The Science Behind the Stain
Gram staining differentiates bacterial species based on the distinct compositions of their cell walls. This technique relies on how these different cell wall structures interact with a series of dyes and washes. The primary difference lies in the peptidoglycan layer, a polymer of sugars and amino acids that forms a mesh-like layer outside the plasma membrane.
Gram-positive bacteria possess a thick peptidoglycan layer, up to 80 nanometers in thickness. This thick layer effectively retains the primary stain. Conversely, Gram-negative bacteria have a much thinner peptidoglycan layer, typically only 2 to 7 nanometers thick.
Gram-negative bacteria also feature an outer membrane external to the peptidoglycan layer. This outer membrane contains lipopolysaccharides and phospholipids, which affect cell permeability. This outer membrane and thin peptidoglycan layer are key to how Gram-negative cells respond during staining.
Gathering Your Materials
Performing a Gram stain requires specific materials. You will need clean microscope slides, a bacterial culture to prepare a smear, a heat source such as a Bunsen burner or a slide warmer for heat fixing, and a microscope for observing the stained slides.
The staining process involves four key reagents. Crystal violet serves as the primary stain. Gram’s iodine acts as a mordant, forming a complex with the crystal violet inside the bacterial cells.
A decolorizer, typically a solution of alcohol or acetone, is used to differentiate between bacterial types. This solution washes away the crystal violet-iodine complex from Gram-negative cells. Safranin is the final counterstain, providing a contrasting color to the decolorized Gram-negative bacteria.
Safety is important when handling bacterial cultures and chemical reagents. Always wear personal protective equipment, including laboratory gloves and eye protection, for safety. Proper disposal of stained slides and used reagents should follow laboratory guidelines.
The Step-by-Step Procedure
Begin the Gram staining procedure by preparing a bacterial smear on a clean microscope slide. If working from a liquid culture, place a small drop onto the slide and spread it thinly; for solid cultures, emulsify a small amount of bacteria in a drop of sterile water. Allow the smear to air dry completely.
Once air-dried, heat fix the smear by passing the slide through a flame two to three times quickly, smear-side up. This step adheres the bacteria to the slide, preventing them from washing off during subsequent steps. Avoid overheating, as this can distort bacterial morphology.
Next, flood the entire smear with crystal violet solution. Allow the crystal violet to remain on the slide for 30 to 60 seconds. After this time, gently rinse the slide with distilled water for about five seconds.
Following the rinse, flood the smear with Gram’s iodine solution and let it sit for 60 seconds. The iodine acts as a mordant, forming a crystal violet-iodine complex within the bacterial cells. After 60 seconds, rinse the slide again gently with distilled water for five seconds.
The decolorization step requires careful timing. Hold the slide at an angle and apply the decolorizer (alcohol or acetone) drop by drop onto the smear. Continue adding the decolorizer until the runoff liquid from the slide appears clear, which usually takes 10 to 20 seconds. Immediately rinse the slide with distilled water.
Finally, flood the smear with safranin, the counterstain, and let it stand for 30 to 60 seconds. Safranin stains any decolorized cells pink or red. After 30 to 60 seconds, rinse the slide thoroughly with distilled water. Blot the slide dry gently with absorbent paper.
Interpreting Your Findings
After completing the staining procedure, observe the prepared slide under a microscope. The color of the stained bacteria reveals their Gram reaction. Gram-positive bacteria will appear purple, indicating that their thick peptidoglycan cell walls successfully retained the crystal violet-iodine complex.
Gram-negative bacteria will stain pink or red. This indicates their thinner peptidoglycan layer and outer membrane allowed the decolorizer to wash out the crystal violet-iodine complex, enabling the safranin counterstain to color them. Observing the colors accurately is the primary step in interpretation.
Beyond color, noting the morphology and arrangement of the bacterial cells is also important. Gram-positive bacteria commonly appear as cocci (spherical shapes), often arranged in clusters or chains, or as bacilli (rod shapes). Gram-negative bacteria also exhibit various morphologies, including cocci, bacilli, and spirilla (spiral shapes), and can be found in diverse arrangements. This visual information aids in characterizing the bacterial sample.