or autolysis may cause Gram-positive bacteria to yield a Gram-negative reaction.