Gram staining is a widely used differential staining technique in bacteriology, and it is also a type of counterstaining method. Unstained bacteria are extremely difficult to observe under a microscope because their refractive index differs minimally from the surrounding environment. After staining, a sharp contrast forms between bacteria and the background, allowing clear visualization of bacterial morphology, arrangement, and certain structural features for taxonomic identification. This staining method categorizes bacteria into two major groups: Gram-positive (G⁺) and Gram-negative (G⁻). The divergent color outcomes stem from differences in cell wall permeability to ethanol and resistance to decolorization, primarily determined by the thickness and structure of the peptidoglycan layer. Cells stained with crystal violet form an insoluble complex upon treatment with iodine solution, which is susceptible to decolorization by ethanol. For Gram-negative cells: ethanol or acetone disrupts the outer cell wall membrane, damages the peptidoglycan layer and cytoplasmic membrane, enabling the crystal violet–iodine complex to leak out of the cells. Subsequent counterstaining with another dye yields a red color. Although the red stain can penetrate pre-stained purple Gram-positive cells, the deep purple masks the red hue completely. For Gram-positive cells: ethanol dehydrates the thick peptidoglycan layer and shrinks its pores. The large molecular size of the crystal violet–iodine complex prevents it from passing through the cell wall, rendering the cells resistant to decolorization and retaining a purple color.
The standard Gram staining solution adopts the classic traditional Gram stain formula. It delivers clean background for direct smears of clinical specimens, strong contrast between cell nuclei and cytoplasm, easily distinguishable intracellular phagosomes, and typical characteristic staining patterns of bacteria.
S774843
| Component
| 4×10 mL
| 4×100 mL
| 4×250 mL
| 4×500 mL
| Storage
|
S774843A
| Crystal Violet Stain Solution
| 10 mL
| 100 mL
| 250 mL
| 500 mL
| RT. Store in the dark
|
S774843B
| Gram’s Iodine Solution
| 10 mL
| 100 mL
| 250 mL
| 500 mL
| RT. Store in the dark
|
S774843C
| Decolorizing Solution
| 10 mL
| 100 mL
| 250 mL
| 500 mL
| RT.
|
S774843D
| Safranin Stain Solution
| 10 mL
| 100 mL
| 250 mL
| 500 mL
| RT. Store in the dark |
Instructions for Use:
1. Smear preparation: Take the test bacteria and spread a thin layer in the center of a glass slide. Alternatively, drop a small amount of sterile water onto the slide, mix the bacteria thoroughly with the water, and spread into a thin film.
2. Drying: Air-dry the smear at room temperature, or heat gently over an alcohol lamp for rapid drying.
3. Fixation: Hold one end of the slide with the specimen side upward. Pass the slide quickly back and forth 3–5 times beside the flame of an alcohol lamp, 1 second each pass. Avoid excessive heat to prevent denaturation of bacterial proteins. Let the slide cool completely before staining. Methanol or ethanol can also be used for fixation.
4. Primary staining: Add Crystal Violet Stain Solution and stain for 1–2 minutes, then rinse off the stain with clean water.
5. Mordanting: Cover the slide completely with Gram’s Iodine Solution, incubate at room temperature for 1–2 minutes, then rinse with water.
6. Decolorization: Apply Decolorizing Solution and rock the slide for 10–30 seconds until the runoff decolorant no longer turns purple. Immediately flush with water to stop the reaction.
7. Counterstaining: Add Safranin Stain Solution, stain for 30–60 seconds, then rinse with water.
8. Drying and microscopic examination: Dry the slide and observe under an oil immersion objective.
Precautions:
1. Draw a circular mark on the back of the slide in advance before smearing to locate the specimen area during subsequent procedures.
2. Wear proper personal protective equipment when handling bacteria. Flame the mouth of the test tube briefly after removing the stopper, and finally sterilize the inoculating loop completely by flaming.
3. Do not hold the slide too close to the flame during heat fixation. The slide temperature should not exceed 60 °C; the back of the slide should feel warm but not hot when touched against the back of your hand.
4. Decolorization control is the critical step for Gram staining, and decoloring duration relies on operational experience. Over-decolorization may falsely render Gram-positive bacteria Gram-negative; insufficient decolorization can misclassify Gram-negative bacteria as Gram-positive.
5. Culture age affects staining results. Gram-positive strains may appear Gram-negative if cultured for too long, or when cells are dead or lysed.
6. Wear a lab coat and disposable gloves throughout operation for personal safety and health protection.
Staining Results:
Gram-positive bacteria
| Blue to purple
|
Gram-negative bacteria
| Red |
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